THE JOURNAL OF BIOLOQ~CAL CHEMISTRY Vol. 239, No. 4, April 1964 Printed in U.S.A.

Protein

Synthesis

I. IKCORPORATION H. K. From the Department

in Plant

OF AMINO DAS,

S.

Ii;.

ACID

CHATTERJEE,

of Biochemistry,

Ir\’ PEI’TIDE AND

Plant mitochondria can incorporate amino acids into protein (1, 2), but the details of the process are lacking, although the situation is somewhat clearer with mitochondria from animal source (3-6). The present paper is concerned with the incorporation of amino acids by the mitochondria from the seedlings of Vigna &ens-is (Linn.) Savi and also with the role of the microsomal fraction in this process. A preliminary report had been published (7). PROCEDURE

Cell Fractionation-Fractionation was done in either the superspeed angle head No. 69404 of the hlajor refrigerator centrifuge (Measuring and Scientific Equipment, Ltd.) or the angle head No. 856 of the model HR-1 centrifuge (International Equipment Company). All cent,rifugal forces hereafter mentioned have been calculated on the basis of the radial distance of the tips of the centrifuge tubes from the rotating axis. In the course of our work, it struck us that some of the preparations made by our original method (8), particularly those isolated in the first mentioned type of centrifuge, were contaminated with whole cells. This result was probably due to the fact that the particles had to travel a greater distance in this centrifuge, especially when the tubes had to be filled to the brim, owing to their smaller capacity. The modifications that had to be adopted are described here. The first centrifugation was done at 1,000 X g for 10 minutes. The supernatant fluid was centrifuged at 20,000 x g, and the sediment was suspended in a solution containing 0.5 M sucrose-O.15 M potassium phosphate buffer, pH 7.4, and was centrifuged at 2,000 x g for 15 minutes. The supernatant fluid was then centrifuged at 20,000 x g to obtain the mitochondria. The supernatant fluid of the first centrifugation at 20,000 x g was diluted with an equal volume of distilled water and spun at 125,600 x g for 3 hours in the Spinco model L centrifuge to sediment the microsomal fraction. The sediment was suspended in 0.25 M sucrose-O.075 M potassium phosphate buffer and recentrifuged at 125,600 X g for 2 hours. The pH 5 enzyme was obtained from the supernatant fluid of the first centrifugation (9). EDTA Titration-The requirement of EDTA (Table I) was determinedby titration (10). Determination of Radioactivity in Protein-For the measurement of radioactivity, protein was processed according to Stachiewiez and Qua&l (11) with two additional steps. The reaction wasstoppedby the addition of trichloroacetic acid to a final concentration of 6%. The precipitate was collected by centrifugation, washedonce with 60/;, trichloroacetic acid, and

January

LINKAGE

S. C. ROY

Calcutta Urkersity,

(Received for publication,

EXPERIMENTAL

Mitochondria

Calcutta 9, India

30, 1963)

then heatedat 95” for 20 minuteswit,h the sameacid. The residue waswashedonceagain with 60/, trichloroacetic acid at room temperature (2530”) and then dissolvedin 0.4 N NaOH with the addition of 25 pmolesof L-glutamate-*%. The solutionwas allowedto stand for 1 hour at room temperature,and the protein wasreprecipitatedwith trichloroacetic acid and washedwith thioglycollate (1ol,) in the cold and then with ethanol at room temperature. (The time during which the solution stood was subsequentlymuch reducedwithout affecting the results.) The protein was warmed to 45-50” with a mixture of ethanol and diethyl ether (1: l), and the centrifuged residuewaswashedwith diethyl ether. The protein was transferred to planchets, and the radioactivity was determined in a windowlessgas flow counter with an M5 semiautomaticsamplechanger(model182X scaler, Nuclear-ChicagoCorporation) and corrected for background and self-absorption. Determinationof Protein--In all cases,exceptin the experiment of net protein synthesis,protein wasdeterminedcalorimetrically by the biuret method (12). Absorbanceat 550rnl.cwasmeasured in a Coleman junior spectrophotometerand compared with valuesfrom a standardcurve madewith crystalline bovine serum albumin. Experiment with Slicesof Seedlings-Healthy seedlingsof uniform size were chosen,kept at &2” for 1 hour, and then sliced in the cold with a safety razor bladeinto sections1 to 2 mm thick. Finer sectionswereavoided in order to leavea greater proportion of the cellsintact. Sectionsfrom four seedlingswere incubated with 3 ml of reaction mixture. The reactionswere stoppedby plunging the flasks into ice, and the contents were ground to a fine slurry in a mortar with seasand at &2”. The slurry was diluted with 3 ml of a solution of 0.5 M sucrose,0.15 M potassium phosphatebuffer, pH 7.4, and 0.02 M EDTA; and the mitochondria and the microsomalfraction were isolated. Radioactivity in thesefractions wasdeterminedafter precipitation of the protein with trichloroacetic acid and washingasdescribedabove. Enzymatic

Determination

of L-Glutamic

Acid in Mitochondria-

Mitochondria were boiled with alcohol (80%) for 10 minutes, and the coagulatedprotein was centrifuged and washed three times. The supernatantfluid with the washingswas evaporated in a vacuum at room temperature,and L-glutamatein the residue wasassayedmanometrically with I-glutamic decarbosylase(13). Performicilcid Treatment-This treatment wasdoneaccording to Petersonand Greenberg(14). Reincubation with 0.02 nl Glutamat0C-Mitochondria were incubated with glutamate-14C,0.05 mM, as describedin Table I, and the incubation mixture was centrifugedat 20,000x g for 15 minutes. The sediment was washed five times with 0.5 M

1126

April

1964

1127

H. K. Das, S. K. Chatterjee, and S. C. Roy

sucrose-O.15 M potassium phosphate buffer, pH 7.0. Half of the mitochondria were then reincubated as described in Table I, escept that glutamateJ4C was replaced by 0.02 M glutamateJ2C, and the residual half of the mitochondria served as the control. Determination of NHz-Terminal Incorporation-The radioactive protein was treated with fluorodinitrobenzene (15), the protein was hydrolyzed, and dinitrophenylglutamic acid was separated from glutamic acid by ether extraction (15). The separation was also achieved by chromatography on Whatman No. 1 paper, with water-saturated phenol as the solvent. Dinitrophenylglutamic acid was prepared according to the method of Sanger (15). Partial Hydrolysis of Radioactive Protein-The radioactive protein was treated with 11 N hydrochloric acid (16) in a sealed tube for 36 hours at 37”. The insoluble matter was centrifuged down, and the acid was removed in a vacuum at room temperature. The peptides were separated by two dimensional chromatography on Whatman No. 1 paper with water-saturated phenol and a mixture of butanol, acetic acid, and water (4: 1: 1) as solvents. The radioactive peptides were eluted with water, hydrolyzed with 5.7 N hydrochloric acid, and rechromatographed. Bacterial Counts-Viable bacteria were counted by the plate and dilution method with Eugonagar medium. Addition of 5 ‘% defibrinated blood produced no improvement. Determination of Endogenous Free Amino Acids in Mitochondria-Mitochondria were boiled for 20 minutes with ethanol (80%), and the coagulated protein was centrifuged and washed three times with the same solvent. The first supernatant fluid and the washings were combined and evaporated in a rotary vacuum evaporator below 40”; the residue was taken up with 1 ml of distilled water and processed according to Spackman (17). Picric acid solution (1%; 5 ml) was added to it, and the precipitated protein was centrifuged. Exactly 5 ml of the supernatant fluid were then passed through a Dowes 2X-10 (Cl-) column, and the walls were washed with five 3-ml portions of HCl (0.02 N). The combined effluent and washings were concentrated in a rotary vacuum evaporator, and the solution was brought to pH 7 to 8 with NaOH and allowed to stand at room temperature for 4 hours to convert cysteine to cystine. The pH was then adjusted to 2, and the solution was increased to 5 ml with the “sample diluting buffer.” The solution was then analyzed (18) in an automatic amino acid analyzer (Beckman, model 120). Determination of Net Protein Synthesis-The method of Lowry et al. (19) was employed for the determination of protein in the The specific reagent was experiments of net protein synthesis. prepared according to Folin and Ciocalteu (20). Absorbance at 750 rnl.c was determined in a Beckman spectrophotometer (model DU) and compared with values from a standard curve made with crystalline bovine serum albumin. Since this method of determination is also very sensitive to tyrosine, the protein was subjected to a process of thorough washing before the assay. The reaction was stopped by the addition of trichloroacetic acid to a final concentration of S%, and the precipitated protein was washed by the method adopted from Stachiewiez and &u&e1 (11) described above. Materials-ATP and GTP were obtained from Sigma Chemical Company, St. Louis, Missouri, fluorodinitrobenzene from Hoffmann-La Roche and Company, Ltd., Basle, Switzerland, Eugonagar as a free gift from Baltimore Biological Laboratory,

TABLE

I

incorporation by isolated particles The incubations were carried out for 2 hours at 37” in 0.05 JI potassium phosphate buffer, pH 7.0, 0.4 M sucrose, 2 mM MgCI?, and 1 mM ATP. L-Glutamate-14C, 0.2 amole containing 33,106 Glutamate-“C

c.p.m., and mitochondria containing 6 to 8 mg of protein mpmoles of endogenous free glutamate per mg of protein employed in a total incubation volume of 0.5 ml.

and 30 were

-

Radioactivity-.

Enzyme

Additions cl

-

1 c.p.nr./mg

Boiled mitochondria Mitochondria Five-times washed mitochondria Microsomal fraction Mitochondria + microsomal fraction Boiled mitochondria

5 183 461 4

GTP, 1 mM, + mixture*, 0.1 mM GTP, 1 mM, + mix. ture, 0.1 mM GTP, 1 mM, + mix. ture, 0.1 mki

Mitochondria Microsomal Mitochondria fraction Mitochondria zymes Microsomal enzymes

fraction + microsomal

+

fraction

pH

135 4 182 5

I GTP, 1 mM, + mix. ture, 0.1 mM

130

GTP, 1 mM, + mix. ture, 0.1 mM

145

5 en.

+ pH

E GTP,

1 mM,

+ mix-

10

ture, 0.1 mM Boiled mitochondria Mitochondria Mitochondria +

7 184 superna-

tantt Mitochondria treated

94 + EDTAsupernatanti

70

* Mixture of L-threonine, L-cysteine, L-leucine, L-proline, Lvaline, L-tyrosine, L-aspartic acid, L-methionine, L-lysine, Larginine, L-alanine, L-tryptophan, L-phenylalanine, L-isoleucine, L-histidine, L-serine, L-hydroxyproline, glycine, L-glutamine, and L-asparagine. t Supernatant fluid after the sedimentation of mitochondria, containing both microsomes and soluble enzymes. $ The heavy metals present had been chelated by the addition of equivalent amounts of EDTA.

Baltimore, Maryland, and the radiochemicals chemical Centre, Amersham, England.

from the Radio-

RESULTS

Site of Amino Acid Incorporation-The microsomal fraction isolated from 4%hour etiolated seedlings of Vigna sinensis (Linn.) Savi has been found to be practically inactive with respect to amino acid incorporation. It was immaterial whether the seedlings were disintegrated with 0.5 M sucrose alone, or whether the medium was supplemented with 2 mM MgC12-0.15 M pota+ sium phosphate, pH 7.4, or with 0.01 M EDTA, or with both. Mitochondria, on the other hand, incorporated appreciable

Protein Synthesis in Plant Mitochondria

1128

TABLE II Distribution of glutamate-% in different cellular fractions Seedlings were homogenized in 0.5 M sucrose, strained, and centrifu#ed at 2ooo X g at 0” for 10 minutes. The super-n&ant fluid, 8.5 ml, was brought to pH 7.0, incubated with 8,270,ooO c.p.m. of glutamate-W in the presence of 2 mM ATP, 2 mM GTP, and 2 rnM MgCl, in a total volume of 9 ml for 2 hours at 37”. The reaction was stopped after 2 hours by chilling, and the particles

were fractionated centrifugally in the cold (O-2’). Fraction

Radioactivity

TABLE IV Centrifugal fractionation of mitochondrial homogenate by layering Mitochondria prepared as described in the text were treated in a Potter homogenizer with 0.5 M sucrose, layered on 1 Y sucrose, and centrifuged at 10,006 X g for 15 minutes. The two layers of sucrose were carefully removed, and the sediment was saved. The 0.5 M layer and the 1 M layer, after dilution to 0.5 M, were separately centrifuged at 20,000 X g, and the sediment was used in the incubations. Conditions of the incubation were as in Table I, except that 132,000 c.p.m. of radioactivity were employed.

c.p.m./mg prolcin

Mitochondria ...................

EWrllle

55 2 1 1 2

Mitochondrial fluffy layer. ...... Microsomal fraction ............. Microsomal fluffy layer .......... Soluble supernatant ............

TABLE III of radioactivity in different cell fraction8 on incubation of 8lice.r of seedlings with L-glutamate-L4C Slices from four seedlings were incubated with 0.5 M sucrose, 0.15 Y potassium phosphate buffer, pH 7.0, and 0.7 mM L-glutamate-r4C containing 362 X 10’ c.p.m. in a total incubation volume of 3 ml. After 2 hours at 37”, the incubation was stopped by chilling, the slices were ground with sand, and the fractions were separated centrifugally.

Distribution

Radioactivity

Fraction

. .. . fraction. . ..

.. .. . .

.. .

. ... .

..

161 5& 34

throughout.

The level of incorporation

in Table

More

or less the same amount

II

was low

comparedto that in Table I, probably owing to the inhibitory effect of the solublefraction. In Table I, 0.1 ml of the supernatant fluid hasproducedabout 50% inhibition of the incorporation into mitochondria obtained from 8 ml of homogenate.

of mitochonclria

C.).rn.

c.p.n.1 PrzL

1800 406 122

838 118 87

2156 2010 2260 was present in

the incubation mixture of Table II, but along with 8.5 ml of the supernatant. Further, dilution of the incubation mixture (9.0 ml instead of 0.5 ml) may be another reason. of Radioact&@

in l?i&mnt

Cdl Fractims on

with L-Glutamde-“C-If slices, 1 to 2 mm thick, of the seedlinga were incubated with L-glutsmateJ4C, the mitochondria obtained from them incorporated more radioactivity into their protein than did the microsomal or the supernatant fractions (Table III). The incorporation waslow comparedto that with isolatedmitochondria (Table I), probably becauseof slow penetration of n-glutamate-*C into the slices. Characterization of Cell Fractions--The microsomalfraction has been found to contain a relatively large amount of RNA, which was readily attacked by ribonuclease. Also, this frection contained appreciable glucose 6-phosphataseactivity.’ Mitochondria were practically free from any glucose 6-phosphataseactivity, and their RNA was un&ected by exogenous ribonuclease,indicating absenceof contamination by the microsomalfraction.’ Electron microscopic studies and biochemical characterizations of our microsomalfraction have indicated that it abounded in free ribonucleoprotein particles and that the occurrenceof membraneswas1imited.r Similar observationwasmadeon pea seedlingsby T’so, Bonner, and Vinograd (22). Electron microscopy of the mitochondrial pellet revealed typical structures of mitochondria with negligible numbers of ribosomesand no microsomalmembranes,whole cells, or nuclei.’ Centrifugal Subfvwtionation of Mftochundr&z-Though centrifugation at 20,000 x g has beenadopted as the routine procedurefor sedimentingmitochondria, most of the incorporating activity canbe sedimentedat 10,ooO X g when the mitochondrial homogenateis layered on 1 Msucrose(Table IV). The particles in the two layers were without influence on the incorporating Incubatzim

radioactivity. Five-times washed mitochondria were 2.5-fold ss active as the once-washedmaterial. With a mixture of mitoehondria and the microsomalfraction, incorporation was lessthan that with mitochondria alone. Addition of GTP, a mixture of amino acids, glutamine, and asparagine,or pH 5 enzymes(9) could not improve the situation. The supernatant fluid after the sedimentationof mitochondria, with or without EDTA, was not only inert, but it produced somedepressionof the glutamate incorporation (Table I). When the whole homogenateof the seedlings,freed from the cell debris, was incubated with glutamate+C and fractionated centrifugally, incorporation was maximal in the mitochondria and negligiblein the microsomalfraction (Table II). This was always so, irrespectiveof whether the whole seedlingswere used or the cotyledons were excepted, whether light was absent or present during germination, whether the period of germination was varied from 48 to 129 hours, or whether 0.15 M potassium phosphatebufIer, pH 7.4, or 2 mM MgCh, or both were included in or omitted from the disintegration medium. The pattern of the results remainedunaltered if seedlingsof mung bean or pea were usedinstead and the cell fractionation technique and incubation conditions of Webster (21) were followed rigorously

Radioactivity

Sediment at 10,CKKl X g.. . . . . . ... ... .. Sediment from 1 M sucrose layer. . . . . . . . . . . . Sediment from 0.5 M sucrose layer. . .. . Sediment at 10,009 X g + sediment from 1 M sucroselayer.................................. Sediment at 10,669 X g + sediment from 0.5 M sucroselayer................................ Sediment at 10,060 X g + sediments from 0.6 M and 1.0 M sucrose layers. . . . . . . , . . . . . . . .

Distributh

c.#.m./mg prokin

Mitochondria Microsomal Supernatant

Vol. 239, No. 4

of f&X?8

of h’dii~s

1 H. K. Das and T. Mukherjee,

personal communication.

April

1964

H. K. Das, S. K. Chatterjee,

activity of the 10,000 x g sediment. It was found on further centrifugal fractionation that the sediment at 4,000 x g - 3,000 x g had the maximal specific activity (Table V). Absence of Release of Labeled Protein from Xitochondria during Zncubation-IVlitochondria from V. sinensis, whether washed once or 5 times, released about half their protein on incubation under the conditions of Table I, but very little labeled protein was released from the mitochondria during incubation (Table VI). Nature of Attachment of GlutamateJ4C with Protein-Incubation of the alkali solution of labeled protein with glutamateJ2C effected very little loss in radioactivity from protein, suggesting that the glutamate-W was chemically bonded with the polypeptide. The possibility of glutathione synthesis from glutamate-W and the subsequent attachment of the former to a protein chain through a disulfide bond may be ruled out, since the performic acid treatment had no effect on the labeled protein. Reincubation of incorporated mitochondria with a large excess of glutamateJC resulted in a loss of only about 8% of the radioactivity (Table VII). Fluorodinitrobenzene treatment yielded only 1 y0 dinitrophenylglutamate-14C (Table VIII), indicating negligible NHz-terminal incorporation or contamination by unreacted glutamate-l*C. Partial Hydrolysis of Radioactive Protein-Partial hydrolysis of the radioactive protein and subsequent paper chromatography TABLE

by different

of mitochondrial

c.p.m./mg protein g g g y

-

2,000 3,000 4,000 5,000

x x x x

g g g g

TABLE

Absence

of

739 995 895 343

VI

release of labeled protein during

from

mitochondria

incubation

L-Glutamate-14C (66,200 c.p.m.) was incubated with mitochondria as described in Table I, and the reaction was stopped by chilling in ice. The reaction mixture was then centrifuged at 20,000 X g for 15 minutes, and the sedimented mitochondria were washed five times with 0.5 M sucrose-O.15 M potassium phosphate buffer, pH 7.0. The supernatant fluid and the washings were combined. The sediment and the supernatant with washings were treated separately as described in the text for counting.

=ercen-

Fraction

Protein

tage of Radiototal activit> protein

at 20,000

X g..

3.55 3.05

Peo;C&$ incorporation

c.p.m.

mg Sediment Supernatant

of glutamate-14C

with

protein

The radioactive protein resulting from incubation I-‘% with mitochondria was subjected to different details of which have been given in the text.

I

Treatment

Alkali and glutamate-W Performic acid. Reincubation for 2 hours glutamate-12C.....................

for 1 hour. with

Radioactivity protein

of glutamatetreatments,

in Loss of radioactivity

Control

Treated

c.p.m./mg

protein

%

458 458

452 462

1.3 0

575

531

7.6

0.02 M

TABLE

NHt-terminal

VIII incorporation

The radioactive protein obtained by incubation of glutamate1°C was treated with fluorodinitrobeneene, and the protein was hydrolyzed. The hydrolysate was fractionated by both ether extraction (15) and paper chromatography. For details, see Table I and the text. Radioactivity

in NHt-Terminal incorporation

Acid

fraction

Glutamate

spot

~~~t~$$~

53.6 46.4

2520 114

C.P.fL

homogenate

Incorporation

Sediment

x x x x

of attachment

V

fractions

Mitochondria prepared as described in the text were treated in a Potter homogenizer with 0.5 M sucrose-O.15 M potassium phosphate buffer, pH 7.0. Particles were then sedimented for 15 minutes at different centrifugal forces and incubated with 132,000 c.p.m. of L-glutamateJ4C. Other conditions of incubation were as in Table I.

3,000 4,000 5,000 2o,oo!l

VII

T.~BLE

A’ature

Ether extract

Zncorporalion

1129

and S. C. Roy

95.7 4.3

63 * The

1

5g70

abbreviation

j used

1570 is: DNP,

1

17

:“:

2,4-dinitrophenyl.

revealed 11 distinct radioactive spots in one experiment, while in another 6 spots were detected. Of the 11 spots, the most radioactive 3 were selected and studied. Spot 1 on elution, hydrolysis, and rechromatography revealed spots of phenylalanine; methionine or valine, or both; tyrosine; alanine; glycine; se&e; glutamic acid; cysteine; and aspartic acid. Spot 8 gave aspartic acid, glutamic acid, serine, glycine, cysteine, and lysine. Spot 10 gave threonine, glutamic acid, glycine, serine, and aspartic acid. The radioactivity in the 11 spots could account for 82% of the total radioactivity put on the paper (Table IX). Bacterial Counts and Radioactive Incorporation-It was felt necessary to ascertain whether glutamate-14C was actually incorporated into mitochondrial protein or whether bacterial contamination was responsible for it. No correlation could, however, be derived between the bacterial content and the radioactive incorporation in different mitochondrial preparations. Further, incorporation in 2 hours was about 10 times that in 0.5 hour, and during this period bacterial counts remained unchanged. Addition of sodium penicillin G (100 units per ml) into Petri culture dishes completely inhibited bacterial growth, but the incorporation of glutamate-14C into protein was unaffected with this concentration of penicillin in the incubation flasks (Table X). Requirement of Other Amino Acids, MgCl?, and ATP for Zncorporation of GlutamaW4C-Incorporation of glutamateJ4C was found independent of other exogenous amino acids. In fact a mixture of amino acids (0.5 mM each) brought about a depression in the incorporation of glutamate-“C into fresh mitochondria.

Protein Synthesis in Plant Mitochondria

1130

TABLE IX Distribution of radioactivity in peptide spots in Fig. 1 Radioactivity in the partial hydrolysate spotted on paper was 217 X lo* c.p.m. All data have been corrected for background counts.

spot No.

Radioactivity c.p.n.

5010 2040 351 373 1950 ,.I_ BlY

1

2 3 4 5 6 7 8 9 10

1550 1950 1220 2170 377

11

TABLE

Radioactive

incorporation

Time of incubation bbefore samplt was drawn

I.

acterial count! in incubation mixture

None Sane Sodium penicillin units/ml*

0.5 2.0

I

were

None Magnesium chloride ATP Other amino acids

3520 3540 3390 4490

Dialyzed

None ATP Other amino acids Other amino acids + magnesium chloride Other amino acids + ATP

3980 3160 3500 3210

mitochondria

,Glutamate-W

incorporation into total protein

TABLE

169 1670

Endogenous

free

Amino

2.0

2770

-

176

2.20 2.16

:.p.m./,mg )rofcm

Fresh mitochondria

C.).rn.

0.36 0.30

ATP

IllCOP poration

Omission

growth

Bacteria

G, 106

XII

amino acids in mitochondria, automatic amino acid analyzer acid

determined

Amount

by an

associated

1660 mpnoles/mg

-

* Bacterial counts become nil if penicillin, was added to the agar plates.

and

‘

millions

0.5 2.0

chloride,

The complete system contained 0.4 M sucrose; 0.15 M potassium phosphate buffer, pH 7.0; 0.1 mM magnesium chloride; 0.5 mM L-glutamate-W containing 687 X lo3 c.p.m.; 2.5 mM ATP; 0.5 mM each of glycine, L-tryptophan, L-lysine, L-histidine, L-arginine, L-threonine, L-serine, L-proline, L-alanine, L-valine, Lmethionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-cysteine, L-aspartic acid, L-hydroxyproline, L-glutamine, and L-asparagine; and mitochondria containing about 8 mg of protein and about 240 mpmoles of free glutamate in a total volume of 0.5 ml. The time of incubation was 2 hours. Dialysis was done against 0.5 M sucrose-O.15 M potassium phosphate buffer, pH 7.4, for 16 hours.

_-

hours

XI

of other amino acids, magnesium for incorporation of glutamate-W

Enzyme

of bacterial

Details of the incubation were as in Table counted as described in the text. Addition

TABLE

Requirement

X

in the absence

Vol. 239, h-0. 4

100 units per ml,

This depression went ta 80%, if the concentration of each amino acid was increased to 20 mM. Magnesium chloride was also found ineffective with fresh mitochondria, whereas ATP was feebly stimulatory. The requirement of magnesium chloride, ATP, and the mixture of amino acids (0.5 mM each), however, was reduced if the mitochondria were dialyzed for 16 hours at 0” against 0.5 M sucrose-O.15 M potassium phosphate buffer, pH 7.4 (Table XI). The activity was destroyed if sucrose was omitted from the dialyzing medium. Endogenous Free Amino Acids in Mitochondria-Since addition of the other protein amino acids was unnecessary for the incorporation of L-glutamic acid into the mitochondrial protein, it might be inferred that these acids were present in the free state inside the mitochondria. Table XII shows the endogenous free amino acid content of mitochondria. E$ect of Potassium Cyani&-Potassium cyanide was inhibitory to the incorporation of L-glutamic acid into mitochondria (Fig. 2). Simultaneous manometric studies have revealed that oxidation of exogenous succinate and glutamate and also of endogenous substrates was inhibited similarly by cyanide (Fig. 2). E$ect of Other Inhibitors-Sodium aside, sodium arsenite, and

Lysine .......................... Histidine ....................... Arginine ....................... Aspartic acid. .................. Threonine ...................... Serine.......................... Glutamic acid ................... Proline. ........................ Glycine ......................... Alanine ......................... Half cystine. ................... Valine .......................... Methionine. .................... Isoleucine. ...................... Leucine ......................... Tyrosine ........................ Phenylalanine. .................. Tryptophan .....................

mirochondrial

)rotcin

2.9 6.0 15.3 6.0 7.4 14.3 30.1 3.5 2.4 15.7 1.4 5.3 1.0 3.5 3.5 4.9 2.4 1.5

2,4-dinitrophenol inhibited the incorporation of L-glutamate into mitochondria (Table XIII). E$ect of ilnaerobiosis-Replacement of air in the incubation flasks by nitrogen resulted in a considerable inhibition of the incorporation (Table XIV). Incorporation of Other dmino Acids-An amino acid mixture

hpril

1964

H. K. Das, 8. K. Chatterjee, and S. C. Roy XIII

TABLE

Effect

of‘ some inhibitors on L-glutamate by mitochondria

L-Glutamate-14C incubated with

(0.2 pmole) containing mitochondria as described

incorporation 662 X lo2 c.p.m. in Table I.

I

Inhibitor

was

Incorporation c.p.m./ng

None ................................... Sodium aeide, 1.0 mM. .................. Sodium arsenite, 1.0 mM. ............... 2,4-Dinitrophenol, 0.1 mM. .............. 2,4-Dinitrophenol, 10 mM ...............

protein

327 63 16 83

.I

4

WATE,Q SA7&!!7ED FIG.

TABLE

Efect

XIV

of anaerobiosis on L-glutamate by mitochondria

L-Glutamate-W incubated with

1131

993 X lo2 c.p.m. in Table I.

Gas phase

of the partial

hydrolysate

of the radio-

active protein on Whatman No. 1 paper. The spots have been identified from the radioautogram. The paper was run up to the

incorporation

(0.2 rmole) containing mitochondria as described

1. Chromatogram

PHENOL

was

edge with water-saturated and was overrun (22 hours) direction of the breadth.

phenol in the direction of the with butanol-acetic acid-water

length in the

Incorporation c.p.m./mg

Air............................. Nitrogen

protein

502 82

TABLE

Net

protein

XV synthesis

The incubation system contained 0.4 M sucrose; 0.15 M potassium phosphate buffer, pH 7.0; 3 mM ATP; 0.1 mM magnesium chloride; 1.0 mM each of glycine, L-tryptophan, L-lysine, L-histidine, L-arginine, L-threonine, L-serine, L-proline, L-alanine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, Lphenylalanine, n-cysteine, n-aspartic acid, L-hydroxyproline, L-glutamic acid, L-glutamine, and L-asparagine; penicillin G sodium (106 units per ml); and mitochondria containing about 1 mg of protein in a total volume of 4.0 ml. The aerobic reaction

at 37” was for 5 hours. Viable bacteria were simultaneously counted with each experiment to ensure that the increase in protein

was not due to bacterial

No. of experimerits done

Protein

at o hr

growth.

Pro~~rsafter I

Increase I m g (average)

1

‘.06’

1

acids,

the incorporation

CYANi!!E @@

(Range~Zk0.072)

failed to step up the incorporation of glutamate-r4C into protein (Table XI). Since this might work against the general nature of the incorporation reaction, the incorporation, if any, of other amino acids was studied. Experiments with 14C-labeled algal protein hydrolysate had revealed that all the amino acids in it-namely, leucine or isoleucine, or both; phenylalanine; valine; proline; tyrosine; alanine; threonine; glycine; lysine; serine; arginine; histidine; and glutamic and aspartic acidswere incorporated into the mitochondrial protein. The labeled protein was hydrolyzed and the r4C-amino acids were detected after two-dimensional paper chromatography. Net Protein Synthesis-In the presence of the other protein amino

AT/ox OF .=VZ4&5fUAI

FIG. 2. Effect of potassium cyanide. Manometric oxidation: 0, endogenous; 0, succinate, 20 mM; A, L-glutamate, 20 mM. Incorporation of glutamate-W into mitochondrial protein: X, L-glutamate, 0.01 mM (21,100 c.p.m.). Concentrations of potassium cyanide have been plotted on a logarithmic scale. Other details of the incubation are described in the text.

21 ‘.020

CONCENTRA

of L-glutamate-14C

into

the

mito-

chondrial protein continued up to 6 hours and sometimes still longer. In fact, it has actually been possible to obtain a net increase (5%) in protein by the incubation of a complete mixture of protein amino acids with mitochondria for 5 hours at 37” (Table XV). The maximal variation in the amount of protein in the different incubation tubes of any single experiment was below

1 To. DISCUSSION

It will appear from Tables I and II that n-glutamate is far more actively incorporated into protein by mitochondria than the microsomal particles obtained from seedlings of V@nusinensis. The latter, even when fortified with all the known factors, fail to respond. The findings were the same with seedlings of mung bean and pea. These results are not in conformity with

1132

Protein

Synthesis

in Plant

the original work of Webster (23) and Raacke (24). We failed to reproduce Webster’s results even by following exactly his cell fractionation technique and incubation conditions (21). Others (25, 26) also could not confirm the findings of Webster and Raacke. In a later communication, Webster, Whitman, and Heintz (27) have reported that the microsomal ribonucleoprotein particles from most of the pea varieties tested by them were unable to incorporate labeled amino acid into protein. It is, however, probable that the protein synthesizing ability of the microsomal particles from higher plants is lost during cell rupture and subsequent fractionation or even that the microsomal particles are incapable of synthesizing protein outside the cell. The results of Table III demonstrate that these particles incorporate ila situ amino acids into their protein, though at a slower rate than mitochondria do. It has not been possible to layer out mitochondria into different species that are likely to be associated with various steps in the incorporation of amino acids into protein (Table IV). The observation that the fractions sedimenting at lower centrifugal force are more active than those at higher force (Table V) indicates that the heavier mitochondria rather than the lighter ones are the seat of amino acid incorporation activity. Table VI indicates that glutamate has been incorporated into the structural protein of the mitochondria and that the protein so formed is not released into the incubation medium (cf. Roodyn, Reis, and Work (3)). The present data satisfy most of the criteria of Hoagland (28) in that they equate amino acid incorporation and protein synthesis. Table VII indicates that the incorporation of glutamateJ4C into mitochondrial protein is mostly irreversible. Fig. 1 and Table IX indicate that the incorporated glutamateJ4C is in true cr-peptide linkage in the mitochondrial protein, since incorporated peptides have been identified by partial hydrolysis of the protein. Some radioactivity might have been lost when the paper was overrun with butanol-acetic acid-water. Tables VII and VIII indicate that glutamateJ4C is located within the peptide chain and not in other positions. Table X eliminates the possibility of incorporation due to bacterial contamination. Inhibition by the mixture of amino acids is possibly due to the formation of glutamateJ*C through transamination and subsequent dilution of glutamateJ4C. However, the experiments with dialyzed mitochondria (Table XI) show that the process of incorporation of glutamateJ4C into protein requires the presence of other amino acids. One of the essential prerequisites for amino acid incorporation and protein synthesis is the dependence of the process on metabolic energy supply. Fresh mitochondria from the seedlings of Vigna sinen&, however, seem capable of incorporating L-glutamic acid into protein without any apparent requirement for ATP (Table Xl). With dialyzed mitochondria (Table XI) the process is actually dependent on energy supply, though the stimulation by ATP is only 20%. The energy dependence of the process also appears from its inhibition by cyanide (Fig. 2), azide, and arsenite (Table XIII), which are well known inhibitors of oxidation, and by 2,4-dinitrophenol (Table XIII), the uncoupler of phosphorylation. This dependence rules out the possibility of incorporation due to transpeptidation, as considered by Suttie (29). Inhibition of incorporation by anaerobiosis (Table XIV) further substantiates the energy dependence of the process.

Mitochondria

Vol. 239, T\‘o. 4

The observation that all the amino acids in algal protein hydrolysate are incorporated into mitochondrial protein proves the general nature of the incorporation and suggests actual protein synthesis. This is also supported from the net increase in protein (Table XV). SUMMARY

Mitochondria isolated from the seedlings of Vigna sinensis (Linn.) Savi incorporate L-glutamateJ4C more actively than the microsomes. Maximal incorporation activity appears to be associated with the fractions sedimenting at lower centrifugal force. That this incorporation is a result of peptide bond formation has been confirmed through different treatments of the radioactive protein with performic acid and fluorodinitrobenzene and also by separation and identification of the labeled peptides from the partially hydrolyzed products. NH*-Terminal incorporation was negligible. Bacterial contamination was not responsible for the incorporation into mitochondria. Incorporation of L-glutamate-14C into fresh mitochondria was not dependent on other exogenous amino acids, adenosine triphosphate, or MgC12, whose requirement was, however, demonstrated to some extent in dialyzed mitochondria. Cyanide, azide, arsenite, and 2,4-dinitrophenol inhibited the incorporation. All amino acids in the labeled algal protein hydrolysate were incorporated. A net increase in protein (about 5%) has been obtained after incubation of mitochondria with amino acids for 5 hours. Acknowledgments-We are grateful to the National Institute of Sciences of India for a fellowship to one of us (H. K. D.), to the Ministry of Scientific Research and Cultural Affairs for some grants, and to Drs. S. K. Roy, N. C. Kar, H. P. Ghosh, and R. N. Dutta, Mr. B. C. Dutta, and Mr. D. D. Roy for their kind help. REFERENCES 1. WEBSTER, G. C., Plant Physiol., 29, 202 (1954). 2. SISAKYAN, N. M., AND FILIPPOVICH, I. I., Biokhimiya, 22, 375 (1957). 3. ROODYN, D. B., REIS, P. J., AND WORK, T. S., Biochem. J., 80, 9 (1961). 4. SIMPSON, M. V.. SKINNER, D. M.. AND LIJCAS, J. M., J. Biol. Chem.,’ 236, Pi=81 (1961)‘. ’ 5. TRUMAN. D. E.. AND KORNER. A.. Biochem J.. 88. 588 (1962). et B&&s. Acta, 69, i84 .(1963)‘. ’ 6. KROON, A. M., biochim. 7. DAS, H. K., AND ROY, S. C., Biochim. et Biophys. Acta, 63, 445 (1961). 8. DAS, H. K., AND ROY, S. C., Sci. and Culture (Calcutta), 26, 317 (1959). 9. KELLER, E. B., AND ZAMECNIK, P. C., J. Biol. Chem., 221. 45 (1956) . 10. DAS,

H. K.,

AND ROY,

S. C., Arch.

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(1962). 11. STACHIEWIEZ. E.. AND QUASTEL, J. H., Can. J. Biochem. and Physiol., Si, 687 (1959). 12. GORNALL. A. G.. BARDAWILL. C. J.. AND DAVID. M. M.. J. Biol. C&em., li7, 751 (1949): ’ W. W., BURRIS, R. H., AND STAUFFER, J. F., Mano13. UMBREIT, metric techniques, Burgess Publishing Company, Minne-

apolis, 1957, p. 208. 14. PETERSON, E. A., AND GREENBERG, D. M., J. Biol. Chem., 194, 359 (1952). 15. SANGER, F., Biochem. J., 89, 507 (1945). 1G. LOCKHART, I. M., AND ABRAHAM, E. P., Biochem. J., 68, 633 (1954). 17. SPACKMAN, D. H., Instruction manual and handbook for Beckman/Spinco amino acid analyzer (model f20), Beckman Instruments, Inc., Palo Alto, 1960, p. 7-4.

April 1964

H. K. Das, S. K. Chaiterjee, and S. C. Roy

18. SPACEMAN. D. H., STEIN. W. H.. AND MOORE. , S.. Anal. Chem.. 30, 1190’(1958).’ ’ ’ 19. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDAM,, R. J.. J. Biol. Chem.. 183. 265 (1951). 20. FoLIN,‘~., AND CIOCA~TEU,. B., j. B&l. Chem., ‘73, 627 (1927). 21. WEBSTER, G. C., Plant Physiol., 30, 351 (1955). 22. T’so, P. 0. P., BONNER, J., AND VINOGRAD, J., J. Biophys. Biochem. Cytol., 2, 451 (1956). 23. WERSTER, G. C., Arch. Biochem. Biophys., 86, 159 (1959). 24. RAACKE, I. D., Biochim. et Biophys. Acta, 34, 1 (1959).

25. LETT, J. T., AND TAKAHASHI, W. N., Arch. Biochem. Biophys., 96, 569 (1962). 26. CAMPAGNE, R. N., AND GRUBER, M., Biochim. et Biophys. Acta, 66, 353 (1962). 27. WEBSTER, G., WHITMAN, S. L., AND HEINTZ, R. L., Ezptl. Cell Research, 26, 595 (1962). 28. HOAGLAND, M. B., in E. CHARGAFF AND J. N. DAVIDSON (Editors), The nucleic acids, Vol. ZZZ, Academic Press, Inc., New York, 1960, p. 351. 29. SUTTIE, J. W., Biochem. J., 34, 382 (1962).