THEJOURNAL OF BIOLOGICAL CHEMISTRY Q 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. 32, Issue of Novemher 15, pp. 1656&16579,1988 Printed in U.S.A.

In Vivo Studies of Cysteine Metabolism USE OF D-CYSTEINESULFINATE,A NOVEL CYSTEINESULFINATE DECARBOXYLASE INHIBITOR, TO PROBE TAURINE AND PYRUVATE SYNTHESIS* (Received for publication, June 7, 1988)

Catherine L. Weinstein, Rudy H. Haschemeyer, and Owen W.GriffithS From the Department of Biochemistry, Cornell University MedicalCollege, New York,New York 10021

Although several pathways contributeto the catab- other potentially toxic, nonenzymatic reactions of cysteine olism of L-cysteine, the products formed are feware minimal in mammalian tissues because intracellular cystaurine + COZ and pyruvate ammonia sulfate. L- teine levels are very low, typically 30-250 PM (6-10). Although Cysteinesulfinate is a key intermediate that is either advantageous in terms of toxicity, maintenance of very low decarboxylated to ultimately yield taurine or transa- cysteine levels might, in principle, compromise the availability minated to yield pyruvate. There is strong evidence of cysteine for necessary metabolic processes. In practice, that pyruvate isalso formed by several cysteinesulfi- cysteine availability is supported by a much larger (0.8-8 mM nate-independent pathways collectively referred toas (11))intracellular pool of glutathione (GSH, y-glutamylcys"cysteine desulfhydrase." The quantitative importanceteinylglycine). In vivo studies clearly show that GSH turnover of cysteinesulfinate-independentpathways of taurine synthesis is less clear, but it has been suggested that plays an important role both in interorgancysteine transport taurine synthesis from the cysteamine released during and in the short-term regulation and maintenance of the phosphopantetheine and CoASH turnover accounts for cellular pool of free cysteine (6, 7, 12, 13). Mammals obtain cyst(e)ine directly from the diet and by the high taurine content of tissues with verylow levels of cysteinesulfinate decarboxylase activity (e.g. skel- synthesis via the transsulfuration pathway from methionine etal muscle and heart). In the present studies, the met- sulfur and serine carbon. Since neither the diet nor transsulabolic flux through each of these pathways was quan- furation is specifically regulated with respect to cysteine actitated in vivo by monitoring the formation of respi- cumulation (8, lo), cellular cysteine levels are controlled ratory 14C02in mice administered L - [ ~ - ' ~ Cor ] - ~ - [ 3 - mainly by the reactions using cysteine-protein and GSH 14C]cyst(e)ine.Mice given 0.05 mmol/kg of L-cystine synthesis, and cyst(e)inecatabolism. In general, the synthetic or 0.5 or 2.5 mmol/kg of L-cysteine catabolize 35, 51, processes contribute littleto thenet change in cysteine stores. and 72% of the dose, respectively, in 6 h; the relative Cysteine used for protein synthesis is balanced by cyst(e)ine contribution of taurine synthesis to total catabolism released by protein degradation. In normally nourished GSHdecreases from 6 3 to 51 to 42%as the L-cyst(e)inedose replete animals, net synthesis of GSH, as distinctfrom GSH is increased. To evaluate therole of L-cysteinesulfinate turnover, is limited by feedback inhibition of y-glutamylcysin taurine synthesis, D-cysteinesulfinate was charac- teine synthetase (14). Thus, under most conditions, tissue terized andused as a metabolism-resistant, potent, and levels of free cysteine and cysteine equivalents (i.e.GSH) are specific inhibitor of cysteinesulfinate decarboxylase. ultimately regulated and limited by the reactions of cysteine Studies with L - [ ~ - ' ~ C ]and - ~-[3-"C]cysteine in the catabolism (8). presence of inhibitor indicate that 85-93% of taurine Although several pathways contribute to cysteine catabosynthesis occurs from L-cysteinesulfinate; the calcu- lism, only two sets of products areformed-pyruvate + sulfate lated contributionof the phosphopantetheine pathway + ammonia and taurine CO,; one intermediate, cysteineis small and may approximate zero. L-Cysteinesulfisulfinate, is central to both pyruvate and taurine pathways. nate transaminationaccounts for 25%of pyruvate synThus, pyruvate is formed by decomposition of P-sulfinylpy(0.05 mmol/kg) but only 11% thesis from ~-['~C]cystine of pyruvate synthesis from ~-['~C]cysteine (2.5 mmoll ruvate, the product of cysteinesulfinate transamination, and kg). Cysteine desulfhydrase reactionsaccount for most also by the cysteine desulfhydrase reaction (actually a composite of several activities (15)). Taurineis formed by oxidaof the pyruvatesynthesis. tion of hypotaurine, an intermediate formed both by cysteinesulfinate decarboxylation and by cysteamine oxidation. Whereas cysteinesulfinate is the main taurine precursor in Cysteine is among the most toxic of the protein amino acids liver, it has been suggested that cysteamine, formed from both as a dietary constituent (1)and asa component of tissue cysteine during phosphopantetheine andCoASH turnover, is culture media (2,3). Although the basis of cysteine toxicity is the major hypotaurine precursor in skeletal muscle and heart not fully defined, formation of reactive oxygen species during (16-18). The lattertissues exhibit high taurine levels but low the auto-oxidation of cysteine (4) and nonenzymatic forma- rates of cysteinesulfinate decarboxylation. Studies with [1-14C]-and [3-14C]cysteineindicate that taution of stable cysteine-pyridoxal 5"phosphate adducts ( 5 ) are thought to be important factors. The rates of these and rine and pyruvate + sulfate synthesis account for 70-85% and 15-30% of cysteine catabolism, respectively, in the male rat * This work was supported in part by Grant DK26912 from the (19-21). Other studieswith ~ - [ ~ ~ S ] c y s t esuggest i n e that >70% National Institutes of Health The costs of publication of this article of cysteine is metabolized to sulfate (19, 22, 23). Although were defrayed in part by the payment of page charges. This article liver contains high levels of the enzymes forming and decarmust therefore be hereby marked "advertisement" in accordance with boxylating cysteinesulfinate, some (24), butnot all (251, recent 18 U.S.C. Section 1734 solelyto indicate this fact. studies with rat hepatocytes support pyruvate + sulfate rather $ To whom correspondence should be addressed.

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Cysteine Metabolism

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than taurine as the major product of cysteine catabolism in that tissue. In the present studies, ~-[‘~C]cyst(e)ine was used to quantitate taurine and pyruvate formation in the intact mouse and to determine the relative importance of various pathways of cysteine catabolism at different cyst(e)ine loads. In some studies, D-cysteinesulfinate, identified as a potent, competitive inhibitor of cysteinesulfinate decarboxylase, was used to block taurineformation from L-cysteinesulfinate. Taurine formation via cysteinesulfinate-independentpathways could then be quantitated in vivo.’ EXPERIMENTAL PROCEDURES AND RESULTS’

I n Vitro Inhibition of CysteinesulfinateDecarboxylase by DCysteinesulfinate-In previous studies, several L-cysteinesulfinate analogs were tested as substrates and inhibitors of rat liver cysteinesulfinate decarboxylase; D-cysteinesulfinate was the most potent inhibitor identified (28).As shown in Fig. 1 (Miniprint), inhibition by D-cysteinesulfinate is competitive HOURS with respect to L-cysteinesulfinate ( A ) and is characterized FIG. 2. Effect of D-cysteinesulfinate on the metabolism of by a Ki of 0.32 mM ( B ) . Similar studies indicate that D- L-cysteinesulfonate. Miceweregiven ~-[l-’~C]cysteinesulfonate cysteinesulfonate is also a competitive inhibitor but with a Ki (0.25 mmol/kg) by subcutaneous injection on the back of the neck. of 2.3 mM (data not shown). Neither D-cysteinesulfinate nor Control mice (closed circles,no inhibitor) received no other treatment; D-cysteinesulfonate is decarboxylated by the enzyme (28). experimental mice (open circles, with D-cysteinesulfinate) were given D-Cysteinesulfinate is not an effective inhibitor of either D-cysteinesulfinate (10 mmol/kg) by intraperitoneal injection 1 h prior to radioisotope administration. Formation of respiratory “COz cytoplasmic or mitochondrial aspartateaminotransferase (Table 1, Miniprint). This finding is consistent with the was monitored as described under “Experimental Procedures.” The points on each line represent averages f S.D. of the results from observation that aspartate aminotransferase is only weakly eight animals. inhibited by D-aspartate (Table 1)and with previous reports that aspartateaminotransferase interacts either not at all (29) hour, metabolism averages only 8 rmol/h. kg, an inhibition of or only very weakly(30)with D-amino acids. Since L-cysteine- 92% (Fig. 2). sulfinatetransamination is catalyzed by aspartateaminoD-Cysteinesulfinate-mediated inhibition of cysteinesulfitransferase (31, 32), the absence of significant aspartate ami- nate decarboxylase persists for several hours. When the innotransferase inhibition supports the view that D-cysteine- terval between D-cysteinesulfinate administrationand Lsulfinate ais specific inhibitor of L-cysteinesulfinate [l-’4C]cysteinesulfonateinjection is extended from 1 to 5 h, decarboxylation. the maximum rate of 14C02formation is decreased by 81% I n Vivo Inhibition of CysteinesulfinateDecarboxylase by D- relative to uninhibited controls (Fig. 3A, Miniprint). The Cysteinesulfinute-Cysteinesulfinate decarboxylase activity extent of inhibition at 5 h is thus only slightly less than seen can be determined in vivo by comparing the extent of 14C02 at 1 h. If ~-[l-’~C]cysteinesulfonate is given 10 or15h (33) following D-cysteinesulfinate administration, the maximum formation from L-[~-’~C]and ~-[3-’~C]cysteinesulfinate or by quantitating the rateof 14C02formation from L - [ ~ - ’ ~ C ]rates of 14C02formation are decreased by 72 and 32%, respeccysteinesulfonate (34). The latter approach, which is both tively (Fig. 3,B and C, Miniprint). Note that the data at 15 h direct andtechnically simple, is based on the observation that shows considerable scatter, suggesting excretion or metabo(i) L-cysteinesulfonate is rapidly decarboxylated by cysteine- lism has reduced tissue D-cysteinesulfinate concentrations to sulfinate decarboxylase in vivo and (ii) that L-cysteinesulfon- nearly noninhibitory levels in some but not all animals. ate transamination, in contrast to L-cysteinesulfinate transIt is probable that theanionic amino acid transport system amination, yields metabolites (P-sulfopyruvate and P-sulfo- mediates both D-cysteinesulfinate and L-[1-“C]cysteinesullactate) that are thermodynamically stable and not fonate uptake (35,36).It is thuspossible that inhibition of Lcatabolized to COz (34). [ 1-’4C]cysteine~ulfonatemetabolism byD-CySteineSUlfinate Mice administered ~-[l-’~C]cysteinesulfonate (0.25 mmol/ reflects inhibition of transport into tissues rather than specific kg) metabolize about 72% of the dose to 14C02in 6 h; the inhibition of cysteinesulfinate decarboxylase. I n vivo studies maximum rate of metabolism occurs in the first hour and comparing the metabolic effects of D-cysteinesulfinate with corresponds to a rate of 95 pmol/h. kg (Fig. 2). Mice given D- D-aspartate, a more effective transport inhibitor, indicate, cysteinesulfinate (10 mmol/kg) 60 min prior to administration however, that little, if any, of the D-cysteinesulfinate-meof L-[1-“C]cysteinesulfonate metabolize only about 16% of diated inhibition of 14C02formation is due to reduced ~ - [ l the dose to l4COZin 6 h. The maximum rate of metabolism is 14C]cysteinesulfonate uptake by tissues (studies described in decreased to 12 pmol/h. kg, an inhibition of 87%. In the first Miniprint). In addition, the finding that urinary excretion of metabolite A preliminary report of some of these studies has appeared (26, P-~ulfo[’~C]lactate,a ~-[l-’~C]cysteinesulfonate formed by the transamination pathway (34),is greatly in27). Portions of this paper (including “Experimental Procedures,” part creased by D-cysteinesulfinate administration indicates that of “Results” and “Discussion,” Figs. 1 and 3-6, and Tables 1-3) are D-cysteinesulfinate alters the metabolic partitioning of radipresented in Miniprint at the end of this paper. The abbreviations olabeled L-cysteinesulfonate without significantly changing used areHPLC, high performance liquid chromatography; AAT, the overall extent of its metabolism (studies described in aspartate aminotransferase; c-AAT, cytoplasmic AAT, m-AAT, miMiniprint). tochondrial AAT; CYSO,, cysteinesulfinate; LSC, liquid scintillation Effect of D-Cysteinesulfinate on@-Sulfopyruvateand P-sulcounting. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition folactate Metabolism-Although neither P-sulfolactate nor 8of the Journal thatis available from Waverly Press. sulfopyruvate is directly metabolized to COz at a significant

16570

Cysteine Metabolism TABLE

4

Metabolism of j%lpyruvate and its precursors Mice were administered radiolabeled pyruvate or a pyruvate precursor in the dose indicated by subcutaneous injection on the back of the neck. The animals were then immediately placed in metabolic chambers, and respiratory “CO2 was collected and quantitated as described under “Experimental Procedures.” The values shown indicate the percent of dose recovered as “COZ after 6 h; in all cases, the rate of ‘“CO2 formation was minimal at that time. Urine was collected for 24 h following injection of radioisotope. L-[1-14C]Alanine, D-[l-“Clalanine, and D-[3,4-‘4C]ghCOSe were used as precursors of [1-“Clpyruvate; L-[3-“Clalanine, D-[3-“Clalanine, and D-[S-“C] glucose were used as precursors of [3-‘4C]pyruvate. The results shown for each compound represent averages f S.D. of data from 8 to 16 mice. Where indicated, D-cysteinesulfinate was given by intraperitoneal injection 1 h prior to the administration of the radiolabeled compound. [1-“C]Pyruvate or precursor [3-“C]Pyruvate or precursor Compound administered % of dose % of dose % of dose % of dose (dose in mmol/kg) recovered as recovered in tn recovered 88 recovered in t* wo* the urine “CO1 the urine Pyruvate (0.1) 81.8 + 3.1 21f 4 5.6 + 2.3 66.5 f 1.4 42 3~ 4 7.4 2 1.5 PyNvate (0.1) + D-CyS80.7 f 4.5 19 f 4 6.4 + 1.3 63.5 f 1.4 41f 4 7.3 f 1.4 teinesulfinate (10) Pyruvate (0.5) 77.5 f 5.6 27f4 6.7 f 1.1 66.5 f 1.8 33k6 7.2 f 1.1 Pyruvate (0.5) + D-cys81.7 + 0.9 21f 2 6.8 f 0.8 66.0 f 2.8 37+5 8.4 f 2.3 teinesulfinate (10) Pyruvate (2.5) 81.1 + 4.0 22 f 2 5.3 f 2.5 62.8 + 3.3 38f 8 7.4 f 0.7 Pyruvate (2.5) + D-cys81.6 + 2.4 6.2 f 0.5 21* 3 63.1 f 1.6 41f 4 8.5 + 1.3 teinesulfinate (10) D-Alanine (0.5) 81.1 + 2.7 3.1 + 1.2 63 f 2 64.1 + 1.6 69 + 1 4.9 + 1.9 D-Alanine (0.5) + D-cvs80.2 + 3.6 3.6 f 0.7 60f 9 67.4 + 1.1 70* 5 5.6 f 0.4 teinesulfinate (10) L-Alanine (0.5) 77.3 + 2.5 30 + 4 2.1 + 0.2 62.2 f 2.0 44 + 2 3.1 + 0.3 L-Alanine (0.5) + D-cys77.1 k 1.6 30 f 4 2.6 f 0.6 59.3 f 0.7 47 f 5 2.1 f 0.2 teinesulfinate (10) Glucose (0.5) 72.2 2 1.5 49 + 4 2.8 f 0.8 65.5 f 3.6 58f 8 2.2 3~ 0.8 Glucose (0.5) + D-cys72.9 f 2.2 51+ 8 2.8 f 0.4 63.3 f 4.4 63 f 4 3.3 f 0.4 teinesulfinate (10)

rate, both are precursors of L-cysteinesulfonate by reversal of the transamination pathway of cysteinesulfonate and cysteinesulfinate metabolism (34). As shown in Fig. 5, A and B (Miniprint), D-cysteinesulfinate (10 mmol/kg) greatly inhibits the metabolism of /3-sulfo[l-14C]pyruvate (0.1 mmol/kg) and /3-sulfo[l-‘4C]lactate (0.025 mmol/kg) to 14C02. Since D-cysteinesulfinate is not expected to inhibit the transport of Bsulfopyruvate or &sulfolactate into cells (37), inhibition of their metabolism to COZ is attributed to the specific inhibition of cysteinesulfinate decarboxylase. Tissue Levels and Metabolism of D-Cysteinesulfinate-In vitro studies indicate that D-cysteinesulfinate is a substrate of D-aspartate oxidase; the presumptive product, P-sulfinylpyruvate, decomposes spontaneously to sulfite and pyruvate (38). Since pyruvate is rapidly metabolized to COZ (see below), the rate of formation of 14C02 from D-[l-'4C]CySteiUeSUlfinate reflects the metabolism of D-cysteinesulfinate by this pathway (and possibly others) in Go. As anticipated from the finding that cysteinesulfinate decarboxylase is inhibited for several hours following D-cysteinesulfinate administration, metabolism of the inhibitor is slow (Fig. 6, Miniprint). In 6 h only 7.5% of an intraperitoneal dose of D-[1'4C]CySteiUeSUlfinate (10 mmol/kg) is metabolized to 14C02; during that period the rate of metabolism is essentially constant at 0.130 + 0.007 pmol/h. kg. This rate may be maximal for D-cysteinesulfinate metabolism since decreasing the dose by 95% to 0.5 mmol/kg reduces the rate of 14C02 formation only 54% (i.e. to 0.06 pmol/h. kg). The central role of D-tISp&td.@ oxidase in D-[l14C]cysteinesulfinate metabolism is supported by the observation that the maximum rate of 14C02 formation is inhibited about 70% in animals given D-aspartate, a preferred oxidase substrate (Fig. 6, Miniprint), and inhibited about 35% in animals given meso-tartarate (10 mmol/kg), a competitive inhibitor of the oxidase (39) (results not shown). It may be

noted

that D-[4-14C]aspartate is metabolized to ‘“COZ much rapidly than is D-cysteinesulfinate; maximum rates observed following intraperitoneal injection at doses of 10 and 0.5 mmol/kg are 2.24 and 0.25 rmol/h. kg, respectively. Tissue, plasma, and urine levels of D-cysteinesulfinate were determined 6 and 24 h after administration of the inhibitor at doses of 0.5 and 10 mmol/kg (Table 3, Miniprint). At the lower dose, the tissue level of D-CySt.eineSUlfinate determined 6 h after injection was reproducibly above the Ki for cysteinesulfinate decarboxylase inhibition only in the kidney; after 24 h, tissue levels were uniformly below 0.1 mmol/kg and were often undetectable. At the higher dose, the concentration of D-cysteinesulfinate 6 h after injection averaged 14 and 16 pmol/g in kidney and liver, respectively. Concentrations above 1 pmol/g were found in skeletal and heart muscle, whereas brain contained 0.42 rmol/g. In most mice, kidney and liver levels of D-cysteinesulfinate remained relatively high (>2 pmol/g) even 24 h after injection, although the range of concentrations observed was large. Interestingly, the brain Dcysteinesulfinate concentration decreased only 19% between 6 and 24 h; by 24 h, skeletal and heart muscle levels decreased >90% to levels below the Ki for decarboxylase inhibition. Chromatography of portions of the kidney and liver homogenates indicated that cysteinesulfinate accounted for >95% of the radiolabeled material present. Plasma levels of D-cysteinesulfmate were low (~0.3 mM) at both 6 and 24 h. Urine levels of D-CysteinesuItinate were high even at 6 h and, when 10 mmol/kg of inhibitor was given, accounted for about 60% of the dose. Chromatography of the urine indicated that about 94% of the radiolabeled material was cysteinesulfinate; the remaining 6% co-eluted with cysteinesulfonate. Effect of D-cysteinesu~finate on the Metabolism of Pyruvate and Pyruvate Precursors-In viva inhibition of cysteinesulfinate decarboxylase is expected to alter the partitioning of Lmore

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FIG. 7. Effect of D-cysteinesulfinate on the metabolism of L-["C]cyst(e)ine to "COZ. A , miceweregiven ~-[l-"C]cystine (circles) or ~-[3-"C]cystine (triangles) by subcutaneous injection on the back of the neck; the dose was 0.05 mmol/kg. Control mice (filled symbols)received no other treatment. Inhibitor-treated mice (open

16571

cysteine between metabolism to taurine and metabolism to pyruvate plus sulfate (see below). Prior to quantitating those changes, it was necessary to determine the extent to which [1-"CC]- and [3-14C]pyruvateare metabolized to 14C02and to establish that D-cysteinesulfinate did not affectpyruvate metabolism. As shown in Table 4, mice given[l-14C]pyruvateby subcutaneous injection at doses of 0.1, 0.5, and 2.5 mmol/kg metabolize about 80% of the dose to 14C02in 6 h; the rate of metabolism (reflected in the ts values), and the fraction of the dose excreted in the urine are unchanged over the range of pyruvate doses tested. D-cysteinesulfinate (10 mmol/kg) affects neither the rate nor extent of [1-"Clpyruvate metabolism. As observed previously (20, 33), [3-14C]pyruvateis less extensively metabolized to 14C02.At all doses tested, about 65% of the administered [3-14C]pyruvate is metabolized to 14C02within 6 h; D-cysteinesulfinate is again without significant effect on the rate or extent of metabolism. To examine the possibility that injected pyruvate is metabolized differently than pyruvate formed metabolically, the formation of 14C02from several precursors of [1-'"C]- and [3-'4C]pyruvatewas also quantitated. In mammals, D-alanine is metabolized efficiently and uniquely to pyruvate by Damino acid oxidase; the yield of "CO2 from D-[l-'4C]alanine and D-[3-l4c]alanine is comparable to that from pyruvate in the presence and absence of D-cysteinesulfinate (Table 4). The averaged results from the pyruvate and D-alanine studies indicate that the yield of 14C02 from C-1 of pyruvate is 80.4 & 1.9% and 81.1 f 0.7% in the absence and presence of Dcysteinesulfinate, respectively. The yield of 14C02from C-3 of pyruvate is 65.0 f 1.8%and 65.0 f 2.1% in the absence and presence of D-cysteinesulfinate, respectively. It is noted that these values are not corrected for the urinary loss of a few percent of the administered radioactivity. If the 14C02data are expressed as a percentage of the dose not excreted in the urine, the yields in theabsence and presence of inhibitor from C-1 andC-3 of pyruvate are 84.8 f 1.7%, 86.0 & 2.0%, 69.7 f 2.4%, and 70.2 & 1.8%,respectively. L-Alanine and glucose are also efficiently metabolized to pyruvate but are subject to alternative metabolisms as well. Although the overall yields are consequently a few percent lower, it is notable that theyield of 14C02from precursors of [3-'4C]pyruvate continues to be about 80% of that from [l-'4C]pyruvate precursors (Table 4). The results support the conclusions that theextent of pyruvate metabolism to COPis not sensitive to the source of the pyruvate and that the rate symbols)were given D-cysteinesulfinate (10 mmol/kg) by intraperitoneal injection 1h prior to administration of radioisotope. The mice were placed in metabolic chambers immediately after ~-["C]cystine injection, and respiratory 14C02was monitored as described under "Experimental Procedures" (Miniprint). The points shown on each line represent averages f S.D.for six to seven animals. Urine was collected for 24h. For mice given ~-[l-"C]cystine, the amount of radioisotope in the urine was 6.7 f 2.7 and 7.0 f 1.8% of the dose administered for control and inhibitor-treated mice, respectively. For mice given ~-[3-"C]cystine, the corresponding values were 10.1 & 1.2 and 8.6 f 2.2%. B , micewere treatedas in A except that the radioisotope given was ~-[l-"C]cysteine (six mice per group) or L[3-"Clcysteine (four mice per group); the dose was 0.5 mmol/kg. In mice given ~-[l-'~C]cysteine, the amount of radioisotope in the urine at 24 h was 3.1 f 1.3 and 3.5 f 0.5% of the dose administered in control and inhibitor-treated mice, respectively. Urine data for mice receiving ~-[3-"C]cysteineis not available. C, mice were treated as in A except that the radioisotope given was ~-[l-"C]cysteine (six mice per group) or ~-[3-"C]cysteine(six mice per group); the dose was 2.5 mmol/kg. In micegiven L-[I-"Clcysteine, the amount of radioisotope in the urine at 24 h was 2.9 +. 0.4 and 3.9 & 0.4% of the dose administered in controland inhibitor-treated mice, respectively. For mice given ~-[3-"C]cysteine, thecorresponding values were 10.0 f 1.2 and 8.9 f 1.1%.

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Cysteine Metabolism TABLE5 Effect of D-cysteinesulfinate on radiolabeled L-cyst(e)ine metabolism Experimental conditions were as shown in Fig. 7. Data are shown for percent of dose recovered as '%02at 6 h. These data and inhibition data from Table 6 were used to calculate the metabolic fluxes for each dose of L-['~C] cyst(e)ine. The calculated metabolic flux values for reaction 8 were tested statistically to determine if they were significantly greater than zero; at L-["C]cyst(e)ine doses of 0.05,0.5, and 2.5 mmol/kg, the correspondingp values are 0.019, 0.086, and 0.042, respectively (see "Numerical Methods" (Miniprint) and the "Discussion"). CYSO,, cysteinesulfinate. % of dose recovered

Compound administered (dose in mmol/kg)

"C02 as

Calculated metabolic

Taurine Taurine TaurinePyruvate Without With D-cysteinesulfinate D-cysteinesulfinate

~-[l-'~C]Cystine (0.05) 32.32 & 0.98 40.80 f 0.88 ~-[3-'~C]Cystine 10.69(0.05) ~-[l-'~C]Cysteine (0.5) 45.81 f 0.67 51.30 35.00f 2.38 ~-[3-'~C]Cysteine (0.5) 19.89 ~-[l-"C]Cysteine(2.5) 63.86 f 0.97 66.45 ~-[3-'~C]Cysteine (2.5) 30.01 f 44.82 2.05

& 2.27 13.1 25.85 f 1.58 f 1.16 f 0.64 & 1.66 f 1.93

f 1.6 (37.5%) 26.9 f 4.2 24.2 (52.6%) 41.5 f 3.8 30.5 (57.6%)

flux as % of dose"

rez~tt:

not from CYS02, reaction 8

CYSOz, from reaction 2

21.8 & 1.7 (62.5%) f 3.5 (47.4%) f 3.4 (42.4%)

18.5 f 3.7 (53.0%) 22.5 f 5.1 (44.0%) 26.6 f 6.2 (36.9%)

3.3 f 3.3 (9.5%) 1.7 & 2.9 (3.3%) 4.0 f 5.1 (5.5%)

Numbers inside parentheses represent % of observed catabolism.

and extent of metabolism are not affected by D-cysteinesul- pyruvate + sulfate + "amm~nia."~ mammals, In taurine is not finate. metabolized further (33), but pyruvate is rapidly metabolized to COZ in moderate to high yield as shown inTable 4. Effect of D-Cysteinesulfimte on L-Cyst(e)ine MetabolismIn vivo inhibition of cysteinesulfinate decarboxylase alters Pathways yielding only pyruvate sulfate (grouped as reacthe metabolic partitioning of cysteinesulfinate in favor of tion 61, pathways yielding only taurine (initiated by reaction pyruvate sulfate transamination. The formation of pyruvate and sulfate is 8),and pathways yielding both taurine and increased, whereas the formation of taurine by the cysteine- (initiated by reaction 1 ) have been proposed. The present sulfinate decarboxylase pathway, but not by other pathways, studies, in concert with previous studies defining the partiis decreased. Since taurine is not metabolized to COz (33), tioning of L-cysteinesulfinate (33), allow the relative metadecreased formation of taurine results inincreased formation bolic flux through the indicated pathways to be quantitated in vivo. of l4COZfrom ~-[Q-"C]cyst(e)ine; littlechange in l4COZforAs examined here, the metabolism of radiolabeled L-cysmation from ~-[l-'~C]cysteine is expected. In mice given L- teine to 14C02is described quantitatively by four equations in [3-'4C]cystine (0.05 mmol/kg) by subcutaneous injection, the four unknowns (Fig. 9,Equations 1-4): Since R4 and R6, the extent of 14C02 formation at 6 h is increased from 10.7 to 25.9%by previous treatment with D-cysteinesulfinate (10 In the formation of pyruvate, the amino group of L-cysteine is mmol/kg) (Fig. 7A). Formation of l4CO2from ~-[l-'"C]cystine either lost directly as ammonia (e.g. by action of y-cystathionase on is increased to a lesser extent (see "Discussion"). Qualitatively L-cystine) or is lost by transamination (e.g. in reaction 4 ) . Similarly, similar results were obtained in studies with L-cysteine at inorganic sulfur is released in various forms by the cysteine desulfhydrase activities and is released as sulfite in the breakdown of Bdoses of 0.5 mmol/kg (Fig. 7 B ) and 2.5 mmol/kg (Fig. 7C). sulfinylpyruvate. In vivo the inorganic sulfur products are metaboThe results are summarized in Table 5 . lized almost completely to sulfate. Since the metabolism of the amino Effect of D-Cysteinesulfimte on L-Cysteinesulfinate Decar- group and inorganic sulfur are not addressed here, the distinction boxylation-Since D-cysteinesulfinate is a competitive inhib- among the various initial products is not considered further. Equations 1-4 rely on several assumptions which are supported itor, the extent of cysteinesulfinate decarboxylase inhibition by the present or previous studies as follows: (i) thecatabolism of Lin vivo will depend on the concentration of both the inhibitor cyst(e)ine to COZ is completely described by the reactions in Fig. 8. and the substrate, L-cysteinesulfinate; the intracellular con- It is noted that no additional, quantitatively significant reactions centration of the lattermay increase with increasing doses of have been documented and that reaction 6 is defined in a manner L-cyst(e)ine. To quantitate the extent of in vivo inhibition that accommodates any L-cysteinesulfinate-independentreaction or pathway leading to pyruvate. (ii) D-Cysteinesulfinate is a specific directly, micewere administeredatracer dose of L - [ ~ ~ S ]inhibitor of reaction 2. Of the enzymes catalyzing reactions 1, 6, and cysteinesulfinate along with unlabeled L-cyst(e)ine in doses 8, only aspartate aminotransferase, a catalyst of L-cysteine transamidentical to the L-['"C]cyst(e)ine doses used in the studies of ination and thus part of cysteine desulfhydrase, normally interacts Fig. 7. After 6 h, the mice were killed, and formation of [35S] with a substrate structurallysimilar to D-cysteinesulfinate.As shown in Table 1, aspartate aminotransferase is not inhibited. In addition, taurine was quantitated in the urine, liver, and remaining the finding that D-cysteinesulfinate increases the yield of l4COz from carcass (Table 6). As anticipated, the extent of inhibition ~-[3-"C]cysteine strongly suggests that reaction 1, a major factor in decreased slightly with increasing doses of unlabeled L- that metabolism, is not significantly inhibited. The finding that 14C02is not inhibited by D-cyscyst(e)ine. The percent inhibition values, with S.D. values metabolism of ~-[l-'~C]cysteine to calculated as described under "Experimental Procedures," are teinesulfinate also supports the view that no quantitatively important pathway other than reaction 2 is inhibited. (iii) D-cysteinesulfinate78.6 f 1.7%, 74.0 k 3.1%, and 72.1 k 2.7% for animals given mediated inhibition of reaction 2 does not influence the partitioning 0.05 mmol/kg of L-cystine, 0.5 or 2.5 mmol/kg of L-cysteine, of L-cysteinebetween reactions 1,6, and8. As noted, direct inhibition of the enzymes catalyzing reactions 1,6, and8 is not likely. Inhibition respectively.

+

+

DISCUSSION

Several pathways and reactions contribute to the catabolism of L-cysteine (Fig. 8).Although a variety of intermediates are formed, the final products are few, taurine + COZ or

of reaction 2 may, however, increase intracellular levels of L-cysteinesulfinate, the product of cysteine dioxygenase (reaction 1 ) . Fortunately, in vitro studies indicate that the cysteine dioxygenase reaction is irreversible and not inhibited by L-cysteinesulfinate (40); partitioning of L-cysteine, as opposed to L-cysteinesulfinate, is thus unlikely to be affected. In fact, since L-cysteinesulfinate is irreversibly transaminated by aspartate aminotransferase (reaction 4 ) , significant

16573

Cysteine Metabolism TABLE6 [j6SlTaurine formation from ~-r’S]cysteinesulfinate Mice were administered a tracer dose of ~-[~‘S]cysteinesulfinate mixed with unlabeled cyst(e)ine in the dose indicated by subcutaneous injection on the back of the neck. After 6 h, the animals were killed, and [=S]taurine was determined in urine, liver, and the remaining carcass as described under “Experimental Procedures.” Where indicated, D-cysteinesulfinate (10 mmol/kg) was given by intraperitoneal injection 1 h prior to administration of radioisotope. 76 of administered “S recovered as [“Sltaurine Metabolites administered D-Cysteinesulfinate mmolkg) (dose in (10mmol/kg) Urine Liver Carcass Total ~

L-Cystine (0.05) + L-[36S] cysteinesulfinate (0.01) L-Cystine (0.05) + L-[”S] cysteinesulfinate (0.01) L-Cysteine (0.5) + Lj3’S] cysteinesulfinate (0.01) L-Cysteine (0.5) + L-[36S] cysteinesulfinate (0.01) L-Cysteine (2.5) + L-[35S] cysteinesulfinate (0.01) L-Cysteine (2.5) + L-[36S] Cysteinesulfinate

~~

No

2.90 f 1.26

11.1f 3.1

37.3 f 2.6

51.3 f 1.9

Yes

0.55 f 0.10

2.6 f 0.5

8.0 f 1.0

11.0 f 0.8

No

1.96 f 1.54

11.0 f 1.1

36.2 f 1.5

49.2 f 1.4

f 0.20

2.7 f 0.5

9.6 f 1.4

12.8 f 1.5

No

4.16 f 1.18

11.8 f 2.1

32.4 f 2.8

49.8 f 1.0

0.82 Yes

f 0.61

3.9 f 1.4

10.1 f 1.0

13.9 f 1.3

0.48 Yes

unknowns representing L-cysteinesulfinate-dependent and -independent pyruvate synthesis, respectively, cannot be independently evaluated in theseequations, only total pyruvate synthesis ( i e . R4 R 6 ) can be determined without additional data on L-cysteinesulfinate partitioning (see below). Similarly, R2 and R 8 , representing L-cysteinesulfinate-dependent and -independent taurine synthesis, respectively, cannot be independently evaluated in Equations 1 and 2. Solution of Equations 1 and 2 does, however, directly establish both the extent of cysteine catabolism and the partitioning of L-cysR 6 ) andtaurine teine between pyruvatesynthesis (R4 synthesis (R2 R8). As shown in Table 5 , mice given Lcyst(e)ine in doses equivalent to 0.1, 0.5, and 2.5 mmol of Lcysteine/kg catabolize 35, 51, and 72%, respectively, of the administered compound to pyruvate or taurine within 6 h. Since little L-cysteine is excreted in theurine ("ofransferase

5 mu IO m M

0.2

mu

0

LO m M

0

D-Aspartate

3

3.8

.

* 0.3 6

f

f

..

0.1 6 1.3 1.3

x x

0.1 6

0.1

x

a/

C y s f e i n e s u l f i n a t e d e c a r h o v y l a s ew a s a s s a y e d i n t h e p r e s e n c e o f 0 . 2 5 m M L ~ i i ' 5 1 ~ y ~ f e i n e s u l f ~ as n~d f ees c r i b e d i n E x p e r i m e n t a l P r o c e d u r e s ;D - a m i n o aclds Y e r e added a t the concentrations indicated. The cytoplasmic and mitochondrial i s o z y m e s of AAT were arrayed spec~rophoforoctricaiiyin the direction The reacof oxaloacefafe synthesis as descrlbed in Experimental Procedures. fian mixtures contained 1.0 m M mM o-ketoglutarate and either 1.0 mM IC-AATl O r 0.3 n M Im-&AT1 I.-aepartafe ; D - a m i n o a c i d sw e r e a d d e d af t h e c o n c e n t r a t i o n s averages I S.D. for triplicate detcrmlnations. shown. The results shown are

..

Since many of the fractions submitted t o LSC were slightly colored, all fractions w e r e inlflally counted and were then nixed with a k n o w n amount o f " 5 a n d r e c o u n t e d . T h e true r a d i o a c t i v i t y o f t h e o r l g i n a i s a m p l e was t h e n calculated on the basis o f the efflciency of Counting the lntcrnal standard. In all cases the corrections were small I < 10 61. As described in Results and I n Discussion. D-cy.teinesuIfina~e-freafed mice form less ['SSIfaurIne from I-IIIS]cysfelnesuifir)afe then do cOnfrol n i c e a n d t h i s c h a n g e was q u a n t i t a t e d t o a l l o w e v a l u a t i e n of I. t h e f r a c t i o n a l inhibition effected by D-oysteinesulfinate. Interpreratian of the results in is equally this manner assumes that the administered L-l,'Slcysteinesulfinate metabolized ~n the control and D - c y a t e i n e s u l t i n a f e - f r F a t e d nice and that only In the partitioning between decarboxylation and transamination Io altered. fact. w e f o u n d t h a t at all doses o f u n l a b e l e d L-cystiellne. t h e f r a c t i o n o f t h e a d m i n i s t e r e d radioactivity r e c o v e r e d i n t h e u r i n ewag i n c r e a s e d 2 - to I-fold by D-cystclneoulfinatc treatment ( n . 6 . this observation is not evident from the results presented in Table 6 because L'iSltaurine accounts for little of t h e radloacfivlfy in the urine of inhibitor-treated mice). Given this finding. if was imporfanf fo establish that D-cysfeinesulfinate did not caUBe a s i g n i f l c a n f u r l n a r y loss o f L-[IISlcysteinesulfinatc b y b l o c k i n g a n i o n i c amino acid transport. TO fhls end. 4 treated and 4 control urine g a m p l e s from the experiments at each cyst[c)ine dose were analyzed by HPLC for metabolites. The column 10.6 x LO c1 . w a s packed with Amlnex anion exchange resin. a n d e l u t i o n was at 1.0 n l l n i n . w i t h a l i n e a r g r a d l e n t of 0 t o 0.4 M l H i P O l , pH 4.25 w i t h o u t a d j u s t m e n t 1 f o r m e dover 6 0 m i n f o l l o w e d b y0.1 H K H 2 P O I f o r 6 0 min. Taurlne. cysfeinesvlfinate. cysfeineaulfonate and sulfate eluted sf 1 3 . 5 4 , 7 0 , a n d 86 mi", respectively. Fractions of 1 n l were coilecfed and s u b m i t t e d t o LSC. None o f t h e s a m p l e s c o n t a i n e d r a d i o l a b e l e d c y s f e i n e s u l contents Yere in accord wlfh finate O r cysteinesulfonate. The I'bSItaYrine t h o s e d e t e r m i n e d o n the nrlxed-bed resin calunna (see a b o v e ) . I ) s S ] S u l f a f e excretion was 2 - t o 3 - f o l d h i g h e r i D n ~ c y s f e i n e s u l f i n a f e ~ f r e a f emdi c e . a n d urine from this difference accounted for the higher total radioactivity ~f the inhibitor-treated nice.

Numerical Uethods. The partitioning of L-cysfleiine among fhe several pathways illustrated ~n Figure 8 w a s calculated by solving the four equations g l v e n In F i g u r e 9 [ S e e D l s c u s s i o n l . T h e a p p a r e n t s t i m u l a t i o n o f total L-cysfelne catabolism by D-cysfeinesulflnafe was accclunfed for by mulfipiying the rlght side of Equations 3 and 4 by the factor (,+SI w h e r e S is t h e "fractional sfimUiafiOD''. Letting Y 1 t o Y4 denote the experlnentally defermlned yield of > ' C O i at 6 h. Equations 1 to 4 (Plgure 9 ) may then be rewritten as follows O.BOIIAB+RII 0.6501R6+R.l 0 IAbtRIl 0 IR64R.l

+ 1.OIRB+AZI + t O.,IR*'R2) +

0 Il+SlII)IR21

+

+

0

IR[t+R?)

0 I1+Sl 0 (,+SI Y1(1+Sl

+

0

+ 0

IR8+R21

t

Y21L+S1

+

+

= Y1

II+S)IIIIR2I = Y 2

+ iD.801-1.0111+S)II)IR21

= Y3 10.65~-0.1111+SIIIIIR2I = Y 1

The above equations were then solved for the UnKnaWns. IRB+RII. IRBIRZ). 11+51 a n d l L + S l I r ) l R 2 ) . 5 1 n c e I 1s e x p e r l m e n t a l l y d e t e r m l n e d ( s e e T a b l e 6 1 . t h e v a l u e s of R 2 and RE4 a r e readily calculated. Note that experimentally dcterm i n e d v a l u e r f a i t h e y i e l d oh.C02 f from ["Clpyruvafe 1i.e. 0 . 8 0 1 and 0 . 6 5 1 w e r e used I " the ~ a l ~ u l a f ~ o n s . For t h e s t u d i e s at each dvse of L-["Clcystleline.an e s t i m a t e of e r r o r ~n t h e c a l c u l a t e d q v a n r l t i e swas m a d e o n t h e b e s i r o f t h e o b s e r v5e .d0 , ' s of the 8 e x p e r ~ m e n t a l i y d e t e r m i n e d v a l u e s : Y 1 to Y 4 [Table 5 1 , the t w o values for ["Sltaurine y>eld required to calculate I [Table 61. and the values far , * C O , I - i3-"Clpyruvate (Table 41. We assumed these experlyleld from i ~ - ' ~ c and mental quantifies to be normally distributed and simulated 2000 d a t a s e t s w i t h each parameter ~ndapendently computed by gencratlon of random numbers with the a p p l i c a b l e m e a n a n d S.D. T h e 5 . 0 . ' ~ o f t h e m e a n v a l u e s ( s e e T a b l e 5 a n d Results1 w e r e t h e n c a l c u l a t e d as t h e S . D . ' s o f t h c 2 0 0 0 r o l u t l o n s to t h e mula la fed equations I n . 6 . t h e m e a n v a l u e s s h o w n a r e exact s o i u t i 0 n 6 t o t h e eq"ations1 ProbabllIfles reported in Table 5 refer to the null hypothesis that the metabolic flux through R e a c t i o n 8 1i.e. R81 1 5 Ze-0 and were computed by appllraflon of e one-failed t-test concerning the mean.

j m a x l m u a Fate decreased by 37 X I whereas D-cysteincsulfinatc has no SlgnifLcant effect on L-aspartate metabolism. Since D-aspartate is not an effective

a c i d transporf~sysfen. The present results Suggest that D-aspartate Is a much m ~ r e effective transportinhibitorthan is D-cystFinesulflnate. Note that D - a s p a r t a t e I S m e t a b o l i z e d to o x a l o a c e t a t e by a s p a r t a t e o x i d a s e a n d t h a t Wasparfate administration may thus significantly reduce the specific activity that oxaloo f [''Cloxaloacetafe formed from L-[~*CIasparfatc. The finding acetate o r a l f e r n a t l v e p ~ e c u r s o r s o f o x a l o a c e t a t e 1e.g. L - m a l a t e ) d o not

lnhibif , ' G o , formation from L-["Claspartate as effectively as does D-asparf a t e indicates that changes 1" a p e c i f l c a c f l v i t y o f o x a l a a c c t a f e Or its metabolites do n o t account for the effect of D-aspartate ldafa not rhownl. A s antlcloated. D-asoarrate doer inhibit L-ll-'.Clcysteinesulfonafe netabollsm to l- 4 C 0 , lpresunably by blocking transpo;ti. but -the n a x i n u n rate of "COi f o r m a t i o n f o l l o w i n g a d m i n i o t r a f l o n o fI O m m o l / k g o f D - a s p a r t a t e (Figure 4 C , Miniprint) is almost 5-fold higher than the maximumrate following 21. Considering administration of 10 m ol/kg of D-cysfeinesulfinate (Figure ? h a t O - a s p a r t a t e 1s t h e n o r e e f f c c t l v e t r a n s p o r t inhibitor. it is probable that little /or none] of fhe effEcf o f D-cyeteinesulfinate On L - I l " . C l c y o f e inesulfonafe metabolism 1s aftrlbutabie to inhibition of L-cyrfeinesulfonafe uptake

I n seoarafe studies. mice w e r e q i v e n either D-cysteinerulfinatc

1 3 not metabolized to '.CO; is excreted in the urine w ~ t h i n24 h. In anlnals not glven D-cysteinesulfinafe, radiolabeled cysteinesulfonate and 8-sulfor e e p e c f i u e l y . o f t h eu r i n a r y r a d i o lactate a c c o u n t for about2 0 X and B O a c f ~ v l t y 18-sulfolactate 1s formed by malate dehydrogenase-nedi~ted reduction

%.

of 8-sulfopyruvafe. the product of A&=-mediated cysfelneoulfonate fransaminaf i o n ; 8 - s u l f o p y r u v a f e is n o t d e t e c t e d i n u r i n e ( 1 . 4 1 . 1 In anlndib glverl D-cysteincrvlfinafe. the fraction o f subsequently adninlsfercd radloacflvlfy e x c r e t e d i n f h e u r i n is e i n c r e a s e d 3.5 to 4.0-faid. but t h e r e l a t ~ v ea m o u n t s of cysteinesvlf~nafe and 8-sulfolacfafe a r e not 5 1 g n i f l c a n f l y c h a n g e d [cysteinesulfonate accounts for 1 5 X and 21 x o f the urinary radloacflvify In experiments Ib and Zb. respectively.) S i m i l a r studies were carrled out u e l n g L-[',Slcysfeinesulfanate ( e x p e r i m e n t s 3 and 4 , T a b l e 2 1 . B e c a u s e the decarboxyiation preduct, ( 2 5 S 1 C a u r l n e , is diluted info large flesue pools and excreted slowly. recovery of total radioacflvify Is much smaller than I " the s t u d i e s w l f hL - I l " l C ] ~ y s f e i n e s u l f o n a ~ e1 i . e . "CO, is e x c r e t e d r a p i d l y a n d common with the "C sfudlcs. excretion of quantitatlvelyl. Nevertheless. in ['lSlcySfeinesulfonafe and 3-["~S]sulfolacfafe in t h e u r i n e i s g r e a t l y increased b y 0 - c y s f e i n e s u l f i n a f e a d n i n l s f r a f l o n i f h e increase 1s 4 . 1 and 5.6-fold i n experiments 3 and 4 , respecfiuelyl. Radiolabeled 8-suifolactafe agaln exceeds cysteinesulfonate in the u r l n e and. most importantly. cysfslnr s u l f o n a t e a s a fraction of cysfeinssulfonate + 8-sulfoiacfafe is Increased o n l y slightly by Prior admlnletration of O-cystelnesuiflnafs ie.g ~n experlm e n f s 3 and 1, cysteinesulfonate increases from I4 6 to 17 x and from 1 1 % t o 1 3 x . respectively) ~~~~~

~~

In summary. the studies reported in Table 2 indicate that D-cysteine5111 finafe adminirtratlon inhibits cysteinesulfonate decarboxylaflon and thus alter6 \he metabolic parfifloning of subsequently admlnlsferedL-cysfeinesulfonate in favor of transamlnafion. R-Sulfopyruvate is f o r m e d in g r e a t e r s n O Y n t S and is metabolized to 8-eulfolacfafe. which 1s excreted in the urlnc. There is n~ evidence of metabolically slgnificant lnhibiflon of L - c y s f e ~ n e s u l fonate transport into tissues. Although urinary cysteinesulfonate excretion is increased several-fold by D-cysfeincsulfinate adminisfraflon. cysfe~nerulfonate excretion relafive t o 8 - s u l f o l a c f a f e e x c r e t l n n is e r s s n f l a l l y unchanged. Previous studies indicated that L - ~ y s f e i n c s u l f o n ~ f e . p - s u l f o p y r u "ate and 6-sulfalactate are reverslbiv lnfercanverted Jn v i v o 1 1 1 . t h e oresenf flndings suggest that metabolic imterkonversion is a t or near equ111br;um and t h a t c y s t e i n e s u l f o n a t e a n d p - s u l f o l a c t a t e a r e lorf i n t h e u r l n e in m o r e o r l e s s constant r e l a t i v e p r o p o r t i o n [ n o t e t h a t t h e r e l a t i v e c o n c e n t r a t i o n s o f c y s t e i n e s u l f o n a t e a n d 3 - s u l f o l a c f a f e~n t i s s u e s or p l a s m a m a y he d i f f e r e n t than that seen In urine. i n v i t r o . the AAT and malate dehydrogenase reactions f a v o r c y a t e l n e s u l f o n a f e a n d 8 - e u i f o l a c f a t e f o r m a t i o n b y m o daenrda t le a r g e factors. rerpecflvely (11.1

16578

Cysteine Metabolism

Effect of D-Cyofeincsulfinat~ on the Formation of U r l n a r y end Respiratory Metabolites from Radiolabeled Cysteinesulfonate*/ "

Radiolabeled EXpt. Na.b/

Percenf of Administered Compound Recovered as:g/

Inhibitor COmDOund Given

HOURS

a/

Mice given inhibitor received D-cysteinesulfonate 110 nmol/kgi by ~ n t r a peritoneal injection. One hour later L-Il-"Clo r L-lI)Slcys~cinesuifonafe wasgivenbysubcufaneou= i n j e c t i o n o n thebackof t h eneck. and themice w e r e Re-piracary >.COI was collected from placed in individual metabolic chambers. m i c e given ["C]cysteine*ulfon.t~ for 6 h; urine was collected far 2 4 h from all animals. Food and water were withheld from all animals after injection of the radiolabeled compound.

b/

Each entry presents data laverage S.D.1 for t w o animals. Studies with the same e x p e r l m e n f n u m b e r w e r e carried out in parallcl andC a n be dlrecfiy compared. The quantitative differences observed between experiments 1 and 2 and between experiments3 and 4 reflect the variability of in v i v o 1fudlc5 carried O u t several weeks apart.

g/

Respiratory T*COi and urinary mefeboliter w e l e analyzed as described i n Experimental Procedures. Figure 2 (Main Text) shows the t i m e course of b 4 C 0 , formation far the animals in e x p e r i m e n t s 1 and 2 plusa third experiment i n which urine was not analyzed.

Tissue

Cancentraflon in Tissue

vase Immol/kg1

after 6

HT.

After 24 hr

~. lYnol/gmJ

Kidney

Liver

Hearc

0 . )

l.5l

10.0

13.9

0.5

0.5

10.0

16.0

0 . 5

I

0.38

* 5.7 ?

0.5

* 5.9

0.07

1 % dose) Of 4.4

1.9

?

.

0 . 8""

0.7

5.7

I

8.6

. 2.9

0.05

0.05

0.55

I#nol/gm)

4.8

f

6.1

0.02

2.6

2.1

.

L.9

0.8

."~

10.0

1.27

0.06 * 0.03

0 . 1 1 t 0.01

10.0

0.42

0.08

0.06 i 0.02

0.34 f 0 . 0 1

Skeletal

10.0

1.20 ? 0 . 6 3

0.12 t 0.05

f

0.5

0.2

I

" " " "

Brain

f

1 % of dose)

""

t 0.8 ""

i o i o

""

""

"uncle Plasma

Urine

Respira-

0.5

0.06

+

0.002

0.37

10.0

0.30 * 0 . 0 4

0 . 5

" "

10.0

""

61.1

0.5

""

58.3

10.0

""

7.5

0.10 3.3

. . . f

+

0.03

0.01

0.01

" "

0 . 0 7 t 0.01

2.8

""

5.5

f

0.6

1.7

""

19.8

f

7..

3.4

""

. . . .

."_

""

tory co, f

""

0.22 f

1.4

~~

M i c e were adminietered D-[I-'.Clcyefeinesulfinete by intraperitoneal Respirafory l * C O was determined at inferinjection at the dose indicated. v a l o for 6 h.. and the a n i m a l s w e r e t h e n either kflled 16 h. tine points) or a l l o w e d t o r e m a i n i n the metabolic chambers for 24 h. and then killed. The tissue disfribufion of radioactivity end its characterization as D-cysteinesulfinate were carrled out as described in Experimental Procedures. a/

Cysteine Metabolism

16579

NO 0

3 HOURS

HOURS

5ot

I

i

I

3 HOURS

INH~BITOR

W ~ T HD-CYSTEINESULFINIT

5

52

1

!

!

i .' 1

1.

2. 3. 4.

5. 6.

l. 8.

9. 10. 11.

5

3

5