Participation of Aminoacyl Transfer Ribonucleic Acid in Aminoacyl Phosphatidylglycerol Synthesis

I. Sl’ECIPIClTY OF LYSYL PlIOSPHATIL)YLGLYCEliOT~ SYXTHETASE*

(Received for publication, January 19,1968)

J. A. NESBITT, 111, AND W. J. LENSA&

From the Department of Physiological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Ala&and 21205

SUMMARY

The biosynthesis of lysyl phosphatidylglycerol by a par- ticulate enzyme from Staphylococcus aurelis involves the transfer of a lysyl group from lysyl-transfer RNA to phos- phatidylglycerol. S-p-Aminoethylcysteinyl-tRNA’Y”, pre- pared enzymatically from the lysine analogue, S-p-amino- ethylcysteine, serves as an effective substrate in the enzymatic formation of aminoethylcysteinyl phosphatidyl- glycerol. In contrast, aminoethylcysteinyl-tRNACY8, chem- ically prepared from cysteinyl-tRNA”r”, is inactive as a sub- strate in aminoacyl phosphatidylglycerol synthesis. These results suggest that phosphatidylglycerol synthetase contains at least one recognition site for a portion of the polyribonucleo- tide chain of lysyl-tRNA.

Recent studies dealing with the enzyme that catalyzes the synthesis of Iysyl l~hosl~hatid~lgl~crrol from phosphatidyl- glycerol and lysyl-transfer RS;\ (Fig. 1) have shown t.hat the enzyme is specific for the lipid substratr, phosl~hatidylglycerol (1). The present. investigation is concerned with the specificity of the enzyme toward the other substrate, lysyl-tRNR.’ The results of this investigation, &s well w the results obtained in a parallel study on alanyl l~hosl~hat.icl~lgl~ccrol synthetase re- ported in the accompanying paper (J), suggest that the trans- ferases involved in aminoacyl l~ho~l~hatitl~lglycerol synthesis manifest q)ctcificity toward both the aminoacyl and polgribo- nuclrotide moieties of that appropriate aminoacyl-tRN.-\.*

* This wvork was supported by Grant AI-06888 from the United States Public llcalth Service.

1 Lederle Medical Faculty Awardee, 1980 to 19M. 1 The ahbreviat.ion, tR.NA, is used to designate unfractionated

transfer HXA, which was utilized throughout these studies. The presence of a superscript following x particnlnr uminoacyl-tRNA, i.e. nminoetl~ylcysteinyl-tRSA~~J, designates the particular amino acid-specific t RNA to which the uminoncyl gro\lp is linked.

2 Brief abstrllcts (GOULD, R., ASD IANNARZ, W. J., Fed. Proc.,

EXPERIMENl’AL PROCEDURE

Nuterials and Methods-T_;niformly labeled 14C-L-amino acids were purch%ed from New England Nuclear. U-14(‘-L-Cystinea wus purified by paper electrophoresis. Unless indicated other- wise, paper electrophoresis on Whatmann No. 3hIhI was per- formed at pH 1.85 t.o 1.90 (2.57; formic acid, 7.57; acetic acid) at 100 volts per cm with either a varsol coolant bath apparatus or a horizontal plate coolant apparatus. Radioactivity measure- ments were performed with a Packard Tri-Carb scintillation counter or a Packard radiochromatogram scanner. S-p- hmitioeth~llcysteirie was a gift from Dr. J. E. Folk, Sational Institutes of Health. Ethylcniminc was purchased from Mathe- son, Coleman, and 13ell. Escherichiu coli p tRSh (deacylated) was lmrchased from General Uiochemicals. Staphylococcus uureus tR?Jh was extracted from intact cells by the phenol method (3).

Enzyme Preparation and 11 sq-- .S. uureus, a penicillin-re- sistant X1,&0 strain obtained from 11. R. Smith in the Depart- ment of JIicrobiology, The Johns Hopkins University Moo1 of Medicine, was grown to the late logarithmic phase (450 Klett units at GGO ITIP) in I-liter cultures at 37” with shaking. The medium consisted of the following components (in milligrams per liter) : nLmethionine, oI,valine, 1)~serine, uL-tryptophan, glycine, I,-isoleucine, L-lcucinc, ])I,-aspartatc, L-alanine, I.- arginine.HCI, 250 of each; nL-threonine, nL-phenylalanine, uL-tyrosine, 125 of each; r,-h~droxyproline, 40; L-glutamic acid, 500; uL-orriithilie.HCl, 50; L-proline, 75; L-cystine, 100; &$O4, 100; NnC’l, 40; FeSOI, 40; JInS04, 150; adcnine, 10; guanine, 10; uracil, 10; santhine, IO; thiaminr, 1; nicotinic acid, 5. Also atldtld were the following components (in grams per liter) : glucose, 10; sodium cit.rate, 5; yrast extract (Difco), 1; and potassium phosphate buffer (1M 7.2), to a final concentration of 0.1 hl. The cells were harvested by centrifugation, washed with 0.02 M Tris-HCl, pH 7.6, and resuspended in the same buffer

.-. 26, 27 (1’967); LENNARZ, W. J., GOULD, K., AND THORKTON,PVI. I’.,

Abstracts Seventh International Congress of Biochemistry, l’okyo,

f967, p. i37) reporting some aspects of this study have been pub- lished.

3 U-“C-L-Cystine refers to the uniformly labeled compound.

by guest on October 30, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Issue of June 10, 1968 J. A. Nesbitt, III, and TV. J. Lennarz 3089

(1 ml of buffer per 5 g, wet weight., of cells). The cells were rupt.ured and the particulate enzyme fract,ion was obtained as previously described (1).

Enzymatic formation of “C-aminoacvl pho~l)hatid?lgl?-cerol was assayed by the following procedure. The components of the assay mixtures used are listed in the legends to the tables. Enzymatic reactions (carried out in a final volume of 0.35 to 0.45 ml) were terminated hy the addition of 5.5 ml of CHCla- CH&II, 2: 1. The mixture was warmed for 5 min at 40-60”, mixed thoroughly on a Vortes mixer, and filtered through a funnel containing glass wool. The incubation tube and fuunel were rinsed with an addit.ional 0.5 1111 of CHCIS--CHaOH, 2: 1. The filt,rate was washed with 2.5 ml of 0.9% NaCl acidified to pH 2.0 with HCI. The two phases were mixed thoroughly on a Vortex mixer and separated by cent.rifugat.ion; the lower phase ws removed, evaporated to dryness in a scintillation vial, and counted.

Prepardion of U-T-L-Lysyl-tR;C’A ‘~“-~J-‘4(“-r.-Lysil~~ was diluted to a specific activity of 18.3 PC per qlole with L-lysine and charged onto B. coli U tRNA by means of a crude super- nataut enzyme prepared from E. coli 13. The cells were grown at 37” in tlgpticase soy broth (30 g per liter) to midlogarithmic phase (300 Klctt units at 660 mp). The cell pellet, ohtained by centrifugation at W x g for 10 min, wm suspended in an equal volume of 0.05 11 ‘l’ris-H(‘l-0.005 M fi-mrrcaptorthanol, pH 7.0, and the cells were ruptured hy sonic disintegration with a Bronwill Kosonik instrument operated at 80 to 9Oyh of masimum intensity. The cell suspension was sonically treated for six l- min periods, care being taken to maintain the temperature of the suspension below 15”. .iftcr centrifugation at 10,000 X g fol 10 min t,he supernatant fluid was subjected to centrifugation at, 100,000 X g for 60 min. The supcrnat.ant fraction was dialyzed overnight at O-5” against 2 liters of 0.05 hi ‘I’ris-HCl-0.005 M mercal)toethanol, pH 7.0. The dialyzed enzyme preparation (16 ml/IO g of cells) had a protein concentration of 13.0 mg per ml. The lysyl-tR~X-sgrlt.hrsizirlg system contained (in micro- moles): Tris-HCl (pH 7.2), 600; ATP (pH 6.9), 40; MgCl,, 75; @-mercaptorthanol, 16; phosphoenolpyruvatc, 180. I:-“C-L-

lysine, 0.55; pyruvate kinase (Sigma), 1.25 mg; tRNi, I50 III~;

and dialyzed act.ivating enzyme, 26 mg, in a final volunlc of 10 ml. ;\fter incubat,ion at 30” for 15 min the reaction was ter- minated by addition of phenol, and the lysyl-tRNh was isolat.ed as described by VOII Ehrenstein (3). .\pproximatcly 50 to 755; of t,hr added ‘W-lysine was recovered charged onto tRNA. The lysyl-tRX.1 was dissolved in 0.001 M potassium acetate buffer, pH 5.5, and stored at -60”. Mild alkaline hydrolysis of the aminoacyl-tRNA, followed by elect.rophoret.ic analysis, indicated that greater than 98Q, of t.he radioactivity in the tHS.1 prrpara- tion was due to ‘4C-lysine.

Preparation of S-/3-A minoeth yl-‘4C-cysteinyl-tRA’.1 ‘us-S-@- .~milloeth3-lc?-rteirlc was prepared by a modification of the method of Cavallini et al. (4). To 1.03 pmoles of IT-“C-L- cystine (2.06 peq of cysteine; sl)ecific activity, 10.85 PC peg

prnolc of cysteine) contained in a small tube were added 0.11 ml of 0.02 M dithiothreitol and 0.03 ml of 0.05 Y NaOll. The tube was flushed with nitrogen and st.oppered. ;\fter 2 min at, room temperature, 0.15 rd of 0.06 M P-bromoeth~lamil~~ was added. The tube was again flushed with nitrogen, stoppered, and heated at 65’ for 10 min and a1 35” for 1 hour. Follon-ing acidificat,ion with 0.01 ml of 1 x HCI, the reaction misture was spotted as a band 19 inches long on Whatmann So. 31111 paper

Adenine , RNA-O-~~OCH~“~n’ne

k-3 H H H,Lrjyl

+ . +

CH2’00CR CH@CR

AHOOCR LHOOCR

I 0 CH20-~~O-CH2~-~H~

I 0 CH20-y-O-CH+H-$H2

b 6 6 1J

FIG. 1. Reaction catalyzed by lysyl phosphatidylglyccrol sgrl-

thetase.

and subjected to electrophoresis at pH 1.9 for 1.25 hours. Ex- aminat,ion of the paper strip 011 the radiochromatogram .scanner revealed ody one “C l)cak corresponding to authentic amino- ethylcystcine. The radioactive aminoethylcysteine was re- covered from the paper by elution 1vit.h H20; the yield, ba.sed on

W-cystine, was greater than 905;. “C-Xminoethylcyst,cillc was charged ont.o lysine-specific tRN:1 by the standard procedure indicated above. Hydrnlysis of the aminoeth~lc?-~;teiI~~l- tRNA’Ys, followed by clcctrophoresis, indicated that greater than 990; of the radioactivity in the aminoacyl-tRS.4 was due to *4C-

aminoethylcysteine. Preparation of Cyskinyl-tR:V:l cYa-Crude rystcinyl-tRNA

synthetasc was prepared from I:‘. coli B according to the procedure of Muench and 13erg (5). The synthctascs were not fractionated on a DE.&-cellulose colunm s described in their procedure, hut the enzyme preparation was Fuhjected to ammonium sulfate fractionation. The aminoaryl-t,RNA synthetasc? act.ivity pre- cipitating bct.ween 45 and 60f>l. saturation was collected h? centrifugalion, dissolved in a minimal volume of 0.05 hr Tris- IICI-0.005 M P-merc:aptoethaliol (pH 7.0), and dialyzed overnight against 2 liters of t.hr same buffer. The protein concentration in the final enzyme extract was adjusted to 6.0 mg per ml hy dilu- tion with 1120. The cysteinyl-t.RS.1”Y” prepared with this enzyme by t.he charging procedure described above had a specific activity of 46 PC per pmolc of cystrine (200 ppmoles of charged cystcine per III~ of B. coli tRS:i).

l’repartion of &‘-p-.-l mi?wethylcysteinyl-tR~~~~~r~“--~ecause cystcine is rapidly cleaved from cysttinyl-tRSAcys under t.he conditions which have been previously described for the amino- ethylation of cystcine residues in protein (6, 7), new conditions had to be devised for the arninocthylation of cysteinyl-tRS.\‘Y”. Preliminary experiment.s were undertaken in order to es- tablish optimal condit.ions (maximal aminoethylation and minimal aminoacyl ester hydrolysis). ‘4C-C3-‘tein~l-tRS.iC’B, 243 ppmoles (19,700 cpm; specific: activity, 46 PC per qlole), in 50 ~1 of 0.001 M pot.a.ssium acetate buffer (pH 5.4), 200 11 of 1 M PO4 buffer (pH i.O), and 20 ~1 of 0.1 hc P-merc:al)toethanol was added to a screw-rapped tube and kept at O--4” for 10 min. Thrre 0.X+1 portions of ethylcniminc wrre then added at 5 min inter- vals. Reaction tubes were incubated at O-5” for the indicated time (see below) after which the rracstion was terminated by addition of sufficient. glacial acetic acid lo lowrr the pIE of the reaction mixture to 5.6. ‘The tR?u’A wm precipitated by atltli- tion of 0.1 volume of 20co potassium acetate and 2 volumes of

by guest on October 30, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Participation of Anzinoacyl-tRNA in Aminoacyl Phosphatidylglycerol Synthesis. I Vol. 243, No. 11

L! I I I I I

REACT& TIME (HOURS) IO

FIG+. 2. Time course of conversion of cysteinyl-tRNAcr# to aminoethylcysteinyl-tRSAoy6. The percent&e of-unhydrolyzed ~4C-aminoacvl-tRNAc~~ is shown bv O-O. The ocrcentaae of the unhydroiyzed aminoacyl-tRNA at each time point which has been converted to aminoethylcysteinyl-tRNAcy* is shown by A-A.

ethanol at - 20”. The precipitate was allowed to stand at -20” for 30 min, collected by centrifugation, and redissolved in 0.5 ml of 0.001 M potassium acetate, pH 5.4.

In order to measure the extent of aminoethylation the radio- active products were analyzed in the following manner. The tRXA was dialyzed at 4’ overnight against 2 liters of acetate buffer and then concentrated to dryness. Carrier ‘*C-amino- ethylcysteine (0.1 pmole), **C-cysteine (2.5 pmoles), and 0.3 ml of 2 x HCl wcrc added to each tube, and the tubes were heat.ed at. 100” for 60 min. The sample was concentrated to dryness under reduced pressure and spotted over 0.75 inch on What- mann No. 3bIM paper. Plate elcctrophoresis at pH 1.9 for 1 hour separated the cysteine and aminoethylcysteine. The relative amount. of each “C-amino acid was determined bz cutting out and neighing the area of the chart paper correspond- ing to the radioactivity obtained by scanning the chromat.ograms. The carrier amino acids were visualized by the ninhydrin rcac- tion. The time course of the aminoethylation of cysteinyl- tRr\‘AcyS under the above condit.ions is shown in Fig. 2.

To prepare a large amount. of 14C-aminoet.hylcysteinyl-tRXAc~a the reaction mixture described above WLS increased M-fold and the reaction was allowed to proceed at O-4” for 6 hours. The reaction was terminated by the addition of 0.25 volume of glacial acetic acid, 0.1 volume of 20% potassium acetate (pH 5.4), and 2 volumes of ethanol at -20”. After 1 hour at -20” the tRNA was collected by centrifugation and dissolved in a mini- mum volume of 0.001 M potassium acetate, pH 5.4. The tRNA was dialyzed overnight against 6 liters of the .same buffer and for an additional 3 hours against 3 liters of fresh buffer. The dialysate was lyophilized to drynes and dissolved in a minimal amount of 0.001 M potassium acetate, pH 5.4. At least 9070 of the radioactivity in the final sample represented charged amino- acyl-tRNA as determined by a minor modification of the disc absorption assay (8). The recovery of W-aminoacyl-tRNA was 48%. :1n aliquot of 14C-an~inoacy1-tRT\‘A (6000 cptn, 78 ppmoles) was hydrolyzed and analyzed by electrophoresis as described above to determine the percentage of the cysteinyl- tRNAcys which had been converted to aminoethylcysteinyl-

t.RKAcY*. The results of this analysis indicated that t.he re- covered 14C-aminoacST1-t.RS-~ was a mixture containing 46 70 aminoethylcystcinyl-tRXAc~a and 54c/, cysteinyl-tRNACYa. This preparation was utilized in all of the st.udies reported in this paper; for purposes of simplicity it is referred to as aminoethyl- cysteinyl-tRN.4”Ya.

RESULTS

General Properties of Lysyl Phosphalidylglycerol Synthetare-.ls previously reported, lysyl phosphatidylglycerol synthetase, when freed of endogenous lipid by extraction with organic solvent, has an absolute requirement for phosphatidylglycerol and lysyl-tRNA (1). The pII optimum of the reaction, as shown in Fig. 3, is approximately 6.9. In early studies with enzyme preparations containing endogenous lipid, a marked stimulation of lysyl phosphatidylglycerol synthesis was observed upon addition of anionic surface-act.ive agents, including the sodium or potassium salts of the branched chain fatty acids of S. aureus or Micrococcus lysodeikttis (9). This stimulatory effect is minimal and somewhat erratic with solvent-extracted enzyme; however, in most of the present studies fatty acid salt has routinely been added to incubation mixtures. Studies on the general effects of surface-active agents on lysyl phosphatidyl- glycerol synt,hesis are in progress and will be reported at a later date.

The extension of early st.udics in which R,fg* ion was found to stimulate lysyl phosphatidylglycerol synthesis has led to the finding that the reaction has a marked requirement for a medium of high salt concentration. Apparently this effect is due solely to ionic strength for it is not ion-specific (Table I). Maximal activity of lysyl phosphatidylglycerol synthesis is observed when the final concentration of salt is 0.1 t.0 0.2 11.

The effect of a variety of potential inhibitors of lysyl phos- phatidylglycerol synthetase has been studied. All of the fol- lowing compounds (tested at the final concentrations indicated)

B z 2

X I I I

5.0 6.0 PH

7.0 8.0 I

Fro. 3. pH dependence of lysyl phosphatidylglycerol synthe- tase. The complete system contained (in micromoles): M&l*, 3.0; KCI, 40.0; fatty acid salt, 0.75; Tris-maleate buffer of the indicated pH, 7.5; and phosphatidylglycerol, 700 pg; solvent- extracted enzyme, 196 pg; and U-W-lysyl-tRNA (specific activity 18.3 pC per pmole), 392 Fpmoles, in a final volume of 0.42 ml. Incubation for 15 min at 30”. Results indicated by l were ob- tained with a different enzyme preparation.

by guest on October 30, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Issue of June 10, 1968 J. A. Nesbitt, III, and W. J. Lennarz

were without inhibitory effect: EDTA (lOma M), chloramphenicol (3.7 X IOea Ii), puromycin (4.7 X IO-’ li), Mracylcline (lOea M),

bacitracin (3.4 X 10-4 MI), penicillin (6.0 X 10e4 M), and DBase (240 c(g per ml). Vancomycin (albamycin), tested at the rela- tively high concentration of lCrs M, caused 60% inhibition. Al- though stimulation of enzyme activit.y by addition of sulfhydryl- containing compounds @-mercaptoethanol, dithiothreitol) to incubation mixtures or to enzyme preparations has not been detected, the results of experiments with sulfhydryl-reactive reagents indicat.c that the enzymatic reaction is inhibited by such compounds (Table II). With the exception of N-ethyl- maleimide, all of the sulfhydryl-reactive compounds t.ested inhibit lysyl phosphatidylglycerol synthesis. Morcol-er, inhibi- tion by p-chloromercuriphenyl sulfonate is abolished by addi- tion of an excess of dithiot.hreitol. Although it seems likely that t.his inhibition is due t.o reaction of these reagents with a reactive sulfhydryl group of the protein, on the basis of the above experi-

TARLE I h’fecl of ionic strength on lysyl phosphalidylglycerol synlhesis

The complete system contained: Tris-maleate (pH 7.0), 7.5 ~molcs; phosphat.itf~lglyc:erol, 600 fig; solvent-extracted enzyme, 571 r~; U-1%.Iysyl-tRNA, 392 ppmoles; and the indicated salt in a final volume of 0.43 ml. Incubation was at 30” for 15 min.

Additions Lysyl phosphatidylglycerol

pjmolcr

None..................... 3.0 KCI, 10 rmoles.. / 27.0 KCl, 40 @moles.. 160.1 NaCl, 40 rmoles.. lM.5 KBr, -10 pmoles.. 160.0

TABLE 11

Inhibition of lysyl phosphalidylglycerol synlhesis by suifhydryl- reaclive compounds

The complete system contained: Tris-maleate (pII 7.05), 10 rmoles; KCI, 60 rmoles; solventcxt.r:Lcted enzyme, 380 pg; and inhibitors to tha final concentration shown below. The mixture (0.355 1111 total volume) was preincubated at 30” for 1 min and then chilled on ice. After the addition of phosphutidylglyc:crol (300 rg), fatty acid salt (0.6 pm&), and U-“C-lysyl-tRNA (580 rpmolc:;), in a final total volume of 0.45 ml, incubation was per- formed for 15 min at 30”.

Incubation conditions Remaining enzymatic activity

o/c C0111p1etc. . w-w”

Complete, boiled enzymtt.. <l Complete, 2.2 X lo-a M p-chloromercuriphenyl-

slllfoIl:~te 6 Complete, 2.2 X 1OP Y p-chloromerruriphenyl-

sulfo11atA?. . . 36 Complete, 2.2 X lo-4 M p-chloromcrcuriphenyl- 1

sulfonate + 10-* JI dithiothreitol _; 119 Complete, 2.2 X 10-d M &SO,. _. Complete, 2.2 X IO-4 M Hg (C2H302)i _’ : : : : 1 : : : :

4 II

Complete, 2.2 X 10-a Y .Y-ethylmaleimidc 72

” One hundred per cent activity represents incorporation of 120 rprnoles of “C-lysine into lysyl phosphatidylglycerol.

I I I I 1000 2cm 3000 4000 5oco 6&o

Ecoli “C-lysyl-tRNA kpm)

FIG. 4. Dependence of lysyl phosphatidylglycerol synthesis on lysyl-tR-UA. Conditions were as in Table I with 40 pmolcs of KC1 and B. coli lysyl-tRNA (specific activity, 3300 cpm/lOO ppmoles) in the indicated amounts.

merits t.he possibil&y t.hat inhibition occurs because of reaction of the sulfhydryl reagents with lysyl-tRNA cannot be excluded.

Aminoacyl-tRN<4 Specifici&-It has been reported previou$ that the aminoacyl phosphatidylglycerol synthetase of S. MLT~US is specific for lysyl-tRNA; only 14C-lysine, in a mixture of 14 different 14C-aminoaryl-tRNAs, was incorporated into phos- phatidylglycerol (10 (cf. references cited in Foot.note 2)). The results of a limited survey of heterologous preparations of lysyl- tRNh suggest that the enzyme is not species-specific with regard to t.he source of lysyl-tRS.4. Thus, lysyl-tRN.4 preparations from (a) S. aureus-activating enzyme and S. aureu.s tRYA, (b) E. coli B-activating enzyme and E. coli IS tRNA, and (c) 2Veurospora crassa enzyme and E. coli B tRNA all serve as effective substrates in Iysyl phosphat.idylglycerol synthesis. Under conditions in which lysyl-tRNA is limiting, the incorpora- tion of lysine into lipid with all of the above substrates was found to bc 75 to 100’j?o. For instance, in the case of lysyl-tR?;A prepared from E. coli-activating enzyme and E. coli tRS.4 the incorporation was linearly dependent on lysyl-tRN.4 and pro- ceeded to the extent of 89yo of the added substrate (Fig. 4). In

view of these findings and the commercial availability of B. coli B tRN.4, all subsequent studies reported in this and the ac- companying paper (2) were performed with aminoacyl-tRS.4 prepared with B. coli-activating enzymes and E. coli tRN.4.

Activity of S-/3-Aminoethylcysteinyl-tRNA’~8 in Synthesis of S-fi-Aminoethylcysteinyl Phosphatidylglycerol-Aminoethylcys- teine, an analogue of Igsine, is incorporated into protein by E. coli and is charged onto tRNA t.o the smne extent u lysine (11). These findings stimulated us to prepare aminoethylcysteinyl- tRSrl’Y” and test it as an analogue of lyr;yl-tRN.4*~B in amino- acyl phosphatidylglycerol synthesis (Fig. 5).

As shown in Fig. 6, aminoethylcysteinyl-tRr\m’Y” does re- place Ivsyl-tRK.l’yB as a substrate in aminoacgl phosphatidyl- glyceroi synthesis. When t.he results shown in Fig. 6 are plotted according to the method of Lineweaver-Burk (Fig. 7), it is evident that the apparent K, values for both substrates are es- sentially identical, although the v,,,,, for aminoethylcysteiny1- tRNA is only one-half that of 1ysyLtRNA. When similar experiments were carried out under conditions in which amino-

by guest on October 30, 2020

http://ww

w.jbc.org/

Dow

nloaded from

3092 Participation of Aminoacyl-tRNA in Aminoacyl PhosphatziQlrJlycerol Synthesis. I Vol. 243, No. 1.1

@NH&H, CH, CH, CH, tH-COOG E. coli ,? @NH,CH2CH2CH2CH2CH-C-tRNA lY6

NH,@ - Enzyme

BfcH2CH2NH2,0NH3CH2CH2SCH2~H-C00Q

I NH,@

4 E. coli Enzyme

@NH, CH2CH@H2CH-8 tRNAtys

HSCHzyH-COO -

NHSQ

E.coli *

Enzyme

I I: CH;-tH2

@NH,CH2CH2SCH2CH- -tRNACYs 8

A&

FIG. 5. Reaction sequences for the preparation of lysyl-tRNA cysteinyl-tRNAc,a.

Iye, aminoethylcysteinyl-tRNAIv*, cysteinyl-tRNAcy#, and aminoethyl-

FIG. 6. Comparison of lysyl-tRNAl”a and aminoet.hylcysteinyl- tRNAlya as substrates in aminoacyl phosphatidylglycerol synthe- sis. The complete system contained (in micromoles): Tris- maleate (pH 7.05), 10; KCI, 60; phosphatidylglycerol, 500 pg; particulate enzyme, 715pg; and nminoncyl-tRNA as indicated in a final volume of 0.43 ml. Incubation was at 20” for 20 min.

0

r o

‘-Aminoethylcys+einyI-tRNA

724 1448 2172 2896

Aminoacyl-tRNA (ppmoles)

Aminathylcyskinyl-tRNA’p KM=2 13~10-6~ -

/

V~@O.4ppnwks/mg.prot./min

VMAX= tObmdes/mg prot./min.

iiX’06 FIG. 7. Determination of the apparent K, and V,.. values for

lysyl-tRNA’y0 and aminoethylcysteinyl-tRNA’y0. Data shown in Fig. 6 were plotted according to the method of Lincweaver- Burk.

by guest on October 30, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Issue of June 10, 1968 J. A. Nesbitt, III, and W. J. Lennarx 3093

ethylcysteinyl-tRNA and 1ysyLtRNA were limiting, in both cases greater than 85 $& of the ‘Gamine acid was transferred from tRNA to phosphatidylglycerol.

In order to characterize the enzymatic product formed from aminoethylcysteinyl-tRNA lY*, the radioactive lipid was isolated from the incubation mixture by extraction and subjected to thin layer chromatography. All of the radioactivity in the lipid was found to be coincident with the endogenous lysyl phos- phatidylglycerol that was also extracted from the enzyme prepara- tion (Fig. 8). Hydrolysis of the enzymatically formed i4C-lipid, followed by electrophoresis, revealed that all of the radioactivity of the product was due to aminoethylcysteine (Fig. 9).

Inactiwity of S-/3-Aminoethylcysteinyl-tRNAc~8 in Synthesis of S-P-Aminoethylcysteinyl Phosphatidylglycero&-In order to in- vestigate further the aminoacyl-tRNA specificity of lysyl phosphatidylglycerol synthetase it was desirable to have as a potential substrate an aminoacyl-tRNA in which either lysine or aminoethylcysteine was linked to a tRNA with acceptor ac- tivity for an amino acid other than lysine. Toward this end cysteinyl-tRNAcys was enzymatically prepared and then chem- ically converted to aminoethylcysteinyl-tRNAeya (Fig. 5).

TLC ANALYSIS

ZCCO- Lipid Product of Incubation

5 with t4C-Lysyl-IRNA’P

4 Doe -

A-- $ @c-a ~3 :z 12 0 1 f

2cc0- rl

Lipid Product of Incubation

z with 14C Amimethylcysteinyl- tRNA’p 0

ORIGIN FRONT

FIG. 8. Tracing of thin layer chromatograms of the enzymatic lipid product formed from lysyl-tRNAiy8 and aminoethylcys- teinyl-tRNA *US. The total lipid was isolated from the incubation mixture and spotted on a silica gel thin layer plate. After elution with CHCla-CH30H-HsO, 65:25:2, the lipids were visualized with rhodamine. The SiOz gel was scraped from the plate in zones 1 to 1.5 cm in width and counted in a scintillation counter.

sine Acid Hydrolysote of ‘%-Lipid Formed

From “C-Aminoethylcysteinyl- tRNAIYS

minoethylcysteine Origin (4 q-ctr

Acid Hydrolysate of “C-Lipid Formed From “C-Lysyl- tRNA’”

FIG. 9. Tracing of the electrophoretogram of the radioactive amino acids obtained by hydrolysis of the lipids enzymatically formed from lysyl-tRNAry* and aminoethylcysteinyLtRNA’y8.

‘NH&“&“&H&“z$H-de tRNA lyL , I

20 60 100 140 180

pprnohs CHARGED t RNA ADDED FIG. 10. Inactivity of &aminoethylcysteinyl-tRNAoy8 in amino-

acyl phosphatidylglycerol synthesis. The activity of the various substrates tested is shown as follows: lysyl-tRNAly6, O---O; aminoethylcysteinyl-tRNAry8, A--A; aminoethylcysteinyl- tRNAcY8, 0-0 ; cysteinyl-tRNAcY8, A-A. The reaction mixture contained the following (in micromoles in a final volume of 0.4 ml) : Tris-HCl (pH 7.05), 7.5; KCl, 60; phosphatidylglycerol, 500 pg; enzyme, 1.81 mg; and i4C-aminoacyl-tRNA as shown. The specific activities (in microcuries per pmole) of the aminoacyl- tRNA preparations were: lysyl-tRNAry#, 13.3; aminoethylcys- teinyl-tRNAiy*, 10.85; aminoethylcysteinyl-tRNAcy8, 46.0; cys- teinyl-tRNAiy8, 46.0. Incubation was at 30’ for 60 min. The values shown were corrected for boiled enzyme control values, which ranged from 1 to 5 rpmoles.

TABLE III

Formation of aminoethylcysteinyl phosphalidylglycerol from amino- ethylcysteinyl-IRNAJUS in presence of aminoelhylcysteinyl-

tRNAcr* Incubation conditions were identical with those in Fig. 10.

Aminoethylcysteinyl- tRNA’ys added

Aminoethylcysteinyl- tRNA’Ys added’

Aminoethylcysteinyl phos- phatidylglycerol formed

LqMw&?s ppnoles p/moles

89 0 82 89 29 80 89 72 81 89 145 74.5

D The aminoethylcysteinyl-tRNA was the standard preparation containing 46yo aminoethylcysteine and 54% cysteine.

When aminoethylcysteinyl-tRNAcy* was tested as a substrate for lysyl phosphatidylglycerol synthetase (Fig. lo), it was found to be totally inactive. Under identical conditions aminoethyl- cysteinyl-tRNAiy* was as effective as lysyl-tRNArys in the formation of the respective aminoacyl phospholipid. These results suggest that the lysyl phosphatidylglycerol synthetase is specific for lysine-acceptor tRNA and therefore has the ability to recognize the polyribonucleotide chain of the aminoacyl- tRNA.

In view of these negative results with aminoethylcysteinyl- tRNACys as a potential substrate in aminoacyl phosphatidyl-

by guest on October 30, 2020

http://ww

w.jbc.org/

Dow

nloaded from

309-l Participation of Aminoacyl-tRNA in Aminoacyl Phosphatidylglycerol Synthesis. I Vol. 213, So. 11

-

//

‘_

g-f,, ,

100 200 300 400

pg of t RNA

FIG. 11. Enzymatic recharging of tRNA obtained by hydrolysis of the aminoacyl groups from aminoethylcysteinyl-tRNAoY8 (A-Al and cysteinyl-tRNAcy8 (O-O). Aminoethyl- cysteinyl-tRNAcYn (11 mg, 4.8 X 10’ cpm) and cysteinyl-tRNAcYg (11 mg, 1.1 X 106 cpm) were incubated in 0.5 M Tris-HCl (pII 9.0) for 30 min at 37”. The deacylated tRNA was precipitated with ethanol at -2O”, dissolved in a minimum volume of 0.001 M K[C!ZH~OS], pH 5.6, and dialyzed overnight. The tRNA was reprecipitated with ethanol and dried under reduced pressure. Each sample was dissolved in 0.3 ml of 0.001 M K[CZH~O~] prior to use. The reaction mixture contained the following (in micromoles in a final volume of 100 ~1) : Tris-HCl (pH 7.2), 3.33; ATl’ (pH 6.8)) 0.2; MgC12, 0.5; mercaptoethanol, 0.66; phosphocnolpyruvate, 0.20; pyruvate kinase, 3 rg; charging enzyme, 60 pg; W-cytseine (specific activity, 46 PC per rmole), 540 prmoles; and tltNA as indicated. The quantity of tRNA was estimated spectrophoto- metrically at 260 mp (t = 15.8 0. D. units per mg per ml). Incubation was at 30” for 15 min.

TABLE IV

Enzyndic incorpordion of aminoelhylcysteinc from utninoelhyl- cy.sleinyl-lK.Y.4C~S into protein

The components and conditions of the cell-free system used were exactly those: described in the accompanying paper (2) escept that arnilloethyl(,gsteirlyl-tKKACUa (containing cysteingl-tRNAC#’ as indicated in the test) was used. The radioactive protein formed ww isolated, hydrolyzed, and analyzed for “(:-amino acids as descrihcd (2)) except t.hst performic acid oxid:rt.ion of t.he protein was omitted. In an experiment carried out >lt, approxi- mately 0.05 the scale shown betow the addition of 1525 cpm of aminoacyl-tltNA resulted in incorporation of 555 cpm of amino acid into protein in the: prcscncc of poly UG and 49 c’pm iu the absence of poly UC.

Input of aminoacyl- tlG\AC)‘8

Incorporation into protein

I cm 1 % I CM Total.. .._.............. j 30,500 1 Cgsteine.. .I Amirloethylcystei~le.

glycerol synthesis, it wa.s essential to establish that the observed inactivity of this compound was solely due t.o the fact. that. the aminoacyl group was attached to cyst&e acceptor tRNA rather t.han to lysinc acceptor tRNh. Conseyurnt,ly, a variety of control experiments to rule out various alternat,& esplana- t.ions for the inactivity of aminoethylcysteinyl-tRS.4’“” were performed.

The experiment, shown in Table III was performed to test whether or not the preparation of amirloethyl(:ysteirl~l-t.lti\rTAC~* contained inhibitors which might interfere with the synthrsis of aminoacyl phospholipids. These results show that no signifi- cant inhibition of aminoethylcysteinyl phosphatidylglycerol synthesis was observed when varying amounts of aminoethyl- cysteinyl-t.RSA c~8 were added to incubation mixtures rontnin- ing a fixed amount of aminoethylcysteinyl-tRKA’~8.

Two experimental approaches were t.aken to excludr the pos- sibility that the inactivity of aminoethylcystcinyl-tHShcs’S was due to modification of the polynucleotidc chain brought about by aminoethylation.

First, aminoethylcysteinyl-tRNAc~8, as well as a control sample of cysteinyl-tRNA’~a, was deacylated at pH 9 and the tRNA was recovered. The relative abilities of the tKNA”ys recovered from arnirlocthyl(:ysteirlyl-tRNAC’a and from cysteinyl- tRKAcy8 to serve as substrates in cysteinyl-tRXA synthesis were tested with a crude enzyme prepared from I?. coli (see “Experi- mental Procedure”). The results indicate (Fig. 11) that the aminoethylation reaction did not alter the structure of the tRK.-1cy8 molecule as t.his structure is perceived by the act.ivatiug enzyme, since bot,h t.RNAcus preparations were equally active as accept.ors of cyst&e under conditions in which tRN.1 was limiting.

A second approach to the question of possible modification of the polynucleotide chain of a~rlinoethylc~stcin~l-iR~j;\c’” was to test this compound ZLS substrate in a polypeptide-s:ynthesizillg system. As shown in Table IV, aminoethylcSsteinyl-~R~~~rY~ does serve as a substrate in the poly ‘IJG-dependent synthesis of polypeptides. Hydrolysis of the sgnthrsizrd polypeptides followed by electrophoretic analyses of the amino acids indicatrd that the aminoethglcysteinc moiety from the mixture of amino- ethylcysteinyl- and cysteinyl-tRNX cY8 was incorporatrtl into pol- ypeptides. Moreover, the proportions of “C-anlinoeth~lcvstcille and ‘Gcysteine incorporated int,o polypeptides exactly parallel the proportion of theye amino acids in the input of W-amino- acyl-tRSA. Thus these results again indicate that aminocthyla- tion has not caused alterations in the polyribonucleotide chain.

DISCUSSION

The present investigation has been undertaken in order to define further the propertics and specificity of the particulate enzyme or enzymes involved in the synthesis of lysyl phospha- t.idylglycerol from lysyl-t.RSA and phosphatidglglycrrnl. Pre- vious studies have revealed that the reaction is specific for phosphatidyl glyccrnl and for lysyl-tRSA (1, 9). The results of the present. study indicate that the pH optimum of the particulate enzyme is approximabely 6.9, and that the react,inu has a marked dependence on ionic strength. The enzymatic rractinn is strongly inhibited by several sulfhydryl-reactive reagents and, at. least in the case of p-chloromercuriphenylsulfonate, this inhibi- tion may be reversed by dithiothrcitol. However, it is not yet clear whether inhibition is due to reaction of the sulfhydryl reagents with the enzyme or with lysyl-tRYA. In view of the presence of sulfhydryl groups in B. coli lysyl-tRSA (12), the latter possibility certainly dwerves further investigation.

Although previous studies have indicated that the enzyme is specific for lysyl-tRN-4, but not species-specific with regard to

the source of lysyl-tRN.4 (lo), no detailed information on the nat.uro of the specificity was available. In order t.o determine n-hether the specificity of the enzyme for the aminoacyl-tRSX is

by guest on October 30, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Issue of Juno 10, 1968 .J. A. Nesbitt, III, and W. J. Lennarz 3095

directed toward the aminoacyl group, the polyribonucleotide chain, or both portions of the subst.rate, it was desirable to have available two potential substrates, one in which lysine was charged onto a tRNA other than lysine-specific tRNA and another in which an amino acid other than lysine w&s charged onto lysine-specific tRNA. The marked amino acid and tRN.1 specificity of the aminoacyl-tRSA synthet.ases precludes the preparation of such components by enzymatic means. Other workers desiring such “mixed” aminoacyl-tRNA preparations frequently have resorted to chemical modifications of the amino- acyl group of an appropriate aminoacyl-tRNA (see the ac- companying paper (2)). However, in the present study, chem- ical conversion of the lysyl m0iet.y of lysyl-t.RNA to another natural aminoacyl group was not feasible.

An alternative approach was suggested by the recent observa- tions of Stern and Xfehler (11) who showed that purified lysyl- tRNA synthetase from E. coli is capable of utilizing the lysine analogue, S-P-aminoethylcysteine, as substrate, with the re- sult.ant formation of S-@aminoethylcysteinyl-tRNA. In the present study enzymatically prepared aminoethylcysteinyl- tRKA1ys (see “Results” and Fig. 5) was found to be an effective substrate for lysyl phosphatidylglycerol synthet.ase. The apparent K, values for 1ysyl-t.RNA’y8 and aminoethylcystcinyl- tRNAIYS were essentially identical (2.4 to 2.8 x lOmE M), al- though the V,,, for anmmcthylcysteinyl-tRNA1~8 was only one-half that. of lysyl-tRNArY*.

In order to obtain as a potential substrate an aminoacyl-tltXA which contained a “suit.able” amino acid (i.e. lysine or amino- ct,hglcysteinc) linked to an acceptor tRKA other than lysine- specific tRNA, we have reversed the sequence of chemical and enzymatic reactions. U-r4C-L-Cyst&c was enzymatically charged onto cysteine-specific tRYA; the result.ing cysteinyl- tRNACY* was chemicallv reacted with ethylenimine to yield aminoethylcysteinyl-tR?;Acy8 (Fig. 5). In cont.rast to the results obtained with aminoethylcysteinyl-tRNA’y8, amino- ethylcysteinyl-tRNA CY* was found to be completely ineffective as a substrate for t.he synthesis of aminoethylcysteinyl phos- phatidylglycerol. Suitable control ex1reriment.s were performed (see “Results”) in order to exclude the possibility that the in- activity of aminoethylcysteinyI-tltNAC~S was due to alterations in its polyribonucleotide chain.

From these results we conclude that lysyl phosphatidyl-

glycerol synthetase possesses a recognition site for at least SOI~C

portion of the polyribonucleotide chain of lysyl-tRK.1. More- over, the observation that the maximum velocity of the reaction leading to aminoacyl phosphatidylglycerol differs by a factor of 2 when the only dXerence in the two active substrates (lysyl- tRNA1ss and an~inoethylcysteinyl-tl~NA’~s) is t.he aminoacyl group suggests that the enzyme also contains a site for recogni- tion of t.he aminoacyl group. This, of course, is not an unreason- able suggestion inasmuch as the aminoacyl group is the moiety undergoing transfer in the enzymatic reaction. Further evi- dence that aminoacayl phosphat.idylglycerol synthetases comain at least. two recognition sites for t.he appropriate aminoacyl- tRKA is reported in the accompanying paper dealing with alanyl phosphatidylglycerol synthetase (2).

~4clino&edggments--It is a pleasure to acknowledge the helpful advice of Drs. 1). Nathans and G. von Ehrenstein during the early phases of this study. We are indebted to Mr. R. Gould, who performed the experiments dealing with protein synthesis.

REFERENCES

1. LESNARZ, W. J., BOXSEIV, 1’. 1’. M., .+~n VAS DEESES, L. L. M., Biochemislry, 6, 2307 (1967).

2. GOULD, R. M., THORNTOS, hI. P., LIEPKALSS, V., ASD LEN- NARZ, W. J., J. Biol. Chem., 243, 3096 (1968).

3. VON ErinENsrmN, G., in 1,. GROSSMAN AZ(I) K. MOLDAVE (Editors), Methods in enzymoZogy, Vol. XII, -4cademic Press, Kew York, 1967, p. 588.

4. CAVALLIXI, I)., DEMAIKO, C., MONI)OVI, B., AND Azzoxs, G. F., Ezperienlia, 11, 61 (1955).

5. MUENCH; K. H., AND B&G, P., in G. L. CASTONI AND D. II. DAVIES (Editors). Procedures in nucleic acid research. Harper and Row, Publishers, 1966, p. 375.

_

6. RAFTMY, M. A., ASD COLE, It. D., J. Biol. Chem., 241, 3457 (1966).

7. SUNO, C. hf., AND FRAESKEL-CONRAT, H., Biochemislry, 6, 2061 (1966).

8. IJOLLIJM, F. J., in G. L. CASTONI AND D. R. DAVIES (Editors), Procedures in nucleic acid research, Harper and Row, Pub- lishers, New York, 1966, p. 296.

9. LESSARZ. W. J.. NESBITT. J. A.. III. and REISS. J.. Proc. Nat. &d. Sci: U. S. A ., 66,934’(1966).

, ,

10. GOULD, R. hl., ANI) LENNARZ, W. J., Biochem. Biophys. Res. Commun., 26, 510 (1967).

11. STERN, R., ANI) MEHLER, A. II., Biochem. Z., 342, 400 (1965). 12. CARBON, J. A., HUNG, L., ANI) JONES, D. S., Proc. Nut. Acad.

Sci. U. S. A., 63, 979 (1965).

by guest on October 30, 2020

http://ww

w.jbc.org/

Dow

nloaded from

J. A. Nesbitt III and W. J. LennarzPHOSPHATIDYLGLYCEROL SYNTHETASE

Phosphatidylglycerol Synthesis: I. SPECIFICITY OF LYSYL Participation of Aminoacyl Transfer Ribonucleic Acid in Aminoacyl

1968, 243:3088-3095.J. Biol. Chem. 

  http://www.jbc.org/content/243/11/3088Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/243/11/3088.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on October 30, 2020

http://ww

w.jbc.org/

Dow

nloaded from