ACID PHOSPHATASES A

REVIEW

BU11NHAM S. WALKER, M.D., HENRY M. LEMON, M.D., M. MOIRA DAVISON, P H . D . , AND MORTON K. SCHWARTZ, P H . D . Departments of Biochemistry and Medicine, Boston University School of Medicine, Boston, Massachusetts

In the classification of phosphatases proposed by Folley and Kay, 60 the phosphomonoesterases are divided into several subclasses according to source, opReceived for publication September 5, 1953. Dr. Walker is Professor of Biochemistry, Dr. Lemon is Assistant Professor of Medicine and Dr. Davison is Instructor in Biochemistry; Dr. Schwartz was Research Fellow, National Cancer Institute from 1950 to 1952. Review articles in the Journal are under the direction of E. A. Gall, M.D., Associate Editor. 807

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For purposes of this review, acid phosphatases are denned as phosphomonoesterases with optimal activities at pH values below 7, and in the majority of instances at pH values between 3.8 and 6.162 Acid phosphatases are found in many microbial species, in plant tissues, in nearly all animal cells, and probably in all such cells at some stage of their lifetime. In the chick embryo acid and alkaline phosphatases are demonstrable at the stage of the primitive streak130 and in all undifferentiated tissues. With later development, the phosphatases may disappear from some cells, and may assume specialized localizations in others. Most early studies of animal tissue phosphatase were in the alkaline pH range and these have been reviewed by Kay.101 A phosphatase of red blood cells, with an optimum pH of 6.0, was demonstrated by Martland and his associates123 in 1924, and corroborated by Roche in 1931.156 Acid phosphatase activity in human urine was first demonstrated by Demuth, 47 whose data also foreshadow the later discovery of tissue acid phosphatases. The acid phosphatases were not clearly demonstrated in animal tissues until 1934, when Davies40 showed that phosphatases exhibiting 2 pH optimums were present in liver and spleen of dogs. One of these optimums was at the expected pH of 9.0; the second was found at pH 4.5 to 5.0. At approximately the same time, Hotta 91 found that kidney phosphatase also had 2 pH optimums, at pH 9.0 and a second at pH 3.2. Bamann and Riedel11 were also among the first to demonstrate that animal tissues that hydrolyzed glycerophosphate in the alkaline range (pH 9.4) possessed a second optimum pH in acid mediums (pH 5.5). They also noted that the alkaline phosphatase was activated by magnesium ions, while the acid phosphatase was not. Bamann and Diederichs10 observed that the acid phosphatase activity of preparations of dried pork liver was not abolished by 6 days in N/20 acetic acid, and that the same treatment rendered alkaline phosphatase distinctly unstable to further storage at 30 C.

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PROSTATIC ACID PHOSPHATASE

Kutscher and Wolbergs"1 found acid phosphatase in larger quantities in urine of young men than in that of women or old men, and observed a high concentration of phosphatase in human ejaculate and prostate, with a pH optimum of about 5, with no activity in the alkaline range and no activation by magnesium ions as with alkaline phosphatase. Interest in acid phosphatase was quickened by the now classical reports of the Gutmans and their collaborators. These authors first demonstrated increased acid phosphatase activity at the site of osseous metastases from prostatic carcinoma.79 Later reports showed elevated acid phosphatase levels in serum of patients with metastatic prostatic carcinoma.12'7S> 78 With this fact established, serum acid phosphatase determinations became an aid to the diagnosis of prostatic cancer. It cannot be assumed that the serum enzyme level is raised because cancerous prostatic tissue produces increased amounts of phosphatase, since Woodard' 95 demonstrated that the activity of this enzyme in carcinomatous prostatic tissue averaged somewhat less than in the normal or hypertrophied gland. When prostatic cancer metastasizes, invasion of lymph or blood channels is accompanied by escape of the prostatic secretion into the circulation.74, 136,196 Operation88 as well as digital manipulation182 of the prostate can cause a transient rise in the serum acid phosphatase. Dean and Woodard41 have proved that it is possible to show whether a tumor does or does not originate in the prostate by measuring the acid phosphatase activity of extracts of tissue removed through the cystoscope or proctoscope. This differentiation is possible because the phosphatase activity of prostatic tissue is 100 to 1000 times as great as that of bladder or rectal carcinomatous tissue. In 1941, Gomori65 showed by histochemical means that acid phosphatase activity is localized in the glandular epithelium of the human prostate. Some years previously, Moore and his co-workers'32 had established that the maintenance of the secretory cells of rat prostate depends upon the presence of androgens. This is in accord with the work of the Gutmans76 who found that the prostate in childhood contains a negligible amount of phosphatase. It also substantiates their report77 that precocious puberty, pro-

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timal pH, and the effect of magnesium ion upon activity at optimal pH. Two classes of acid phosphomonoesterases of animal origin are designated by these authors. Class A II has an optimal pH between 4.5 and 5, is not activated by magnesium ion, and hydrolyzes beta-glycerophosphate more rapidly than the alpha isomer. This enzyme had been reported at that time from mammalian spleen, liver, pancreas and kidney. Class A IV, found in mammalian erythrocytes, has an optimal pH of 6, is activated by magnesium ion and hydrolyzes alpha-glycerophosphate more rapidly than beta-glycerophosphate. We shall limit our discussion to acid phosphatases of man, with only such references to other species as may help clarify relationships to human metabolism and human disease. An earlier review by Sunderman184 cites many clinical reports not mentioned by us.

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duced in the rhesus monkey by the injection of testosterone propionate, causes a maturation of the prostatic epithelium accompanied by a rapid increase in acid phosphatase concentration to adult levels. These facts are the basis of the work of Huggins and Hodges,95 demonstrating that castration or injection of large amounts of estrogen causes a sharp reduction of the serum acid phosphatase level in the blood of patients with prostatic cancer. A significant but more gradual decrease follows bilateral adrenalectomy.94 In 11 dogs, Hertz and his group87 were unable to produce significant elevation of serum acid phosphatase by surgical grafting of autologous prostatic tissue. Upon histologic examination, the grafts appeared to be well vascularized and actively secreting. Intensive administration of androgens to the animals bearing the grafts also failed to increase the serum acid phosphatase. These observations in dogs are in contrast to the human cases studied by Woodard and Dean,196 where elevation of serum acid phosphatase occurred in the presence of small outgrowths of malignant prostatic tissue. No pure crystalline preparation of prostatic (or other) acid phosphatase has yet been reported. Partial purification has been accomplished in several different ways. Kutscher and his co-workers109 subjected ejaculate or autolyzed prostatic tissue to electrodialysis, adsorption on alumina, elution with citrate and ammonium sulfate fractionation. This procedure resulted in 100- to 120-fold concentration. Dmochowski and Pracowity48 and Anagnostopoulos4 have also reported preparations by ammonium sulfate fractionation. Hudson and Butler92 used 35 per cent ethanol to precipitate acid phosphatase from human ejaculate at pH 3 and 0 C. The precipitate was extracted with water, the extract adsorbed on alumina and eluted with acetate buffer at pH 3. The product contained about 40 per cent of the original activity and had a purity index of 1000 times that of the starting material. A simple method of partial purification has been developed by one of us (MMD) in our laboratory. Slices of prostatic tissue were extracted overnight with 0.85 per cent NaCl at — 7 C. After dialysis against tap water the extract was lyophilized. Active precipitates were obtained when an aqueous solution of dried powder was subjected to ammonium sulfate or ethanol fractionation at 0 C. Finally, inert protein was removed by precipitation at pH 4.9. This procedure produced a 4-fold increase in the activity/nitrogen ratio as compared to the original extract. Electrophoretic analysis defined 3 major components of which the active fraction was about 10 per cent of the total material and had an isoelectric point calculated at pH 4.5, comparable to the value of 4.4 reported by the Kutscher group. Some of this work is reported in 2 recent papers (see addendum references Nos. 2 and 3). Transphosphorylation to biologic alcohols from organic phosphate donors occurs in the presence of prostatic 6 or seminal acid phosphatase. 70 This direct transfer of phosphate groups seems to require that the phosphate donor and acceptor react simultaneously with the same enzyme molecule. In similar reactions utilizing purified citrus phosphatase, Axelrod8 was able to demonstrate, by the use of tagged phosphorus, that the transported phosphate does not go through the state of phosphate ion.

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The prostatic secretion is at least 50 times more potent in acid phosphatase activity than any other normal or abnormal body fluid (including purulent exudates) studied by Hansen, 8 ' who based a medicolegal test for identification of seminal material upon this fact. Normal human ejaculate averages 5.5 X 103 King-Armstrong units of acid phosphatase per ml.120 The medicolegal test can be applied conclusively to seminal stains after 14 months 66 or 2 years' 02 of storage under ordinary conditions. ACID P H O S P H A T A S E S O F BODY F L U I D S

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The urinary excretion of acid phosphatase in girls and women of all ages averages about 50 King-Armstrong units per 24 hours. Young boys have a similar rate of acid phosphatase excretion, but at puberty a sharp increase occurs. Maximal excretion occurs during the fourth decade, reaching a maximum of about 350 units per day.27 In general, adult men have approximately 5 times the urinary acid phosphatase output of adult women.175'192 The excess of acid phosphatase excretion in men originates from prostatic secretion, as indicated by a number of indirect evidential points. Prostatic fluid, as obtained by massage, increases in acid phosphatase concentration during puberty. 106 Psychosexual stimuli increase the acid phosphatase output in the urine of men, but not of women.32 Urine obtained from adult male subjects by catheterization contains much less acid phosphatase than urine voided normally by the same subjects.'75 Courtois and his collaborators have found no relationship between the phosphatase activity of serum at pH 5.2 and that of urine. Courtois and Plumel37 have presented evidence of 2 distinct phosphatase fractions in male urine: the first was precipitated at 60 to 66 per cent saturation with ammonium sulfate and inactivated by higher salt concentrations; the second fraction was precipitated by ammonium sulfate only in the neighborhood of saturation. It is probable that the first fraction is of prostatic origin, since the urine of women does not show the first optimum of precipitation, but does show it upon addition of prostatic secretion. The origin of the nonprostatic portion of urinary acid phosphatase from red cells was originally proposed by Waldschmidt-Leitz and Nonnenbruch. 190 The rate of destruction of red cells was found adequate to explain the amounts of acid phosphatase in the urine of women, but not of men.104 Inconsistently with this concept, the acid phosphatase in the urine of women is inhibited by M/1.0 tartaric acid, and not by 0.5 per cent formalin, behaving toward these reagents exactly like the acid phosphatase of male urine.44 Colling and Rossiter34 reported both alkaline and acid phosphatases in human cerebrospinal fluid. The activity of alkaline phosphatase was correlated with the protein concentration and with the cell count of the fluid. Such correlation did not hold for acid phosphatase. Increases of both phosphatases occurred in cases of poliomyelitis, and very striking increases in meningitis. Increases in syphilis were smaller and of doubtful significance. Acid phosphatase activity has also been demonstrated in saliva61 and in amniotic fluid.176

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The serum acid phosphatase level of women is approximately the same as that of men. The acid phosphatases of serum and of prostate react similarly to most inhibitors and activators. The chief difference is the inhibition of prostatic acid phosphatase, but not of normal serum acid phosphatase, by alcohol and certain other narcotics, first noted by Kutscher and Worner" 2 and utilized by Herbert86 in her method for the estimation of prostatic phosphatase in serum. In the same paper, Herbert summarized the early work on the utilization of determinations of serum acid phosphatase in the diagnosis of prostatic carcinoma, and indicated the value of alcohol inactivation, particularly in cases where the phosphatase elevation was minimal. Bensley and his co-workers20 found 1 to 4 Gutman units78 of serum acid phosphatase in 63 adults with no demonstrable disease of bone, liver, biliary tract or prostate. In 242 adults without disease of the prostate (but including the 63 "normals"), 21 instances were noted of elevation above 4 Gutman units, and up to 8 units. Ten of these 21 persons had carcinoma not involving the prostate, 6 hepatic disease, 2 osteitis deformans, 1 multiple myeloma, 1 cholelithiasis with obstructive jaundice and 1 chronic rheumatoid arthritis with osteoporosis. Out of 145 persons with benign prostatic adenoma or hypertrophy, 5 had values of serum acid phosphatase slightly above normal (maximum, 6 Gutman units). In 51 patients with prostatic carcinoma without evidence of metastases, 36 normal serum acid phosphatase values were observed, 9 values being between 5 and 8, and 6 having values of 9 or above. Of 40 patients with prostatic carcinoma with metastases, 11 had norma) values, 10 values between 5 and 8, and 19 values of 9 or above. Woodard195 found a range of 0.00 to 0.89 units, equivalent to Bodansky units,24 of serum acid phosphatase in 103 women and a range of 0.00 to 0.98 units in 389 men without disease of the prostate or nearby structures. This author reported no increase above these levels in any persons studied other than those with prostatic carcinoma or having conditions such as trauma that might result in escape of prostatic secretion into the blood. Not all patients with prostatic carcinoma had values of serum acid phosphatase above the normal range. Herbert, 86 and the Bensley group,20 used the Gutman method, which involves the use of phenylphosphate. It should be noted that workers who have used this technic have found moderate elevations of serum acid phosphatase in a small number of patients without prostatic carcinoma. Many of these patients had carcinoma of nonprostatic origin or bone disease or liver disease. Woodard, on the other hand, used the Bodansky method, which utilizes glycerophosphate as the substrate. Her studies made it clear that when beta-glycerophosphate was used as the analytic substrate, increased acid phosphatase activity was seldom observed in human blood plasma except in cases of prostatic malignancy, trauma or similar conditions permitting leakage of prostatic secretion into the circulation.' 96 Elevations of beta-glycerophosphatase activity have not been observed in women. The beta-glycerophosphatase activity in healthy women (mean value 0.36 units) was only slightly less than in healthy men (mean value 0.39 units). The units were modified Bodansky units.194

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Abul-Fadl and King2 have proposed differentiation of plasma acid phosphatases of prostatic, erythrocytic and other origins on the basis of differences in inhibition by 0.5 per cent formaldehyde. Kintner 106 recommends the routine use of formaldehyde inhibition, which reduces the activity of nonprostatic acid phosphatase, but does not affect enzyme of prostatic origin. Alcohol, contrariwise, inhibits prostatic acid phosphatase completely, and some other phosphatases partially. Inhibition by L-tartrate has been successfully utilized by Fishman and Lerner58 to characterize the prostatic portion of serum acid phosphatase. This differentiation is not perfect,3 but is adequate to distinguish cases of prostatic cancer from those cases where serum acid phosphatase is elevated as a result of other causes. ACID PHOSPHATASES OF NONPROSTATIC CELLS AND TISSUES

Acid phosphatase is demonstrable consistently and in appreciable concentration in adult epithelium and in numerous other tissues. In embryonic tissues it is also consistently present. These conclusions are based in part upon the specific catalytic effects observed in tissue extracts, and in part upon histochemical methods with the observation of reaction products by means of the microscope. The best known histochemical technic for acid phosphatase is that of Gomori.66 The method involves incubation of microtome-cut sections at 37 C. and pH 5 in a solution of lead alpha-glycerophosphate. The lead phosphate deposited by acid phosphatase activity is made visible by conversion to lead sulfide. By this procedure Gomori demonstrated acid phosphatase consistently and in large quantity in human prostate (dog prostate was sometimes negative) and in spleen of all species studied (man, dog, gopher, guinea pig, cat, mouse and ground hog). The blood cells and lymph nodes of all species were negative. Gastric mucosa was negative in dog, cat and rat, but usually positive in man, with a patchy distribu-

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When, on the other hand, phenylphosphate is used as the analytic substrate, osseous metastases of breast or thyroid cancer may also give rise to definite elevations of serum acid phosphatase activity. In addition to the evidence reported by the Bensley group,20 and already summarized, our own experience has been that nearly 40 per cent of patients observed with advanced cancer of the breast, totaling over 180 in number, have had transient or persistent increases in serum acid phosphatase, above 4 Gutman units. With extensive bone disease of nonneoplastic origin, we have seldom found acid phosphatase activity above normal levels. Occasionally one notes elevations in Paget's disease or in hyperparathyroidism, in myelosclerosis associated with idiopathic myeloid metaplasia, or in nephritis, acute hepatitis or hepatic necrosis. Neither in our observations nor in the reports of Herbert or the Gutmans has there been any correlation between acid and alkaline phosphatase activity in the serum. A survey of our studies of metastatic cancer (excluding prostatic cancer) over a 4-year period indicates that high serum acid phosphatase is associated with an advanced or rapidly advancing stage of the disease and with extensive metastases, usually osseous. Some few patients with initial high phosphatase demonstrated a drop to lower levels accompanying a remission induced by roentgen, hormone or combined therapy.

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tion involving all types of cells in a non-uniform manner. The intestine stained faintly, the liver variably, sometimes showing a positive response only in the Kupffer cells. Kidney was uniformly positive, but varied in the distribution of the stain. Adrenal medulla was consistently positive, the cortex positive only in man and the guinea pig. Negative reactions occurred consistently in pancreas, lungs, bronchi, male genital system (exclusive of the prostate as noted above, and exclusive of seminiferous tubules in cat), female genital system (except corpora lutea of dog and guinea pig), muscles, bones and skin. Increases of acid phosphatase were observed in chronic inflammation and in some, but not all, malignant epithelial tumors. Certain modifications of technic were made later by Gomori.66 He also emphasized that the method often yielded patchy results, and occasionally failed to indicate activity in tissues where the enzyme was definitely known to be present. Wolf and his associates'93 made technical modifications in the Gomori method and obtained consistently positive results in nuclei and in hepatic cells. Wachstein189 reported acid phosphatase as predominating over alkaline phosphatase in the cytoplasm of mammalian liver cells. Nuclei possessed both types of phosphatase activity, increasing during mitosis. Acid phosphatase activity was decreased in protein-depleted animals. Areas of guinea pig colon heavily invaded with Endamoeba histolytica show strong acid phosphatase activity by the Gomori technic, as do the amebae themselves.28 Dalgaard39 has added to the Gomori method the quantitation of the deposited phosphate by utilizing a glycerophosphate substrate containing radiophosphorus. Seligman and Manheimer177 developed the use of calcium alpha-naphthyl phosphate as a substrate in the histochemical demonstration of acid phosphatase. Alpha-naphthol, liberated by phosphatase action, was coupled in situ with anthraquinone-1-diazonium chloride, yielding an insoluble azo dye. Their results were similar to those obtained by others with the Gomori stain, except that dog prostate was positive. A qualitative difference could be shown between (a) the acid phosphatase of normal serum and skeletal muscle and of the nuclei of prostatic epithelial and stromal cells, which was not inhibited by 4 per cent tartaric acid, and (b) the acid phosphatase of prostatic epithelial and stromal cytoplasm, which was completely inhibited. False-positive results with the Gomori technic have been reported,13' m particularly in neural tissues. Such results have been explained on the basis of nonenzymatic hydrolysis of organic phosphates. Holter 90 has reported a growing body of criticism of the strictly histochemical methods applied to the localization of enzymes within cells. Rabinovitch and his colleagues150 reviewed the Gomori technic step by step, and compared the results of staining rodent liver with those obtained by direct estimation by the method of Gutman and Gutman. 78 The steps in the histochemical method up to and including embedding were found to have a notable inactivating effect upon acid phosphatase. In spite of this handicap, the modified technic was found to be specific, although not quantitative.

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In the cells of the anterior pituitary gland of guinea pigs, Abolins and Abolins1 demonstrated at least 2 acid phosphatases. One with optimal pH between 5.5 and 5.0 was typical of the nucleus, and another, most effective at pH 6.6, was found in cytoplasm and on cell surfaces. Some less definite evidence suggested a third enzyme in both locations, with a 4.3 pH optimum. A modification of the Gomori method was used in these studies. Goetsch and his co-workers63 dismissed as artifacts most demonstrations by the Gomori method of nuclear acid phosphatase. They incubated the paraffin ribbons directly in the substrate, thus avoiding the loss and spread of phosphatase activity during deparaffinization. With this technic, nuclei of rat pituitary gland and spleen, and of human bladder papilloma showed no acid phosphatase staining.181 Stafford and Atkinson179 had previously reported notable loss, up to 95 per cent, of acid phosphatase activity of tissues during the processes of fixation and imbedding. The nuclear localization of both acid and alkaline phosphatase is supported by the work of Rabinovitch and Andreucci,149 who worked with human bone marrow cells, using the Gomori technic with certain modifications including fixation in formol vapor. Cytoplasmic reaction for acid phosphatase in these cells was nearly negative. Nonspecific and eosinophilic granules were also negative, but neutrophilic granules were variable. Nucleoli were stained lightly or negatively for acid phosphatase, but were surrounded by deeply staining areas corresponding to the "nucleolus-associated chromatin" of Caspersson.29 The specific glucose-6-phosphatase of liver has an optimal pH of about 6.5 and therefore might be classified as an "acid phosphatase." Active preparations of glucose-6-phosphatase were found slightly active against glycerophosphate, with

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Employing their own modification of the Gomori technic, Montagna and Noback129 found abundant acid (pH 4.7) phosphatase in the mast cells of rats. Its distribution was limited to cytoplasmic granules, with none in the nucleus. In the cells of a benign mastocytoma, and in the cultured cells from this tumor, acid and alkaline phosphatases were also observed in the nuclei.143 Deane and Dempsey42 found concentration of acid phosphatase, as well as of alkaline phosphatase, in the Golgi region of liver, kidney and intestinal cells of several mammalian species, and of the uterine epithelium of the pregnant cat and sow. Newman and his associates137 have indicated an artifact in the Gomori technic resulting from the precipitation of lead in the absence of substrate. This effect is only occasional at pH 4.7 or below, but is maximal and constant at pH 5.6, decreasing at higher pH values. This finding does not invalidate the technic if adequate controls are used. In fresh frozen sections of adrenal medulla, Eranko 66 found no nuclear acid phosphatase by the Gomori procedure, but observed a strong cytoplasmic reaction. In sections fixed in 4 per cent formaldehyde for several hours, this rinding was reversed, showing a definite nuclear but only a weak cytoplasmic darkening. Tsuboi187 found no difference in acid (pH 5.5) phosphatase activity between normal and regenerating livers in mice. The activity was concentrated in the microsomal fraction of normal liver, but in the nuclear fraction of regenerating liver.

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an optimum between pH 6 and 7.185 The extreme lability of this enzyme and its optimum so different from those of the usual acid phosphatases, indicate that it is a different enzyme. A nonspecific acid phosphatase, in addition to glucose-6-phosphatase, is demonstrable in liver tissue. Berthet and Duve22 presented evidence that the nonspecific acid phosphatase in rat liver was bound by a labile linkage to mitochondria. Phosphatase so bound was inactive against beta-glycerophosphate, but activity could be demonstrated after aging in the cold, after freezing and thawing several times, and most simply by 3 minutes in the Waring blendor. The mitochondrial fraction, separated by differential centrifugation and washed once, contained 55 to 60 per cent of the total acid phosphatase of the original homogenate prepared in isotonic sucrose by the Potter-Elvehjem method. The remaining acid phosphatase activity (measured at pH 5) was divided between smaller granules and the final supernate. Only 4 to 6 per cent of the total enzyme was found bound to the nuclei. In a further study of the nonspecific acid phosphatase of rat liver, Berthet and his associates21 concluded that the cytoplasmic granules, which bind the phosphatase, possessed a semipermeable membrane and could be disrupted in hypotonic mediums. The slowest release of phosphatase observed was in 0.25 molar sucrose at 0 C , increasing rapidly with increase of temperature. Full activation occurred after 2.5 hours at 38 C , or within 1 hour after addition of 15 to 25 volumes of distilled water. In salt solutions, the enzyme was liberated, even in hypertonic mediums. The mitochondrial membrane was therefore considered permeable to salts and water, impermeable to sucrose. Other experiments indicated impermeability to glycerophosphate, and prompt activation in the presence of glycerol, saponin or desoxycholate. Between pH 5.5 and 6.5, stability was maximal. Acid phosphatase may remain adsorbed to disintegrated particles, or may even be adsorbed onto intact mitochondria, but such adsorption does not modify enzyme activity. This differentiates the original or "native" linkage, in which the enzyme is inactive, from adsorption. Using methods involving direct separation of cell structural components, Novikoff and his co-workers140 demonstrated the presence of 35 to 50 per cent of the acid phosphatase of liver in the ground plasm, 35 to 40 per cent in the large granules and 10 to 28 per cent in other cell constituents. The same authors found the distribution of alkaline phosphatase to be even more predominantly (55 to 70 per cent) in the ground plasm. Drabkin and Marsh 51 found very consistent levels of acid (and alkaline) phosphatase activity in liver homogenates of normal rats. Significant increases were demonstrated in both types of phosphatase activity when rats were made diabetic with alloxan, followed by a return to normal with insulin therapy. Rat liver, in the process of regeneration following partial surgical removal, showed increased acid phosphatase activity in the nuclei of hepatic cells, but not in the cytoplasm.183 In normal liver cells, acid phosphatase activity by the method of Wolf and his collaborators193 was found chiefly in chromatin and nucleoli, but in regenerating liver all parts of the nucleus showed increased acid (and alkaline)

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Our experience with the Gomori method indicated, in agreement with Wolf and his group193 that the reaction was localized primarily in the nuclei of most cells, using unfixed tissues preserved by freezing at —70 C. and short incubation periods of 15 to 30 minutes for the development of the reaction. In cancerous tissues, only the nuclei were heavily stained. In nonneoplastic tissues, variable amounts of precipitate appeared in the cytoplasm. In prostatic epithelium and in neurons the cytoplasmic reaction was rapid and intense, obscuring the nuclear reaction. Distinct granular cytoplasmic precipitation was typical in bronchial mucous glands, and in specimens of benign hyperplasia of corpus luteum, adrenal medulla and parathyroid gland. Fixation by organic solvents destroyed much of the enzyme, so that longer incubation periods were required, and there was greater opportunity for migration of enzyme or products. Ohlmeyer142 described a chemical bonding between prostatic acid phosphatase and desoxypentose nucleic acids below pH 5. This could be displaced by substitution with certain proteins. Progress in the purification of acid phosphatases of organs other than the prostate has been limited. Ruffo167 purified the acid phosphatase of dog liver by the use of 50 per cent ethanol to extract the enzyme from a homogenate. Inert material was removed at pH 4.5 to 5.0. Alkaline phosphatase was then precipitated in the cold at a concentration of 70 volumes per cent of ethanol. Acid phosphatase was precipitated in the cold at a concentration of 34 volumes of acetone, following the removal of an inactive fraction precipitated at a concen-

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phosphatase activity. Norberg138 found that acid phosphatases regenerated proportionally to the liver mass, with a sudden increase on the third day of regeneration. Dounce49 isolated nuclei of rat liver cells and found in these a ratio of acid phosphatase to alkaline phosphatase activities of about 1.5, as compared to ratios as high as 13 in whole liver. In the acid phosphatase determination (pH 5) nuclei were not broken, whereas at the higher pH of alkaline phosphatase nuclear disruption did occur. It was noted that the acid phosphatase activity may have been limited by the rate of diffusion of substrate into the nuclei. Phenyl phosphate was the substrate used. A similar separation of rat liver cells into nuclear and cytoplasmic fractions was carried out by Palade.144 He found, using betaglycerophosphate substrate, that over 90 per cent of the total liver enzyme was in the cytoplasmic fraction. Histochemical studies on samples of the same tissues by the improved Gomori method demonstrated the usual nuclear localization. Palade attributed the divergent results of the histochemical studies to differences in phosphate affinity among the cell organs, and pointed out that the lead phosphates were sufficiently soluble, and were formed sufficiently slowly in the histochemical procedure, to permit their diffusion. Eger and Geller,54 however, in a histochemical study of frozen sections of rat liver, found that in these unfixed sections the earliest acid phosphatase reaction occurred in the nucleolus, followed by granular staining of the nucleus, eventually becoming generalized. In human placenta acid phosphatase becomes demonstrable near the end of gestation, localized in the nuclei and perinuclear cytoplasm of the syncytium.191

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tratioii of 26 volumes per cent. The product obtained was described as containing the acid phosphatase in a most remarkable grade of purity. Perlmann and Ferry146 used ammonium sulfate to fractionate beef kidney extracts. They found that the acid phosphatase was a water-soluble protein and the alkaline phosphatase, a water-insoluble, salt-soluble globulin. The fraction separating at 2.7 M showed both acid and alkaline activity, while that separating at 3.0 M showed only acid activity. Sables168 was unable to separate the acid and alkaline phosphatases of dog liver or kidney by the method of Perlmann and Ferry. The suggestion has been made that "acid" and "alkaline" phosphatases are not distinct entities, but that the pH of optimal activity is determined by changes in the equilibrium between prosthetic ions or groups of the enzyme.169 Effects of altering zinc and magnesium concentrations produced effects on the phosphatase of Penicillium chrysogenum, Q-176, which tend to bear out this suggestion. The author admitted that application of this theory to the enzymes of higher animals entailed further complications. In direct opposition to this concept, chromatographic separation of acid and alkaline phosphatases of liver and of serum has been reported.62 Blood cells. Although Gomori, in his first publication on the histochemical method for acid phosphatase, 66 reported none found in blood cells, the acid phosphatase of red cells had already been described.123' 166 In later studies, Roche and his co-workers163 identified 2 acid phosphatases with different pH optimums in the red cells of rats and cattle. These results were confirmed by Abul-Fadl and King3 and extended to other species. In human erythrocytes, the optimums lay at 5.3 and 4.4, with some variation among individuals. Rossiter166 demonstrated that surface-active substances liberated large quantities of alkaline phosphatase from rabbit polymorphonuclear leukocytes into the surrounding fluid. This phosphatase could not be measured if estimations were done on suspensions of intact cells. Using saponin to extract the enzyme and disodium phenylphosphate and alpha- and beta-glycerophosphate, as substrates Cram and Rossiter38 identified and studied the acid and alkaline phosphatase of rabbit polymorphonuclear leukocytes. The mean concentrations of alkaline and acid phosphatase were 207 ± 68 and 7.8 ± 3.7 King-Armstrong units per 1010 cells. Because of the difficulty in obtaining pure lymphocytes from thoracic duct cannulations, no data are available on the phosphatase distribution in lymphocytes. However, by statistical analysis of phosphatase activity of mixed leukocytes, it was found that alkaline phosphatase was confined chiefly to the polymorphonuclear leukocytes and the acid phosphatase chiefly to the lymphocyte.80 The human lymphocytes have twice the acid phosphatase activity of the rabbit lymphocytes. Beck and Valentine 16, 188 have reported on the phosphatase activity of human leukocytes in health and in various blood diseases. It was shown that neutrophilic leukocytosis was accompanied by extremely high alkaline phosphatase levels per leukocyte, but normal acid phosphatase. On the other hand, the leukocytes of chronic myelocytic leukemia contained low levels of alkaline and normal to high acid phosphatase, and those of chronic lymphatic leukemia and acute

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blastic leukemia showed low levels of activity of both acid and alkaline phosphatase. In cases where the leukocyte levels were brought to normal with therapy, the phosphatase activity approached normal levels. They concluded that alkaline phosphatase resides chiefly in the cells of the granulocytic series, and that the acid phosphatase is probably uniformly distributed throughout both the lymphatic and granulocytic cells. Nervous system. According to Wolf and his collaborators,193 acid phosphatase is present in the axis-cylinders of both central and peripheral neural tissue, but absent from the myelin sheaths. Lassek and Hard115 concluded from a study of cats of various ages, "that variations in the time of appearance of the enzyme in the axons are to be correlated with the phylogenetic background of the several nerve tracts as well as the ontogenetic age of the animal." The ventral and dorsal roots, the sensory tracts within the cord, the medial longitudinal fasciculus and tecto-spinal tracts in the brain stem, all gave evidence of the enzyme in newborn kittens. The higher brain centers and the pyramidal tract reacted weakly or not at all to acid phosphatase tests at birth, but progressively showed an increased reaction reaching the adult state in the cat at an age of about 70 days. Following removal of the cells of origin of the pyramidal tract in cats, acid phosphatase disappeared from the degenerating axons within 3 days, and did not reappear.82 The situation in monkeys differed somewhat; approximately 15 days elapsed between cortical extirpation and the disappearance of axonal acid phosphatase in the pyramidal tract. The enzyme disappeared more quickly from the larger axons, while the glial cells in the degenerated field showed notable increase in acid phosphatase activity.116 The reliability of the histochemical methods used in the above work has been questioned13' U4 Assay for acid phosphatase in sciatic nerves of cats by direct incubation of the tissue with phenyl phosphate 85 confirmed the presence of the enzyme. In the peripheral stumps of transected sciatic nerves, increases in acid phosphatase activity occurred for the first 3 weeks, after which activity was comparable to that of normal sciatic nerve. Bodian and Mellors26 accomplished fixation of spinal cord in monkeys by intravascular perfusion of about 1 liter of chilled acetone through the anesthetized animal. Tissue specimens were then removed promptly and kept in chilled acetone overnight. After 2 24-hour passages through absolute alcohol and 2 through toluene, the tissues were imbedded in paraffin. Such preparations retained phosphatase activity for 3 months or more. The staining procedure used was similar to that of Wolf and his co-workers,193 with certain modifications. These included several alternative substrates—sodium glycerophosphate (52 per cent alpha), calcium hexosediphosphate, adenylic acid, yeast nucleic acid and a mixture of pentose nucleotides. All yielded positive results, with sharper staining from the substrates of adenylic acid and pentose nucleotide. This was taken as an indication of ribonuclease activity in addition to acid phosphatase. Employing these special technics, the acid phosphatase of anterior horn cells of rhesus monkeys was studied during regeneration following axon amputation. The maximal acid phosphatase activity was observed when Nissl-body breakdown was at its maxi-

JULY 1954

ACID PHOSPHATASES

819

SUBSTRATE

SPECIFICITY

The question whether the acid phosphatases of different tissues, or even within the same cell,1 are the same enzyme has attracted the interest of many workers. If phosphatases differ, they should have different substrate specificities as well as different pH optimums and different responses to the presence of activators and inhibitors. Many substrates have been employed in work on acid phosphatase. Hotta 91 phosphorylated aliphatic and aromatic alcohols and tested these as substrates for the acid phosphatases of kidney. He found that esters of aliphatic alcohols were hydrolyzed more rapidly than those of aromatic alcohols. Hotta also worked with pyrophosphates and N-phosphorylated compounds, and

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mum. However, high activity was maintained until the later stages, when Nissl bodies became normal again. These workers tentatively suggested a correlation of acid phosphatase activity with rate of nucleoprotein synthesis in the regenerating cells. The acid phosphatase activity of cat sciatic nerve increased after the nerve was sectioned or crushed. This reached a maximal value in 16 days and then slowly decreased. These studies, confirming the reports of Bodian and Mellors,25 were not histochemical, but were made on homogenates, using phenyl phosphate as a substrate. 89 The increase in acid phosphatase accompanied an increase in concentration of desoxypentosenucleic acid. Josephy98 has reported high acid phosphatase activity in the senile human brain, both in cells and fibers, and particularly in senile plaques. Following the method of Wolf and his group,193 he observed staining of nucleus and nucleolus of individual nerve cells, with diffuse cytoplasmic staining. McNabb,127 confirming studies done by earlier workers utilizing ox brain, found in dog brain that the activity of both acid and alkaline phosphomonoesterases in gray matter greatly exceeded that in white matter. The measured ratio of activity (gray matter:white matter) was 3.4 for acid phosphatase. The activity in gray matter was 2.58 mg. of phenol liberated per 100 mg. of dry weight per hour, after the procedure of Colling and Rossiter.34 Naidoo and Pratt, 135 working on frozen sections of rat brain, found that acid phosphatase staining was chiefly in the cytoplasm of nerve cells when glycerophosphate was used at pH 5.3 as the substrate. With adenosine triphosphate at pH 6.5, nuclei of glial cells and neurons stained preferentially, and with adenosine monophosphate at pH 6.5, the nerve fibers. The method was based upon the Gomori principle, but was quite different in its technical details. Applying the Dempsey and Deane46 modifications of the Gomori method to frozen sections of choroid plexus of rats, Leduc and Wislocki117 demonstrated acid phosphatase activity in both nuclei and cytoplasm of the epithelial cells with 5-hour incubation. With longer periods of incubation, intense nuclear reaction could be demonstrated in the pineal parenchyma, and moderate nuclear, cytoplasmic and interstitial reaction in the neurohypophysis. Variable intracellular reaction was observed in other structures forming the blood-brain barrier, except the walls of the cerebral vessels.

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It is unlikely that any specific phosphoprotein phosphatase exists, in the sense of an enzyme that would hydrolyze the phosphate groups from all known phosphoproteins. Enzymes or enzyme systems capable of splitting phosphate from casein have been reported in frog eggs,53 pig kidney119 and in a variety of tissues of rat,139 hamster, guinea pig and rabbit,57 but not in frog.103 Other investigations 5, 100' 154, 172 showed no action of phosphatases when casein was the substrate. Peptic or tryptic digestion of casein liberates phosphopeptones148 • 153 with phosphate groups readily split by phosphatases. Perlmann145 found that when casein was fractionated into alpha and beta caseins, the alpha casein lost about 40 per cent of its phosphorus in the presence of prostatic phosphatase, with optimal activity at pH 5.6. Beta casein was resistant to the enzyme, and actually appeared to inhibit its action upon alpha casein. Prostatic phosphatase also split about 46 per cent of the phosphorus from ovalbumin. Tests on synthetic substrates 146 showed that prostatic phosphatase catalyzed the hydrolysis of —0—P— linkages only. On the other hand, intestinal phosphatase, usually regarded as an "alkaline" phosphatase, hydrolyzed —N—P—• linkages at pH 5.3 and at pH 9, with only slight hydrolysis at pH 7, and with action on —0—P— linkages in esters of low molecular weight only in the alkaline range. Intestinal phosphatase liberated phosphate from ovalbumin at pH 5.3 and at pH 9. Crystalline rennin, when activated by ultrafUtrate of milk, liberated phosphate from phosphopeptone but not from casein or glycerophosphate.124 Hashimoto84 investigated the hydrolysis of both alpha- and beta-glycerophosphates by monoesterases with pH optimums of 3.2, 5.5 and 9.0. He found that the enzyme having a pH optimum at 3.2 hydrolyzed beta esters more rapidly

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concluded that different phosphatases were specific as phosphomonoesterases, pyrophosphatases or phosphamidases. The phosphamidases (phosphatases splitting a P—N bond) were originally studied by Ichihara, 96 who synthesized many amino phosphoric acid compounds, including phosphoric acid monamide, phenyl phosphoric acid monamide, and phenyl phosphoric acid anilide. He found that kidney extracts hydrolyzed these substrates equally. The hydrolysis occurred optimally at pH values of 9 and 3.3. The P—'N bond is not very stable in acid medium, and Ichihara was forced to establish his optimum pH as one where there was the greatest difference between the amount of phosphate liberated in the presence of the enzyme and the amount hydrolyzed spontaneously. He was able to make a partial separation of phosphomonoesterase from phosphamidase, since at a pH of 3.0 the phosphomonoesterase was completely adsorbed on a kaolin column. The phosphamidase was only partially adsorbed, most of it passing through the column. Gomori67 devised a histochemical method for the demonstration of sites of phosphamidase activity. Small amounts were present in many normal tissues; large amounts were found in the gray matter of the central nervous system and in certain malignant epithelial tumors. No clear physiologic function has been established for phosphamidase. It may be involved in tissue proteolysis and protein synthesis. It is also apparent 23 that the — S ^ P — bond can be enzymatically hydrolyzed, since s-phosphocysteine is hydrolyzed by extracts of rat kidney.

JULY

1954

ACID

PHOSPHATASES

821

Mononucleotides (3-adenylic and 3-guanylic acids) were rapidly hydrolyzed in the presence of prostatic phosphatase at pH 5.3, but there was no significant hydrolysis of ribonucleic acid under these conditions.170 Reis151 reported that the phosphatase of prostate and semen was composed of a powerful 5-nucleotidase and, to a lesser extent, of a glycerophosphatase. This conclusion could not be confirmed histochemically by Gomori.68 In his later investigation of specific 5nucleotidase, Reis152 compared the actions of adenylic acid nucleotidase having a pH optimum of 7.5 with acid phosphatase (pH 5.5) and alkaline phosphatase (pH 9.0). He used phenyl phosphate as substrate and compared the tissue distribution of enzymes acting at these 3 pH values on phenyl phosphate and at pH 7.5 on adenylic acid. He found 500 times more acid phosphatase (pH 5.2) in prostate tissue than in kidney cortex, the next richest tissue. Acid phosphatase was found in thyroid gland, testes, brain cortex, optic nerve, retina, posterior and anterior lobes of the pituitary gland, liver, lung, kidney, cartilage and

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than alpha esters. Enzymes operating optimally at pH 5.5 and 9.0 split the 2 substrates equally well. Hashimoto also demonstrated that erythrocyte acid phosphatase hydrolyzed alpha-glycerophosphate more rapidly than the beta isomer. The activity of red blood cell phosphatase upon alpha-glycerophosphate, but not upon beta-glycerophosphate, was shown to decrease after refrigeration. Splenic acid phosphatase hydrolyzed beta-glycerophosphate more rapidly than the alpha isomer. This is contrary to the situation with the red cell enzyme.40 A very extensive survey of substrate specificity was made by Fleury and his associates,69 who used kidney, bone and intestinal alkaline phosphatase, prostatic acid phosphatase, the phosphatase found in the urine of females and various vegetable acid phosphatases. The following substrates were used in the study: aminoethanol monophosphate, dimethyl aminoethanol monophosphate, diethyl aminoethanol monophosphate, morpholine ethanol phosphate, choline phosphate, benzyl phosphate, glucose-1-phosphate, methyl naphthohydroquinone diphosphate and beta-glycerophosphate. In summarizing their work, Fleury and his group concluded that although phosphatases from various sources could be grouped according to pH optimum and inhibitor effects, they were all broad in their action and overlapped in their effects on various substrates. The differences seen in the action of urinary and prostatic phosphatase indicated that these enzymes were not identical. Urinary acid phosphatase in the human male is considered to be chiefly prostatic in origin,31, 175 although the evidence is indirect. The smaller amounts of acid phosphatase demonstrable in the urine of women are inhibited by M/10 tartaric acid and not by 0.5 per cent formalin, behaving toward these reagents exactly like the acid phosphatase of male urine.44 Scott and Huggins175 measured phosphate liberated from phenyl phosphate by urine that had been previously dialyzed. The same principle was utilized by Delory and Hetherington.43 Burgen27 eliminated the preliminary dialysis and measured the liberated phenol with diazotized p-nitroaniline. Values by this method were shown to be misleading as a result of the work of Robinson and Warren,156 who demonstrated an apparent increase in phosphatase activity upon dilution of urine specimens.

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The action of prostatic phosphatase on phosphorylcholine was further studied by Hudson and Butler.92 These authors found that phosphorylcholine was not hydrolyzed by perfusion through the prostate, nor in the presence of a purified acid phosphatase preparation. However, phosphorylcholine was rapidly split by diluted unfractionated semen at a p l i greater than the optimum pH of prostatic acid phosphatase. These facts seem to indicate that human semen contains more than the 1 phosphatase of prostatic origin. These authors also investigated the action of purified acid phosphatase and semen on adenosine triphosphate, glucose-1-phosphate, glucose-6-phosphate, hexose diphosphate and fructose-6-phosphate. Semen and purified prostatic phosphatase acted similarly although there were differences in pH optimums among the various substrates. Acid phosphatases of liver, kidney and prostate hydrolyzed phosphorylcholine less rapidly than glycerophosphate, and phosphorylethanolamine still less rapidly, according to Roche and Bouchilloux.159 The alkaline phosphatases of liver and kidney were more effective than acid phosphatase in the hydrolysis of the latter 2 substrates, and catalyzed their synthesis in vitro, which acid phosphatase failed to accomplish. Pyrophosphorylcholine is not the coenzyme of muscle acid phosphatase.186 Active fractions of acid phosphatase preparations, separated by acetone and ammonium sulfate precipitation, differed in their relative activity toward different phosphate monoesters.164 Prostatic acid phosphatase does not act as a diesterase,171 and has been found not to hydrolyze diphenylphosphoric acid, glycerylphosphorylcholine, glycerylphosphorylethanolamine or the monophosphatides. Prostatic enzymes will cleave the monoester linkage of triphosphopyridine nucleotide,99 and will catalyze the phosphorylation of glucose and fructose.128 From the evidence cited above, it can be stated with certainty that "acid phosphatase" or acid phosphomonoesterase is not a single enzyme in a given spec-

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duodenal and jejunal mucosa. Little 5-nucleotidase was found in prostatic tissue and in intestinal mucosa. In all other tissue, the 5-nucleotidase was several times more active than the acid phosphatase. Lundquist121 found that the inorganic phosphate content of semen increased on standing. He also demonstrated that if the alcohol-soluble organic phosphate fraction of semen was treated with prostatic secretion, the organic phosphate compound was split into choline and inorganic phosphate. Human semen contains large amounts of choline and large amounts of phosphatase. The semen of bulls, which is low in phosphatase, is also low in choline content. From those observations Lundquist concluded that phosphorylcholine was the natural substrate of prostatic phosphatase and the function of the enzyme was to split this substrate. In a later work, Lundquist122 compared the action of prostatic phosphatase on phosphorylcholine, beta-glycerophosphate, and phenyl phosphate. These 3 substrates were found by him to be similarly attacked by the prostatic enzyme. The effects of inhibitors were similar, pH optimums were almost identical, and the heat of activation of the substrates as calculated by Lundquist was 11,300 cal./mol. for beta-glycerophosphate, 11,800 cal./mol. for phenyl phosphate, and 11,700 cal./mol. for phosphorylcholine.

JULY 1954.

ACID PHOSPHATASES

823

ies, but is an inclusive group, including enzymes of different substrate specificity, unified only by their characteristic catalytic effect, exerted in an acid environment, upon the hydrolysis of phosphoric monoesters. ACTIVATORS AND INHIBITORS

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Magnesium ion, which activates many alkaline phosphatases, is without effect upon the acid phosphatases of prostate,112 of liver158 and of muscle,110 but does activate the acid (pH 5.6) phosphatase found in the total homogenate of the adult worker honey bee.165 The activation of red cell acid phosphatases by magnesium ions has been reported several times 40, 97 ' 163 and at least once16 denied. The most recent report3 confirms magnesium activation of beta-glycerophosphate hydrolysis by the red cell enzymes, no effect with alpba-glycerophosphate and inhibition of phenyl phosphate hydrolysis. The acid phosphatases of liver and kidney are inhibited by fluoride, oxalate and trichloroacetate.17,18 Prostatic acid phosphatase activity is inhibited 82.6 per cent by 0.01 M sodium fluoride.112 Since this enzyme is inhibited by fluoride ion the presence of magnesium is suspected; however, analysis of ashed samples has not substantiated this hypothesis.160 Kutscher and Wiist113 postulated that if an element such as magnesium141 was a part of the enzyme molecule it would serve as the point of attachment between the enzyme and substrate. They showed that the usual phosphatase substrates, phenyl phosphate and beta-glycerophosphate, formed nondissociating compounds with magnesium in a manner similar to the fluoride-magnesium combination. The inhibition therefore was considered to be competitive between fluoride and the substrate for the magnesium attachment. Mattocks and Holtan126, however, demonstrated noncompetitive inhibition by fluoride of the acid phosphatase of sheep brain. Courtois and Anagnostopoulos36 reported that fluoride inhibition was related to the concentration of fluoride, and was the result of a reversible combination between the enzyme and the fluoride ion. Substances capable of combining with fluoride diminished the inhibition. The phosphatase of white mustard was completely inhibited by fluoride. However, it could be reactivated by dialysis, precipitation of the enzyme by acetone or ammonium sulfate, or by adsorption of the enzyme on alumina and then elution at alkaline pH. Fluoride did not act by precipitating metallic ions necessary for the activity of phosphatase, since calcium fluoride, magnesium fluoride and zinc fluoride exerted the same inhibition as sodium fluoride. The problem of differentiating erythrocyte acid phosphatase from the acid phosphatases of other tissues is of practical importance, since tissue enzyme preparations usually contain blood, and therefore erythrocyte phosphatase. Roche and Bullinger161 found that the acid phosphatase of erythrocytes (pH 6.0) was not affected by magnesium, fluoride or oxalate. Abul-Fadl and King2 proposed the use of specific inhibitors that would allow the study of either red blood cell or prostatic acid phosphatase in the presence of the other. These authors showed that 0.5 per cent formaldehyde had no effect on prostatic phosphatase but would completely inhibit the action of red cell phosphatase. It was also shown that fluoride almost completely inhibited the prostatic enzyme (97

824

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Prostatic acid phosphatase was inactivated by certain alcohols and by ureIhane.112 The principle of alcohol inhibition of prostatic phosphatase was applied to clinical estimations in serum by Herbert. 86 The question of alcohol inhibition is complicated by the phenomenon of transphosphorylation. 6 Sizer178 investigated the action of oxidants and reductanls on the phosphatases of beef lung and kidney at pH 4.5. He found that all reductants and mild oxidants were without effect, but that phosphatase activity was greatly decreased by the addition of strong oxidants. This inhibition was reversed by the addition of a reductant. The action of phlorizin on the acid phosphatase activity of kidney cortex and intestinal mucosa extracts at pH 5.0 was investigated by Beck,14 who found the kidney enzyme was markedly inhibited regardless of substrate. The intestinal enzyme was inhibited only when hexose phosphate was the substrate and not when glycerophosphate was used. Acid phosphatase of rat liver was inhibited by alloxan.147 Dumazert and his co-workers52 showed that substances that formed metallic complexes accelerated the action of liver phosphomonoesterase at pH 5.4. Bossard26 found that phosphatases of all types were strongly inhibited by molybdate ion. Courtois and Anagnostopoulos35 found that liver and kidney acid phosphatase at pH 5.2 was markedly inhibited by molybdic, phosphomolybdic, tungstic, phosphotungstic, metavanadic and uranylacetic acids. The necessity of free amino groups for acid phosphatase activity was investigated by Baccari and Auricchio.9 Nitrous acid, formaldehyde and phenylisocyanate all inhibited acid phosphatase activity. These substances are all known to react with free amino groups. Roche and Abul-Fadl157 confirmed this work using phenylisocyanate or formaldehyde to block the amino groups. Anagnostopoulos4 investigated the action of amino blocking reagents on acid phosphatase of prostate and beef liver. He found that although alkaline phosphatase and liver acid phosphatase were completely inactivated by ketene, nitrous acid, formaldehyde and phenylisocyanate, the acid phosphatase was slowly inhibited by nitrous

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per cent) but inhibited the red blood cell enzyme only slightly (10 per cent). In a later investigation,3 they found that ^-tartrate completely inhibited prostatic or liver acid phosphatase but had no effect on the erythrocytic enzyme or on the acid phosphatase of bile. The maleic acid radical activated the red blood cell and prostatic phosphatases slightly. Lactic, pyruvic, malic, malonic, mucic, fumaric, succinic and tartronic acid radicals were without effect. Red cell acid phosphatases were notably inhibited by cwpric ions, whereas the prostatic enzyme was slightly activated by 0.001 M copper sulfate. (For practical applications in diagnosis, refer back to the section on prostatic acid phosphatase, and also see Delory and his associates45 and Bensley and his co-workers.19) The acid phosphatase of human endometrium resembles that of prostate in that it was nearly 100 per cent inhibited (with respect to glycerophosphate) by 0.02 M tartrate. 64 When phenyl phosphate substrate was used, the inhibition was notably less. The acid phosphatase of rat adrenal cortex69 was inhibited by copper ion, formaldehyde, ethyl alcohol and tartrate, but not by ferrous ion.

JULY 1954

ACID PHOSPHATASES

825

MECHANISM AND KINETICS 33

Cohn was able to show that the places where phosphoric acid esters were split differed in acid hydrolysis and acid enzyme hydrolysis. According to Cohn, the acid hydrolysis of a phosphoric acid ester proceeded in the following manner: OH

OH

I

I

HO—P ( + ) —O-i-R + OH2 - * HO—P + ROH 2 +

I o

'

I o

ROH 2 + ->• R—OH + H+

water reaction

or OH HO—P< +) —0—R ^ 0(->

OH OH HO—P (+ >—O-i-R -» HO—P—0 +

OH R (+) + H 2 0 - • ROH + H+

R (+)

OH acid catalyzed

The enzymic mechanism was shown to proceed in a slightly different manner with splitting of the P—0 bond rather than of the 0—R bond: OH

OH

I

I

HO—P (+ >—0—R + H+ ?± HO—P OH

I

OH OH

OH

I

I

HO—P(+>—OR + H 2 0 ?± OH—P-!-OR ^ HO—P (+) —0 ( -> + H+ + ROH

I

I

'

I

OH OH OH Sch0nheyder173 investigated the kinetics of acid phosphatase action. He used unprocessed seminal plasma as his source of enzyme and phenyl phosphoric acid

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acid and ketene and only partially inactivated by phenylisocyanate. Anaenostopoulos concluded that either free amino groups were not necessary for the action of acid phosphatase or that the reactive centers of acid and alkaline DhosDhatase were different. Mattocks and Holtan125 investigated the action of various inhibiting substances on the acid phosphatase of sheep brain. They found that the phosphatase was inhibited competitively by sulfate, pyrophosphate and diisopropylfluorophosphate. Fluoride inhibited the enzyme in a noncompetitive manner. Cyanide, iodoacetate, alanine, glutamic acid, para-aminobenzoic acid, creatine, cystine, cysteine, glutathione, ascorbic acid, acetylcholine, carbamylcholine, choline, urethane, tetraethylpyrophosphate, triethylpyrophosphate, tri-o-cresyl phosphate, myanesin, procaine, sulfanilamide and pilocarpine had no effect on the enzyme.

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AL.

as the substrate. All reactions were carried out at 22 C. rather than at physiologic temperature. As the substrate was split, the mediums became more alkaline, since phenyl phosphoric acid is a stronger acid than the liberated phosphoric acid. Sch0nheyder added acid to maintain the pH at 5.62, and recorded time and acid added at each time. The progress of the reaction was neither zero order nor first order. The reaction followed the rate equation: dS dt

aS b + cS

which when integrated is:

t = A In f° + B(S0 - 8) o The values of A and B were determined by graphical solution of simultaneous equations: A = —=- when B = 0 1

ln

OO

s-

B = j^

gr when A = 0

The kinetics of acid phosphatase are more complicated than would be expected from the Michaelis-Menten enzyme theory. According to Michaelis and Menten: Km t =

KEinS

So ,

1 ,0 +

es

KE{&°-S)

where K equals the velocity constant of the breakup of the enzyme substrate complex, Km is the Michaelis constant. The Sch0nheyder equation differs from the Michaelis-Menten equation, since A varies with substrate concentration while Km IKE does not. The difference, according to Sch0nheyder, may be due to inhibition of acid phosphatase activity by the products of the reaction. Sch0nheyder demonstrated experimentally that phenol had no effect on the reaction but that inorganic phosphate inhibited the reaction. With increased phosphate added to the substrate, the A value increased linearly but the B value was not affected.

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at = b In § + c(So - S) o where S is concentration of substrate at time I, So is concentration at zero time, a is an empirical constant that depends only upon enzyme concentration, b is an empirical constant independent of enzyme concentration but a linear function of initial substrate concentration So, and c is an empirical constant whose value is independent of both enzyme and substrate concentration. Sch0nheyder allowed A = b/z and B = c/a, and his rate equation became:

JULY

1954

ACID PHOSPHATASES

827

PHYSIOLOGIC FUNCTIONS OF ACID PHOSPHATASE

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Little is known definitely about the physiologic importance of prostatic acid phosphatase. Lundquist121 suggested that the splitting of phosphoryl choline, known to be present in human semen, was the sole function of prostatic phosphatase. Hudson and Butler92 found that their purified preparations of human ejaculate phosphatase failed to hydrolyze this substrate, hence they suggested that more than one phosphatase existed in ejaculate. Appleyard,6 basing her work on the findings of Axelrod,7 demonstrated that a partially purified extract of human prostate catalyzed the transfer of phosphate between pentasodiumphenolphthaleindiphosphate and certain aliphatic alcohols. The presence of these alcohols increased the liberation of free phenolphthalein without appreciably affecting the formation of free phosphate. Appleyard's assumption was that 2 enzymes were involved—a phosphatase and a transphosphorylase—and that the latter competed for the phenolphthalein more effectively than did the phosphatase. Transphosphorylases or phosphotransferases are enzymes capable of phosphate transfer without participation of ATP. Axelrod reported that kidney alkaline phosphatase was inactive as a transferase. Aryl phosphates were good donors; aliphatic alcohols were satisfactory acceptors. The ratio of phosphate transferred to substrate cleaved increased within limits with decreasing pH; it was independent of donor concentration but increased with acceptor concentration. By using labeled P, Axelrod8 was able to show that the phosphate transferred did not pass through the state of free phosphate ion. The phosphatases of the kidney are concentrated in the cells of the proximal convoluted tubules, which is the chief site of glucose reabsorption. Drabkin 50 summarized the literature on this and other relationships of phosphatases to diabetes mellitus. While the renal alkaline phosphatase is concentrated on the brush borders of the cells, the acid phosphatase is observed throughout the cell structure. During alimentary glycosuria in rats an average increase of 55 per cent occurred in the acid phosphatase activity of renal tissue, accompanied by a significant increase in alkaline phosphatase. Phlorizin, both in vitro and in vivo decreased the activity of both types of phosphatase. Without attempting to differentiate the separate functions of acid and alkaline phosphatase, Drabkin proposed that the renal threshold for glucose expressed in part the limit to which phosphatase activity could be raised. The total acid phosphatase in the kidneys of castrated rodents is decreased in proportion to the decrease in kidney size. Testosterone and other steroids that increase kidney size also increase the total acid phosphatase content of the kidney in proportion.107 Alkaline phosphatase of the kidneys of these same animals behaves quite differently, being increased in concentration by castration and decreased in concentration and total amount by testosterone. The acid and alkaline phosphatases of rat seminal vesicle and prostate decrease together following castration, and are increased together by testosterone injections 8 days after castration.180 Kroon108 measured acid and alkaline phosphatase activities by chemical means in 3- to 7-day chick embryos, medullary bone in the process of formation in the

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ACID P H O S P H A T A S E A N D CANCER

The acid phosphatase content of cancerous prostatic tissue, and the significance of serum acid phosphatase in cancer diagnosis, have been discussed in earlier portions of this review. Greenstein71 reviewed this earlier work on acid phosphatase (and other enzymes) in tumors compared with homologous normal tissue. Morel and his colleagues,133 in a review article, concluded that many phosphatases were present in elevated concentrations in many cancerous tissues, but were decreased in the necrotic areas of tumors. Schoonover and Ely174 reported a higher glycerophosphatase activity at pH 5 to 6 in the red cells of cancer patients as compared to normal subjects. Greenstein and Thompson73 found higher activities of both alkaline and acid phosphatases in fetal rat liver and in transplanted hepatoma than in adult liver. This was not the case with other enzymes observed in.the same study—-including arginase, catalase, xanthine, dehydrogenase and zj-amino acid oxidases. In each enzyme system studied, the direction of change in hepatoma was the same as in fetal liver, as compared with the normal. In a later paper, Greenstein and other co-workers72 reported acid phenylphosphatase as occurring in about equal amounts in extracts of normal mouse liver and of mouse hepatoma, while the activity of acid beta-glycerophosphatase was greater in normal liver than in hepatoma. Liver tumors induced in rats by feeding of carcinogens were found by Mulayand Firminger134 to have about half the mean acid phosphatase activity of control normal rat liver tissue. Gomori68 found acid phosphatase in cancerous tissue of the bladder, stomach, bronchus, prostate, bone and occasionally in the colon. Woodard194 extracted human tissues with water. Using these filtered extracts in determinations of acid phosphatase activity, she found acid phosphatase in normal kidney, liver and prostate. Phosphatase was found also in an adenoid cystic adenocarcinoma of

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pigeon and activated oviducts of the pigeon. Correlated with this, determinations of glucosamine were made as a measure of mucopolysaccharides present. In growing bone, alkaline phosphatase activity preceded the appearance of mucopolysaccharides. The activation of oviducts by follicle maturation or by estrogen injection was accompanied hy an increase of acid phosphatase activity, coincident with the appearance in the glandular cells of droplets of secretion that gave a strong McManus reaction,126 and therefore contained protein-mucopolysaccharide complexes. The author concluded that phosphatases were important in mucopolysaccharide formation, since they liberated from hexosephosphates the necessary sugars in a nascent state and at the site. The hexosephosphates were assumed to originate by the phosphorylytic degradation of glycogen. Norberg139 showed that liver regeneration in the rat was accompanied by a large increase in the several acid phosphatases present. This suggested a relation between these enzymes and protein synthesis. Evidence for such a function for acid and alkaline phosphatases has been reviewed by Moog,131 together with additional evidence as to their importance in the chemistry of differentiation.

JULY 1954

ACID PHOSPHATASES

829

REFERENCES 1. ABOLINS, L., AND ABOLINS, A.: Different kinds of acid phosphatases in various cytological structures of t h e anterior p i t u i t a r y of t h e guinea pig. N a t u r e (London), 164: 455-456, 1949. 2. ABUL-FADL, M. A. M . , AND K I N G , E . J . : The inhibition of acid phosphatases by formaldehyde and its clinical application for t h e determination of serum acid phosphatases. J . Clin. P a t h . , 1: 80-90, 1948. 3. ABUL-FADL, M. A. M., AND K I N G , E . J . : Properties of the acid phosphatases of e r y t h rocytes and of t h e human prostate gland. Biochem. J . , 46: 51-60, 1949. 4. ANAGNOSTOPOULOS, C : Action compared de quelques r&ictifs des functions amines sur diverses phosphatases acides et alcines. Biochem. e t Biophys. Acta, 4: 584-595, 1950. 5. ANAGNOSTOPOULOS, C , PACHT, M . , B O U R L A N D , E . , AND G R A B A R , P . : P h o s p h o r y l a t i o n

6. 7. S. 9.

par le phe'nylphosphodichlorure de groupements hydroxyles. Action des phosphatases sur diffe>ents protides phosphorylds. Bull. Soc. chim. biol., 33: 699-704, 1951. APPLEYARD, J . : The effect of alcohols on t h e hydrolysis of sodium phenolphthalein diphosphate by prostatic extracts. Biochem. J . , 42: 596-597,1948. AXKLROD, B . : A new mode of enzymic phosphate transfer. J . Biol Chem., 172: 1-13, 1948. AXBLROD, B . : A study of the mechanism of "phosphotransferase" activity by use of radioactive phosphorus. J . Biol. Chem., 176: 295-298,1948. BACCARI, V., AND AURICCHIO, G . : Importanza dei gruppi aminici liberi per I'azione della fosfatasi acida. Boll. Soc. ital. biol. sper., 22: 49-50, 1946.

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salivary gland origin and in the lymphomatoid tissue of a patient with Hodgkin's disease. Lemon and Wisseman118 used frozen tissue sections for the simultaneous quantitative estimation and cytologic localization of the enzyme. These authors measured acid phosphatase activity in human neoplasms and compared the activity with that found in the normal tissues of origin. Acid phosphatase was found in both normal and malignant tissue of breast, bronchus, skin, bladder, esophagus, stomach, colon and rectum. In all cases greater amounts of enzyme were found in the neoplastic than in the normal tissue. However, Lemon and Wisseman did not study malignant prostatic tissue. Huggins93 found that malignant prostatic tissue, on the other hand, contained less enzyme than normal tissue of origin. Lemon and Wisseman suggested that the increased acid phosphomonoesterase activity of human cancer cells was related to the known rapid uptake and retention of labeled phosphorus by malignant tissue. Changus and Dunlap 30 found an acid phosphatase activity greater than 10 units (by the method of Gutman and Gutman78) per 100 ml. in the gastric aspirates of 24 of 25 patients with gastric carcinoma. In only 1 of 10 control patients without gastric cancer did the gastric juice show acid phosphatase activity over 10 units. No cases of early cancer were included in this series. In another communication63 high acid phosphatase activity was reported in neoplastic gastric tissue and in the surrounding mucosa. Beck and Valentine15 reported high acid phosphatase activity per unit number of leukocytes in chronic myelocytic leukemia, and low alkaline phosphatase activity as compared with normal leukocytes. Chronic lymphatic leukemia, and acute leukemia, were characterized by decrease of both types of phosphatase. In an article seen by us only in abstract, Yanagisawa197 reported enhancement of activity of serum acid phosphatase by 0.01 to 0.04 M cobalt nitrate in serums of patients with cancer, as opposed to inhibition by the same reagent in normal serums. Comparable results were reported in ascites and organs of rats with Yoshida sarcoma.

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17. B E L F A N T I , S., C O N T A R D I , A., AND E R C O L I , A . : Studies on t h e p h o s p h a t a s e s . I . T h e k:

influence of some electrolytes on t h e phosphatases of animal tissue. Phosphatases of the liver, kidney, serum, and bones of t h e rabbit. Biochem. J . , 29: 517-527, 1935.

18. B E L P A N T I , S., CONTARDI, A., AND ERCOLI, A.: Studies on t h e phosphatases. I I . I n a c t i -

vation and reactivation of t h e phosphatases of animal organs. Biochem. J., 29: 842S46, 1935. 19. BENSLEY, E . H . , WOOD, P . , AND LANG, D . : Estimation of acid phosphatase of hemolyzed serum b y t h e formaldehyde inactivation technic. Am. J . Clin. P a t h . , 18: 742744, 1948. 20. B E N S L E Y , E . H . , WOOD, P . , M I T C H E L L , S., AND M I L N E S , B . : E s t i m a t i o n of serum acid

phosphatase in t h e diagnosis of metastasizing carcinoma of t h e p r o s t a t e . Canad. M. A. J . , 58: 261-264, 1948. 21. B E R T H E T , J . , B E R T H E T , L., A P P E L M A N S , F . , AND D U V E , C. D E : Tissue

22. 23. 24. 25. 26. 27. 28. 29. 30.

fractionation

studies. I I . N a t u r e of t h e linkage between acid phosphatase and mitochondria in rat-liver tissue. Biochem. J., 50: 182-189, 1951. B E R T H E T , J . , AND D U V E , C. D E : Tissue fractionation studies. I. T h e existence of a mitochondria-linked, enzymatically inactive form of acid phosphatase in rat-liver tissue. Biochem. J . , 50: 174-181, 1951. B I N K L E Y , F . : Preparation and properties of S-phosphocysteine. J . Bio!. Chem., 195: 283-285, 1952. BODANSKY, A.: Phosphatase studies. I I . Determination of serum phosphatase. F a c t o r s influencing t h e accuracy of t h e determination. J . Biol. Chem., 101: 93-104, 1933. BODIAN, D . , AND MELLORS, R . C : T h e regenerative cycle of motoneurons, with special reference to phosphatase activity. J . Exper. Med., 81: 469-488, 1945. BOSSARD, M . : Action des molybdates sur divers enzymes. Bull. Soc. chim. biol., 29: 218-221, 1947. BURGEN, A. S. V.: Urinary excretion of phosphatases in man. Lancet, 1: 329-331, 1947. CARRERA, G. M . : Acid phosphatase activity in t h e intestinal wall in experimental amebic colitis. Proc. Soc. Exper. Biol. &'Med., 7 3 : 682, 1950. CASPERSSON, T . : T h e relations between nucleic acid and protein synthesis. Symp. Soc. Exper. Biol., 1: 127-151, 1947. CHANGUS, G. W., AND D U N L A P , C. E . : Acid-phosphatase activity in t h e gastric cont e n t of patients with carcinoma of t h e stomach. J . N a t . Cancer Inst., 10: 481-487, 1949.

31. CLARK, L. C , J R . , B E C K , E . , AND THOMPSON, H . : T h e excretion of acid phosphatase as

32. 33. 34. 35. 36.

a measure of prostatic development during pubescence. J . Clin. Endocrinol., 11 84-90, 1951. CLARK, L. C , J R . , AND TREICHLER, P . : Psychic stimulation of prostatic secretion. Psychosomatic Med., 12: 261-263, 1950. C O H N , M . : Mechanisms of cleavage of glucose-1-phosphate. J . Biol. Chem., 180: 771781, 1949. COLLING, K . G., AND R O S S I T E R , R . J . : Alkaline and acid phosphatase in cerebrospinal fluid; d a t a for normal fluids and fluids from patients with meningitis, poliomyelitis, or syphilis. Canad. J . R e s . , Sect E, 28: 56-68, 1950. COURTOIS, J . , AND ANAGNOSTOPOULOS, C : Action de quelques acides mineraux form a t e u r s de complexes sur les phosphatases. Compt. rend. Soc. de biol. 226: 523-524, 1948. COURTOIS, J . , AND ANAGNOSTOPOULOS, C : Recherches sur l'inhibition des phosphatases acides p a r les fluorures. I . Influence du fluorure de sodium sur la cinetique de

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10. BAMANN, E . , AND D I E D E R I C H S , K . : T r e n n u n g der beiden isodynamen Phosphoesterasen tierischer Organe durch ein selektives Inaktivierungs-Verfahren. Ber. deutsch. chem. Gesellsch., 67: 2019-2021, 1934. 11. BAMANN, E . , AND R I E D E L , E . : Uber das Verkommen zweier durch das p H Wirkungsoptimum unterschiedbaren Phosphoesterasen in tierischen Organen. Ztschr. p h y s iol. Chem., 229: 125-150, 1934. 12. BARRINGER, B . S., AND WOODARD, H . J . : Prostatic carcinoma with extensive intraprostatic calcification. T r a n s . Am. Assoc. Genito-Urinary Surg., 3 1 : 363, 193S. 13. BARTELMEZ, G. W., AND B E N S L E Y , S. H . : "Acid p h o s p h a t a s e " reactions in peripheral nerves. Science, 106: 639-641, 1947. 14. BECK, L. V . : Action of phlorizin on acid phosphatase activity and on glucose phosphorylation of kidney cortex extracts. Proc. Soc. Exper. Biol. & Med., 49: 435-439, 1942. 15. BECK, W. S., AND VALENTINE, W. N . : Biochemical studies on leucocytes. I I . Phosphatase activity in chronic lymphatic leucemia, acute leucemia, and miscellaneous hematologic conditions. J . L a b . & Clin. Med., 38: 245-253, 1950. 16. BEHRENDT, H . : Phosphatase activity of human erythrocytes. Proc. Soc. Exper. Biol. & Med., 54: 268-270, 1943.

JULY 1954

37. 38. 39. 40. 41. 42. 43. 44.

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831

45. D E L O R Y , G. E . , S W E E T S E R , T . H . , AND W H I T E , T . A . : T h e use of formalin and alcohol

in t h e estimation of prostatic phosphatase. J . Urol., 66: 724-733, 1951. 46. D E M P S E Y , E . W., AND D E A N E , H . W . : T h e cytological localization, s u b s t r a t e specificity, and p H optima of phosphatases in t h e duodenum of t h e mouse. J . Cell. & Comp. Physiol., 27: 159-179, 1946. 47. D E M U T I I , F . : Uber Phosphatstoffwechsel. I . Mitteilung: Uber Hexosephosphatasen in menschlichen Organen und Korperfliissigkeiten. Biochem. Ztschr., 159: 415-423, 1925. 48. DMOCHOWSKI, A., AND PRACOWITY, M . : Die Ammonsulfatmethode fiir Reineinung d e r Njakulat und-Prostataphosphatase. Acta biol. exper., 11: 11-12, 1937. 49. DOUNCE, A. L . : Enzyme studies on isolated cell nuclei of r a t liver. J . Biol. Cheni., 147: 685-698, 1943. 50. DHABKIN, D . L . : Hyperglycemia, glj'cosuria and dephosphorylation—the role of phosphatases. Proc. Am. Diabetes A s s o c , 8: 171-212, 1948. 51. D H A B K I N , D . L . , AND M A R S H , J . B . : Increased liver phosphatase activity in alloxandiabetic rats. J . Biol. Chem., 171: 455-462, 1947. 52. DUMAZERT, C , LEVY, M., AND MARSZAK, I . : Action de quelques formateurs de complexes mdtalliques sur diverses phosphoesterases e t pyrojjhosphatases. Compt. rend Soc. de biol., 139: 99-101, 1945. 53. D U N L A P , C. E . , AND CHANGUS, G. W.: Acid phosphatase activity of t h e gastric contents of patients with carcinoma of t h e stomach. Federation P r o c , 8: 353, 1949. 54. E G E R , W., AND G E L L E R , H . F . : Zum Nachweis der alkalischen und siiuren Phosphatase in der Leber am nativen Gefrierschnitt. Virchows Arch. p a t h . Anat. u. Physiol., 322: 645-661, 1952. 55. E R A N K O , 0 . : Demonstration of acid phosphatase in tissue sections. Ann. med. exper. et biol. Fenniae, 29: 287-289, 1951. 56. FAULDS, J . S.: Phosphatase in dried seminal stains. Edinburgh M . J . , 58: 94-9S, 1951. 57. F E I N S T E I N , R . N . , AND VOLK, M . E . : Phosphoprotein phosphatase in mammalian tissue. J . Biol. Chem., 177: 339-346, 1949. 58. FISHMAN, W. H . , AND L E R N E R , F . : A method for estimating serum acid phosphatase of prostatic origin. J . Biol. Chem., 200: 89-97, 1953. 59. F L E O R Y , P., C O U R T O I S , J . , ANAGNOSTOPOULOS, C ,

AND D E S J O B E R T , A . : Action do

phosphatases de tvpes distincts sur differents esters phosphoriques. Bull. Soc. chim. biol., 32: 77117S3, 1950. 60. FOLLEY, S. J . , AND K A Y , H . D . : T h e Phosphatases. Ergebiiisse der Enzymforschung Band V. 159. Akademische Verlagagesellschaft, Leipzig, 1936. 61. G n u , K . V . : Uber Spcichol-Phosphatase. Biochem. Ztschr., 285: 306-310, 1936. 62. G I R I , K . V., P R A S A D , A. L. N . , D E V I , S. G., AND R A M , J . S.: A t e c h n i q u e for t h e iden •

tification and separation of enzymes by paper chromatography. Biochem. J . , 51: 123-12S, 1952. 63. GOETSCH, J . B . , R E Y N O L D S , P . M . , AND B U N T I N G , H . : Modification of Gomori m e t h o d

for alkaline and acid phosphatase avoiding artefact staining of nucleus. P r o c Soc. Exper. Biol. & Med., 80: 71-75, 1952. 64. GOLDBERG, B . , AND J O N E S , H . W., J R . : Acid phosphatase in human female genital tract, a histochemical and biochemical study. P r o c Soc. Exper. Biol. & Med., 8 3 : 45-50, 1953. 65. GOMORI, G.: Distribution of acid phosphatases in t h e tissues under normal and under pathologic conditions. Arch. P a t h . , 32: 1S9-199, 1941. 66. GOMORI, G.: T h e studv of enzymes in tissue sections. Am. J . Clin. P a t h . , 16: 347-352, 1946.

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la reaction. Bull. Soc. chim. biol., 31: 1494-1503, 1949. I I . Essais d'inhibition e t de reactivation dans diverses conditions. Ibid, 1504-1509. COUBTOIS, J . , AND PLUME L, M . : Essais de purification de la phosphatase urinaire Son hetdrogeneitc Bull. Soc. chim. biol., 31: 165-170, 1949. CRAM, D . M . , AND ROSSITER, R. J . : Phosphatase of rabbit polymorphonuclear leucocytes. Canad. J . Research., Sect. E , 27: 290-307, 1949. DALGAARD, J . B . : Tracer phosphatase determination in tissue sections. N a t u r e , 162: 811, 1948. D A V I E S , D . R . : Phosphatase activity of spleen extracts. Biochem. J . , 28: 529-536, 1934. D E A N , A. L., AND WOODARD, H . Q : Differential diagnosis of tumors of t h e p r o s t a t e and adjoining areas of t h e rectum and bladder by chemical analysis. J . U r o l . , 57: 172-174, 1947. D E A N E , H . W., AND DE.MI'SEY, E . W . : T h e localization of jjhosphatases in t h e Golgi region of intestinal and other epithelial cells. Anat. R e c , 93: 401-417, 1945. D E L O R Y , G. E . , AND HETHERINGTON, M . : T h e determination of urinary acid phosphatase. Canad. J . Med. S c , 30: 1-3, 1952. D E L O R Y , G. E . , AND HETHERINGTON, M . : T h e acid phosphatases of human urine. Canad. J . Med. S c , 30; 4-9, 1952.

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67. GOMORI, G . : Histochemical demonstration of sites of phosphamidase activity. Proc. Soc. Exper. Biol. & Med., 69: 407-409, 1948. 68. GOMORI, G . : Histochemical specificity of phosphatases. Proc. Soc. Exper. Biol. & Med., 70: 7-11, 1949. 69. GORDON, J . J . : Characterization and assay of enzymes in rat adrenal cortex. I. Esterase and phosphatase activities. Biochem. J . , 51: 97-103, 1952. 70. G R E E N , H . , AND M E Y E R H O P , O . : Synthetic action of phosphatase. I I I . Transphosphorylation with intestinal and semen phosphatase. J . Biol. Chem., 197: 347-364, 1952. 71. G R E E N S T E I N , J . P . : Recent progress in tumor enzymology. Advances Enzvmol., 3 : 315-348, 1943. 72. G R E E N S T E I N , J . P . , C A R T E R , C. E . , AND L E U T H A R D T , F . M . : A c t i v i t y of

73.

75. 76. 77. 78.

79. GUTMAN, E . B . , SPROUL, E . E , AND GUTMAN, A. B . : Significance of increased phos-

80. 81. 82. 83. 84. 85. 86.

phatase activity of bone a t t h e site of osteoplastic metastases secondary t o carcinoma of t h e prostate gland. Am. J . Cancer, 28:485-495,1936. H A I G H T , W. F . , AND ROSSITER, R . J . : Acid and alkaline phosphatase in white cells; d a t a for lymphocyte and polymorphonuclear leukocyte of man and rabbit. Blood, 5: 267-277, 1950. H A N S E N , P . F . : Determination of t h e " a c i d " prostatic phosphatase as a new method for medicolegal demonstration of sperm spots. Acta path, e t microbiol. Scandinav., 23: 187-214, 1946. H A R D , W. L . , AND LASSEK, A. M . : T h e pyramidal t r a c t . Effect of maximal injury on acid phosphatase content in neurons of cats. J . Neurophysiol., 9 : 121-126,1946. H A R R I S , D . L . : Phosphoprotein phosphatase, a new enzyme from t h e frog egg. J . Biol. Chem., 165: 541-550, 1946. HASHIMOTO, K . : t l b e r Hydrolyse des a l p h a - u n d beta-Glycerophosphates. J . Biochem. ( J a p a n ) , 26:.137-159, 1937. H E I N Z E N , B . : Acid phosphatase activity in transected sciatic nerves. A n a t . R e c , 98: 193-207, 1947. H E R B E R T , F . K . : T h e estimation of prostatic phosphatase in serum and its use in t h e diagnosis of prostatic carcinoma. Q u a r t . J . Med., 15: 221-241,1946.

87. H E R T Z , R . , W E S T F A L L , B . B . , B A R R E T T , M . K . , AND T U L L N E B , W. W . : T h e effect of

ectopic autologous grafts of androgen-stimulated prostate upon the serum " a c i d " phosphatase of the dog. J . N a t . Cancer Inst., 10: 61-66, 1949. 88. HOCK, E . , AND T E S S I E R , R . N . : Elevation of serum acid phosphatases following prostatic massage. J . Urol., 62:488-491,1949. 89. H O L U N G E B , D . M . , R O S S I T E R , R . J . , AND U P M A L I S , H . : Chemical studies of peripheral

90. 91. 92. 93. 94. 95.

nerve during Wallerian degeneration. IV. Phosphatases. Biochem. J . , 52:652-659, 1952. HoLTEB,H.: Localization of enzymes in cytoplasm. Advances Enzymol., 13:1-20, 1952. HOTTA, R . : tlber die Spezifitilt der Phosphatase. J . Biochem. (Japan), 20:343-360,1934. HUDSON, P . B . , AND B U T L E R , W. W. S., I I I . : A study of t h e enzj'me acid phosphatase and its possible role in intermediary carbohydrate metabolism of t h e prostate gland and its secretion in dog and man. J . Urol., 63: 323-333, 1950. H U G G I N S , C . : T h e physiology of t h e prostate gland. Physiol. Rev., 25: 281-295, 1945. H U G G I N S , C , AND BERGENSTAL, D . M . : Inhibition of human mammary and prostatic cancers by adrenalectomy. Cancer Research, 12:134-141,1952. H U G G I N S , C., AND HODGES, C. V.: Studies on prostatic cancer; effect of castration, of estrogen and of androgen injection of serum phosphatases in metastatic carcinoma of prostate. Cancer Research, 1: 293-297, 1941.

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74.

phosphatases

in fresh and dialyzed extracts of normal mouse liver and of mouse hepatoma. J. N a t . Cancer Inst., 7 : 47-49, 1946. G R E E N S T E I N , J . P . , AND THOMPSON, J . W . : Enzymatic activity of normal adult, regenerating, fetal, and neoplastic hepatic tissues of t h e r a t . J . N a t . Cancer Inst., 4: 271-274, 1943. GUTMAN, A. B . : Serum " a c i d " phosphatase in patients with carcinoma of the prostate gland; present s t a t u s . J.A.M.A., 120:1112-1116, 1942. GUTMAN, A. B . , AND GUTMAN, E . B . : An " a c i d " phosphatase occurring in the serum of patients with metastasizing carcinoma of t h e prostate gland. J . Clin. Investigation, 17: 473-478, 1938. GUTMAN, A. B . , AND GUTMAN, E . B . : " A c i d " phosphatase and functional activity of the prostate (man) and preputial glands (rat). Proc. Soc. Exper. Biol. & Med., 39: 529-532, 193S. GUTMAN, A. B . , AND GUTMAN, E . B . : Adult phosphatase levels in pre-pubertal rhesus prostate tissue after testosterone propionate. Proc. Soc. Exper. Biol. & Med., 4 1 : 277-281, 1939. GUTMAN, E . B . , AND GUTMAN, A. B . : Estimation of " a c i d " phosphatase activity of blood serum. J . Biol. Chem., 136: 201-209, 1940.

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96. ICHIHARA, M . : tlber die Phosphamidase. J . Bioehem. ( J a p a n ) , 18: S7-106, 1933. 97. J E N N E R , H . D . , AND K A Y , H . D . : T h e phosphatases of mammalian tissues. I I I . Magnesium and t h e phosphatase system. J . Biol. Chem., 93: 733-748, 1931. 98. JOSEPHY, H . : Acid phosphatase in t h e senile brain. Arch. Neurol. & Psychiat., 61: 164-169, 1949. 99. K A P L A N , N . 0 . , COLOWICK, S. P . , AND C I O T T I , M . M . : E n z y m a t i c

100. 101. 102. 103. 104. 105.

dcamination

of

106. K I R K , J . E . , E I S E N S T E I N , A., AND M A C B R Y D E , C. M . : T h e acid phosphatase concentra-

107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117.

HS. 119.

tion of t h e prostatic exprimate during normal puberty. J . Clin. Endocrinol. & M e t a b . , 12: 338-345, 1952. KOCHAKIAN, C. D . : T h e role of hydrolytic enzymes in some of the metabolic activities of steroid hormones. Recent Progress in Hormone Research, 1:177-216,1947. K R O O N , D . B . : Phosphatase and t h e formation of protein-carbohydrate complexes. Acta anat. 15: 317-328, 1952. KUTSCIIER, W., AND PANY, J . : Prostataphosphatase. Ztschr. f. physiol. Chem., 255: 169-189, 1938. KUTSCIIER, W., AND SCHREIER, K . : tlber die Magnesium Aktivierung dor sauren M u s kel-phosphatase. Naturwissenschaften, 35: 255-256, 1948. KUTSCIIER, W., AND WOLBERGS, H . : Prostataphosphatase. Ztschr. f. physiol. Chem., 236: 237-240, 1935. KUTSCIIER, W., AND WORNER, A.: Prostataphosphatase. 2. Mitteilung. Ztschr. f. physiol. Chem., 239: 109-126, 1936. KUTSCIIER, W., AND W U S T , H . : tlber die Fluoridhemmung der Phosphatase m i t sauren pl-I-Optimum. Bioehem. Ztschr., 310: 292-301, 1942. LASSEK, A. M . : T h e stability of so-called axonal acid phosphatase as determined by experiments in its " s t a i n a b i l i t y . " Stain Technol., 22: 133-138, 1947. LASSEK:, A. M . , AND H A R D , W. L . : Acid phosphatase in growing axons and degenerated nerve tissue. Science, 102: 123-124, 1945. LASSEK, A. M., AND HARD, W. L . : T h e pyramidal t r a c t . T h e relation of axonal diameters to the rate of degeneration as revealed by t h e acid phosphatase method in the monkey. J . N e u r o p a t h . & Exper. Neurol., 5: 374-379, 1946. LEDUC, E . H . , AND WISLOCKI, G. B . : T h e histochemical localization of acid and alkaline phosphatases, non-specific esterase, and succinic dehydrogenase in t h e structures comprising t h e hematoencephalic barrier of t h e r a t . J . Comp. Neurol., 9 7 : 241-279, 1952. LEMON, II. M . , AND WISSEMAN, C. L . : Acid phosphomonoesterase a c t i v i t y of human neoplastic tissue. Science, 109: 233-235, 1949. LOEOREN, T . : Inverkan av fosfatas p a Kasein. Svenska K e m . Tid., 57: 95-101, 1945.

120. I.OMIIOLT, E . , RASMUSSEN, E . S., AND SCHO'NIIEYDER, F . : On t h e content of acid phos-

phatase in ejaculate and prostatic secretion. Acta Dermatovenereol., 26: 269-2S1, 1946. 121. LUNDQUIST, F . : Function of prostatic phosphatase. N a t u r e , 158: 710-711, 1946. 122. LUNDQUIST, F . : Studies in biochemistry of human semen. Some properties of prostatic phosphatase. Acta physiol. Scandinav., 14: 263-275, 1947. 123. MARTLAND, M., HANSMAN, F . S., AND ROBISON, R . : Phosphoric esterase of blood. Bioehem. J., 18: 1152-1160, 1924. 124. M A T T E N I I E I M E R , H., NITSCHMANN, H S . , AND Z A H L E R , P . : D a s L a b und seine W i r k u n g

auf das Casein der Milch. VI. tlber die Phosphatase wirkung des Labs. Helvet. chim. acta, 35:1970-1983, 1952. 125. MATTOCKS, A. M., AND HOLTAN, S. D . : Inhibition of acid phosphatases of sheep brain. J. Pharmacol & Exper. T h e r a p . , 96: 114-118, 1949. 126. M C M A N U S , J. F . A.: Histological and histochemical uses of periodic acid. Stain Technol., 23: 99-108, 1948. 127. M C N A B B , A. R . : Enzymes of gray m a t t e r and white m a t t e r of dog brain; distribution of certain nonoxidative enzj'mes. Canad. J . M. S c , 20: 208-215, 1951.

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adenosine derivatives. J . Biol. Chem., 194:579-591,1952. K A Y , H . D . : Kidney phosphatase. Bioehem. J . , 29:791-811,1926. K A Y , H . D . : Phosphatase in growth and disease of bone. Physiol. R e v . , 12: 3S4-422, 1932. K A Y E , S.: T h e acid phosphatase test for seminal stains. A s t u d y of reliability of aged stains. Am. J . Police Sc. (In J . Criminal Law Criminol.), 41: S34-835, 1951. K I N D , C. A., AND MACCHI, M . E . : Phosphatase activity in the tissues of the frog, Rana pipiens. J. Cell. & Comp. Physiol., 39: 153-159, 1952. K I N G , E. J . , WOOD, E . J . , AND D E L O R Y , G. E . : Acid phosphatase of t h e red cells. Bioehem. J . , 39: Proc. Bioehem. S o c , xxiv-xxv, 1945. K I N T N B R , E . P . : T h e estimation of prostatic phosphatase using a modified King-Armstrong method by determining t h e formaldehyde stable and alcohol labile fraction of serum acid phosphatase. J . L a b . & Clin. Med., 37: 637-642, 1951.

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128. M E Y E R H O F , 0 . , AND G K E E N , H . : S y n t h e t i c action of phosphatase. I . E q u i l i b r i u m of biological esters. J . Biol. Chem., 178: 655-667, 1949. 129. MONTAGNA, W., ANDNOBACK, C . R . : A c i d phosphatase and lipids in the mast cells of the r a t . Science, 106:19,1947. 130. M O O G , F . : Localizations of alkaline a n d acid phosphatases in t h e early embryogenesis of t h e chick. Biol. Bull., 86: 51-80, 1944. 131. M O O G , F . : Alkaline and acid phosphomonoesterase activity in chick embrvos. J . Cell. & Comp. Physiol., 28: 197-208, 1946. 132. M O O R E , C. R . , P R I C E , D . , AND GALLAGHER, T . F . : R a t - p r o s t a t e cytology as a t e s t i s -

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