Amino Acid Catabolism and Malic Enzyme in Differentiating

Transcription

1 JOURNAL OF BACTERIOLOGY, My 1979, p /79/5-467/8$2./ Vol. 138, No. 2 Amino Acid Ctbolism nd Mlic Enzyme in Differentiting Dictyostelium discoideum JOANNE K. KELLEHER,t PATRICK J. KELLY, AND BARBARA E. WRIGHT* Deprtment of Developmentl Biology, Boston Biomedicl Reserch Institute, Boston, Msschusetts 2114 Received for publiction 2 Jnury 1979 Amino cids produced from protein degrdtion re the mjor energy source for differentition nd ging in Dictyostelium discoideum. Considering the rections involved in the conversion of mino cids from n verge protein into tricrboxylic cid cycle intermedites, route from cycle intermedite (probbly mlte) to cetyl coenzyme A is required for the complete utiliztion of mino cids. Citrte ws isolted from cells pulse-lbeled with "C-lbeled mino cids nd ws cleved with citrte lyse. When cells were pulse-lbeled with [U-'4C]- glutmte the specific rdioctivity of the cette nd oxlocette portions of citrte were consistent with the conclusion tht one-third of the crbon flowing through the tricrboxylic cid cycle is removed for the synthesis of cetyl coenzyme A. The dt were lso consistent with the ptterns of crbon flux required to mintin stedy-stte levels of cycle intermedites in cells ctbolizing mino cids. It is suggested tht the mlic enzyme (EC ) ctlyzes the synthesis of cetyl coenzyme A from mlte nd is responsible for the observed citrte lbeling pttern. In cell extrcts the ctivity of this enzyme incresed mrkedly with the onset of differentition. The properties of prtilly purified (4-fold) mlic enzyme isolted t culmintion indicted tht the enzyme ws llosteric nd ws positively ffected by sprtte nd glutmte. Thus, mino cid production from protein degrdtion would stimulte rection essentil for the efficient utiliztion of these mino cids for energy. Differentition in the cellulr slime mold Dictyostelium discoideum occurs in - strving system. Therefore, endogenous reserves of protein, crbohydrte, RNA, nd lipid must supply ll the precursors for the synthesis of new mterils nd for energy genertion over the 24-h differentition period. As differentition occurs, totl crbohydrte remins essentilly constnt (15), nd totl lipid chnges re reltively smll (12). However, protein decreses by pproximtely one-hlf (6, 24). A quntity of mmoni comptible with the mount of protein degrded is relesed from the differentiting orgnism (2). In ddition, there is evidence tht the conversion of mino cids to crbohydrtes is not significnt (2). Tken together, these results strongly suggest tht crbon supplied by protein degrdtion is the mjor energy source for differentition nd ging in this model system. The utiliztion of mino cids s n energy source hs been documented in severl other systems. Spore germintion is often ccompnied by rpid decrese in free mino cids. For exmple, in germinting Bcillus megterium t Present ddress: Deprtment of Physiology, The George Wshington University Medicl Center, Wshington, DC spores, it hs been estimted tht mino cid degrdtion genertes significnt mount of 4TP in the period 5 to 15 min fter the onset of germintion (17). Strving muscle tissue lso metbolizes mino cids, lthough complete oxidtion of ll mino cids to CO2 my not occur in this system (3). Goldstein nd Newsholme (5) hve stressed the necessity of pthwy from tricrboxylic cid cycle intermedites to pyruvte nd hence cetyl coenzyme A (CoA) for complete oxidtion of mino cids vi the tricrboxylic cid cycle. Their rguments pply to ny mino cid-ctbolizing system where complete oxidtion occurs. Figure 1 presents scheme for the degrdtion of mino cids from typicl cell proteins. The flux rtes (in prentheses in Fig. 1) were clculted ssuming rte of protein degrdtion t culmintion of.4 mmol of mino cid residues per min, vlue which is consistent with the rtes of 2 utiliztion (11), mmoni production (2), nd protein degrdtion (24). This rte ws broken down into flux rtes into the tricrboxylic cid cycle t vrious points by fctoring in the mino cid composition of typicl cell proteins (21). The vlues were further corrected for the fct tht some mino cids feed the tricrboxylic

2 468 KELLEHER, KELLY, AND WRIGHT J. BACTERIOL. AMINO ACIDS CO2 (.2) CH3-CO-SCoA j i ACETYL CoA ASPARTATE PYRUVATE \ (.7/ (.3) ALANINE, / etc. OXALOACETAT I 1 COOH 2 CH2 3 HOC-COOH 6 4 CH2 5 COOH CITRATE \ Co2 CITRATE (.13) MALATE (.5) COOH 2 5 \ \CH 3 4 N CH 4 3 COOH COON l 2 CO I (.5) 3 CH2 * 4 CH2 5 COOH KETOGLUTARATE FUMARATE SUCCINYL COA (.2) j(3) SUCCINATE AMINO ACIDS AMINO ACIDS FIG. 1. Digrm of the tricrboxylic cid cycle, including mino cid ctbolism. Vlues in prentheses in millimoles per minute, clculted from rte ofprotein degrdtion nd mino cid content of re fluxes, cell protein. (See text) cid cycle t more thn one point (e.g., phenyllnine is broken down to fumrte nd cetyl CoA). Complete oxidtion of mino cids to CO2 ws ssumed. The resulting fluxes re only pproximte, yet, s is pprent from Fig. 1, the supply of crbon must be such tht the flux through cetyl CoA must equl tht through oxlocette (millimoles per minute) for the citrte synthse rection to operte nd mintin stedy-stte levels of cycle intermedites. However, blnce between these two fluxes will not exist unless there is route which converts excess tricrboxylic cid cycle intermedites to cetyl CoA, since mny of the mino cids produced from typicl cell proteins re converted to C4 nd C5 tricrboxylic cid cycle intermedites. The mjor enzymes involved in metbolizing tricrboxylic cid cycle compounds to produce precursors of cetyl CoA re phosphoenolpyruvte crboxykinse, which utilizes oxlocette s substrte, nd the mlic enzyme, which utilizes mlte. Although both of these rections re known to function in vivo in the direction of cetyl CoA formtion, we will present isotopic evidence tht mlte rther thn oxlocette is the tricrboxylic cid cycle compound used, s 1 COOH 2 HC-NH2 l - 3 CH2 l 4 CH2 5 COON GLUTAMATE shown in Fig. 1. Thus, we hve isolted mlic enzyme from Dictyostelium nd hve evluted its kinetic properties, in view of its possible role in mintining stedy-stte levels of tricrboxylic cid metbolites s mino cids re ctbolized. The crbon toms hve been numbered in Fig. 1 so tht specificlly lbeled molecules my be followed through the tricrboxylic cid cycle. Although this numbering system is internlly consistent, it violtes the R-S rules for the citrte molecule (18). Thus, it should be noted tht, in this study, Cl nd C2 of citrte re derived from the crboxyl nd methyl groups of cette, respectively. MATERIALS AND METHODS Cells. The moebe of D. discoideum strin NC-4 were grown in ssocition with Escherichi coli on nutrient gr, hrvested, wshed free of bcteri, nd plced on non-nutrient gr pltes contining phosphte buffer nd EDTA s described previously (11). These pltes were incubted t 23 C until the desired stge of differentition ws reched. The cells were then hrvested for pulse-lbeling or enzyme purifiction. Pulse-lbeling. Freshly hrvested cells were

3 VOL. 138, 1979 shken in 1 ml of phosphte buffer (5 mm, ph 7.) t 23 C nd pulse-lbeled with 8,uCi of [U-'4C]lnine, [U-14C]sprtte, [U-_4]glutmte, or [2-'4C]glutmte. At pproprite time intervls, liquots (1 ml) were frozen in liquid nitrogen nd 25 ml of 95% ethnol ws dded. Citrte ws isolted nd purified by ionexchnge chromtogrphy (Dowex AG1-1 x 1), procedure described in more detil elsewhere (Kelly, Delleher, nd Wright, mnuscript in preprtion). Citrte clevge. The isolted [14C]citrte ws quntitted by using fluorometric enzymtic ssy (13). Rdioctivity ws mesured by liquid scintilltion counting. A second liquot of citrte ws treted with citrte lyse to cleve the C2-C3 bond. The rection mixture consisted of pproximtely 1 nmol of lbeled citrte in.5 M Tris buffer (ph 7.5) contining 1. mm EDTA, 1. mm MgCl2, 1. mm NADH, nd.1 U of citrte lyse. After 15 min of incubtion t 23 C the rection mixture ws plced on Dowex AGl-1 x 1 ion-exchnge column (1 by 4 cm; formte form). Acette nd mlte were recovered by step elution with formic cid ( to 1. M), nd the rdioctivity in ech frction ws counted. Isoltion of mlic enzyme. Approximtely 18 g (wet weight) of cells ws hrvested into Tris buffer (5 mm, ph 7.5) contining 1 mm dithiothreitol, repetedly frozen nd thwed (three times), nd centrifuged t 4, x g for 15 min. The enzyme in the superntnt ws precipitted t concentrtion of (NH4)254 between 35 nd 45 g/1 ml of cell extrct. This precipitte ws solubilized in Tris buffer (5 mm, ph 8.1) nd frctionted on Sephdex G-2 column (2 by 6 cm). The enzyme ws eluted with 5 mm Trishydrochloride (ph 8.1) contining 1. mm dithiothreitol. The ctive frction from this column ws further frctionted on DEAE-cellulose equilibrted previously with 5 mm Tris-hydrochloride (ph 8.1). The column ws eluted by pplying 5-ml liner grdient from 5 mm Tris-hydrochloride (ph 8.1) to 5 mm Tris-hydrochloride (ph 7.4). The results of this purifiction scheme re shown in Tble 1. Mlic enzyme ctivity ws ssyed in the direction of mlte decrboxyltion. One unit of ctivity is defined s tht mount of enzyme ctlyzing the reduction of 1,umol of NADP per min t 25 C. Mximum enzyme ctivity ws ssyed in.5 M Tris (ph 8.1) contining 1 mm MgCl2, 15 mm mlte, 1. mm dithiothreitol, 1. mm EDTA, nd.1 mm NADP. Chemicls. [U-'4C]lnine (.17 Ci/mmol), [U- TABLE 1. Purifiction scheme for mlic enzyme from D. discoideum Sp ct Frctin Prf- % Re- Vol tein of ction covery Frction Pro- Ulg (mil) (mg/ ofepro Crude extrct (NH4)2S4 pre cipittion Sephdex G DEAE-cellulose See text for detils. AMINO ACIDS AND MALIC ENZYME IN DICTYOSTELIUM 469 '4C]sprtte (.19 Ci/mmol), [U-14C]glutmte (.24 Ci/mmol), nd [1,5-_4C]citrte (8.2 mci/mmol) were obtined from New Englnd Nucler Corp. [2-'4C] glutmte (4.3 mci/mmol) ws obtined from ICN Phrmceuticls, Inc. Enzymes nd metbolites were obtined from Sigm Chemicl Co. [3,4,5,6-'4C]citrte ws synthesized from [U-'4C]sprtte in the presence of glutmte oxlocette trnsminse, citrte synthse, nd excess cetyl CoA nd -ketoglutrte, ccording to the procedure for rdiochemicl ssy of cetyl CoA (16). The resulting rdioctive citrte ws purified by ion-exchnge chromtogrphy s described bove. RESULTS Whet nd Ajl (23) demonstrted tht the stereospecificity of citrte lyse (EC ) ws the sme s tht for citrte synthse. We hve verified tht commercilly produced citrte lyse (isolted from Enterobcter erogenes) cleves citrte exclusively t the C2-C3 bond nd tht the products, fter tretment with mlte dehydrogense, chromtogrph s cette nd mlte. This ws ccomplished by incubting [1,5-14C]citrte or [3,4,5,6-14C]citrte with citrte lyse nd mlte dehydrogense, followed by ion-exchnge chromtogrphy (see Mterils nd Methods). The results (Tble 2) indicte tht citrte ws broken down to its originl C2 nd C4 moieties under these conditions. Citrte isolted from cells pulse-lbeled with [2-14C]glutmte ws lbeled only in the mlte moiety (see below). In the pulse-lbeling with U-_4C-mino cids it is importnt to note tht the exogenous concentrtion of mino cids in the cell suspension, pproximtely.8,um, ws fr below the in vivo levels of these compounds (pproximtely 1 mm). Thus, the cellulr metbolite levels were not perturbed, nd the system ws ssumed to be in metbolic stedy stte. It ws not expected tht the system would ttin isotopic stedy stte under these conditions. In fct, the ctul specific rdioctivity of most of the metbolites in Fig. 1 continued to chnge over the course of TABLE 2. Distribution of lbel in citrte fter clevge with citrte Iyse After citrte lyse nd mlte dehy- Originl drogense tret- Smple citrte ment (cpm) Acette Mlte (cpm) (cpm) [1,5-_4C]citrte stndrd 8,9 4,48 4,51 [3,4,5,6-14C]citrte stndrd 9,5 9,45 [2-14C]glutmte 5-min pulse min pulse 9,99 1,1 3-min pulse 8,5 1 8,3

4 47 KELLEHER, KELLY, AND WRIGHT 9 CN 8 c 7. 6._S 5 -J v 4 *> 3 2~ I Time in Minutes FIG. 2. Percent totl citrte rdioctivity found in Cl plus C2 (cette moiety) when cells were pulselbeled with U-14C-lbeled mino cids for 3 min. Citrte ws isolted from cells t vrious timne points nd cleved t the C2-C3 bond s described in the text. 3-mim pulse-lbel (dt to be presented elsewhere). Figure 2 grphs the frction of totl rdioctivity in the cette (Cl nd C2) portion of citrte isolted from cells pulse-lbeled with three different - ANI~~~~ANNE - t X GLUTAMATE A A ASPARTATE 32 U-14C-lbeled mino cids: lnine, glutmte, nd sprtte. The lbel distributions shown in Fig. 2 my be interpreted in ccordnce with the scheme in Fig. 1, s discussed below. Glutmte lbeling. When cells from the preculmintion stge of development were pulse-lbeled with [U-"4C]glutmte, rdioctivity ppered in both the cette nd oxlocette portions of citrte. This result indictes tht some metbolic route other thn glutmte -. -ketoglutrte CO2 vi the tricrboxylic cid -- cycle must be in opertion since this pthwy would produce lbel only in C3, C4, C5, nd 6 of citrte. Two simple explntions for this result re: (i) cetyl CoA is synthesized from some tricrboxylic cid cycle intermedite lbeled in the glutmte pulse experiment ( plusible route involving the mlic enzyme is digrmmed in Fig. 1); (ii) the uniformly lbeled glutmte is converted to [1,2,3,4,5-'4C]citrte by reversl of the citrte-to-glutmte rections in the cell cytoplsm. This pthwy is possible since there is cytoplsmic glutmte dehydrogense in Dictyostelium (1), nd the enzymes conitse nd isocitrte dehydrogense which ctlyze revers- J. BACTERIOL. ible rections re found in the cytoplsm. Obviously these two explntions re not mutully exclusive; both pthwys my be in opertion. Cells t the culmintion stge of differentition were pulse-lbeled with [2-14C]glutmte to exmine the relevnce of the second explntion. [2-'4C]glutmte would produce [2-14C]citrte if cytoplsmic enzymes generte citrte from glutmte (Fig. 1). Clevge of citrte produced in this mnner t the C2-C3 bond would generte ['4C]cette. Alterntively, [2-'4C]glutmte would not yield lbeled cette vi decrboxyltion of mlte nd pyruvte with mlic enzyme nd pyruvte decrboxylse, since [2-14C]-glutmte produces [1,4-14C]mlte in the tricrboxylic cid cycle (see Fig. 1). The crboxylic cid groups re relesed s CO2 in the synthesis of cetyl CoA from mlte vi the mlic enzyme nd pyruvte dehydrogense. The nlysis of lbel distribution in citrte for the [2-'4C]glutmte pulse-lbeling experiments is presented in Tble 2. At the three experimentl time points essentilly ll citrte rdioctivity is locted in the four-crbon mlte frction, indicting tht the conversion of lbeled glutmte to citrte by the -ketoglutrte-to-citrte rections is not prominent in Dictyostelium t the preculmintion stge of differentition. This conclusion is supported by erlier studies (1) demonstrting tht, throughout differentition, the mechnism of CO2 formtion from glutmte is oxidtive, with glutmte nd -ketoglutrte decrboxyltion proceeding t the sme rte. Uniformly lbeled glutmte lbels the cette nd oxlocette portions of citrte t constnt rtio over the course of the 3-min pulse-lbel. Approximtely 15% of the totl citrte rdioctivity is locted in Cl nd C2. The expected lbel distribution in citrte my be clculted from the dt in Fig. 1. Assuming tht Fig. 1 is essentilly correct, the expected specific rdioctivity of ny pool cn be clculted by dividing the product of the flux of lbeled mteril entering the pool times its specific rdioctivity by the totl flux of mteril entering the pool. If the specific rdioctivity of mlte is, the expected specific rdioctivity of the oxlocette frction of citrte is (.37/.4), or.92, vlue slightly less thn the mlte specific rdioctivity, due to isotope dilution from sprtte degrdtion. The specific rdioctivity of the cette frction of citrte is estimted s 1/2 (.13/.4) =.16, where the fctor 1/2 represents decrboxyltion of uniformly lbeled mlte t the phosphoenolpyruvte crboxykinse nd pyruvte dehydrogense rections. Thus, the percentge of the totl citrte lbel

5 VOL. 138, 1979 in the cette frction, C1 nd C2, should be (.16 / ) = 15%. In clculting this reltionship it is ssumed tht the specific rdioctivity of the mlte pool in the tricrboxylic cid cycle is the sme s tht used by the mlic enzyme. Tht is, either the mlic enzyme is locted in mitochondri, shring the sme pool s mlte dehydrogense, or two pools of mlte (i.e., cytoplsmic nd mitochondril) rpidly exchnge with ech other. It is relevnt to note tht the frction, 15%, is expected to remin constnt nd is not influenced by vrition in the ctul specific rdioctivity of mlte which occurs over the course of 3-min experiment. Furthermore, the clcultion is not dependent on the ctul rte of protein degrdtion but is contingent on (i) the mino cid content of degrded cell protein in Dictyostelium being representtive of typicl cell proteins (21) nd (ii) the complete oxidtion of these mino cids. The ctul frction of totl citrte lbel in Cl nd C2 of citrte in the uniformly lbeled glutmte experiment is in excellent greement with this clcultion (see Fig. 2). Asprtte lbeling. [U-14C]sprtte-lbeled cells lso produced citrte lbeled in both the cette nd oxlocette portions. As with the glutmte experiment, the frction of totl lbel in the cette crbons did not vry over the pulse-lbeling period, indicting tht the precursors for both prts of the citrte molecule follow similr lbeling ptterns. The reltive mount of lbel in the cette crbons, 3 to 4% of the totl, is lower thn tht found with the glutmte lbel. This result indictes tht the tricrboxylic cid cycle precursor for cetyl CoA synthesis (presumbly mlte) is lower in specific rdioctivity thn the oxlocette pool used by citrte synthse. Alnine lbeling. In contrst with the sprtte nd glutmte pulse-lbeling, the clevge of citrte produced in the lnine pulse-lbel indictes tht the reltive specific rdioctivity of the two precursors of citrte chnge over the course of the pulse-lbel (Fig. 2). On first entering the tricrboxylic cid cycle lnine lbels only the cette crbons of citrte. At lter time points incresing rdioctivity ppers in the oxlocette frction, indicting tht citrte produced by lnine degrdtion is further metbolized by the tricrboxylic cid cycle to yield oxlocette. The low rte of lbeling of the oxlocette frction of citrte is evidence tht isotopic stedy stte hs not been ttined. Mlic enzyme from Dictyostelium. (i) Generl properties. Mlic enzyme ws isolted nd purified 4-fold from culminting Dictyostelium s shown in Tble 1. The enzyme AMINO ACIDS AND MALIC ENZYME IN DICTYOSTELIUM 471 requires NADP nd ws ssyed in-the direction of mlte decrboxyltion. Enzyme ctivity ws not detected when NAD ws used s the pyridine nucleotide substrte. Unlike other NADPrequiring mlic enzymes (EC ), decrboxyltion of oxlocette could not be detected t cid ph. Divlent ctions (t lest 1. mm Mg2" or.1 mm Mn2+) were required for mxinum enzyme ctivity in the bsence of EDTA. (ii) Enzyme kinetics. A double-reciprocl plot of enzyme ctivity versus mlte concentrtion t ph 8.1 ws nonliner, indicting llosteric chrcteristics (Fig. 3); the Hill coefficient ws 1.8. However, the degree of nonlinerity ws ph dependent; t ph 7., the kinetics were nerly Michelis-Menten. In ddition to ph, the kinetic properties of this enzyme were ffected by the presence of dicrboxylic cids. The nonlinerity of the reciprocl plot ws bolished with 1 mm sprtte (Fig. 3). The influence of vrious mino cids, tricrboxylic cid cycle intermedites, nd denine nucleotides on the rection rte t substurting mlte concentrtions is presented in Tble 3. These dt indicte tht high levels of certin mino cids nd compounds preceding mlte in the tricrboxylic cid cycle tend to positively ffect the rection rte, wheres inhibition occurs in the presence of 1. mm oxlocette nd cetyl CoA. A double-reciprocl plot of enzyme ctivity versus NADP concentrtion displyed norml Michelis-Menten kinetics with Km for NADP of pproximtely 1,uM (iii) Enzyme ctivity during differentition. The ctivity of mlic enzyme in crude extrcts from cells over the course of differenti- 1/MALATE (mm)- FIG. 3. Double-reciprocl plot ofmlic enzyme ctivity versus mlte concentrtion in the bsence (-) ndpresence () of 1 mm sprtte. Assyed t ph 8.1 s described in the text.

6 472 KELLEHER, KELLY, AND WRIGHT TABLE 3. Effect of tricrboxylic cid cycle were mixed together before cell lysis nd enzyme intermedites, mino cids, nd denine ssy. The enzyme recovery in this experiment nucleotides on mlic enzyme ctivity ws 1 ± 1%, indicting tht the growing Effectorb Reltive rte moebe did not contin n inctivtor of enzyme ctivity. None Asprtte Glutmte DISCUSSION Alnine...1. Amino cid pulse-lbeling. We hve dem- Serine onstrted the existence of pthwy from the Succinte. 1.5 Fumrte tricrboxylic cid cycle to the cette moiety of Citrte citrte during differentition nd ging in Dic- 1 tyostelium under conditions of ctive protein Oxlocettte.. Acetyl CcA..68 degrdtion. The method used for this purpose, Pyruvte.96 clevge of lbeled citrte produced from l- ATP beled mino cid precursors, should be pplic- ADP ble in other systems, provided tht the gener- 5'-AMP...1. tion of lbeled citrte by the -ketoglutrte-to- 3',5'-cAMP.1. citrte rection is not significnt. Pulse-lbeling Enzyrme ssyed in 5 mm Tris buffer (ph 8.1) with [2-'4C]glutmte my be used to rule out contininm g.3 mm mlte,.1 mm NADP, 1. mm tht possibility, s in this study. The citrte EGTA [e-thyleneglycol-bis(,8-minoethyl ether)-n,n- clevge method my be used to evlute qun- cid], nd 1 mm MgCl2. tittively the contribution of the two different tetrcetiic b All efl 3- c z ~ 5- FIG. 4. I cid oxidtion during development nd ging in.~ IDictyostelium, t lest in terms of reltive fluxes into citrte. Similr clcultions cnnot be mde for sprtte nd lnine pulse-lbeling becuse both of these compounds contribute lbel to citrte from source other thn mlte (i.e., oxlocette nd cetyl CoA). The reltive contribution HOURS OF DEVELOPMENT of lbel from two or more sources cnnot be Mlic enzyme ctivity in crude extrcts of quntitted if isotopic stedy stte is not t- D. discoiudeum over the course of differentition. The 4, x g superntnts were ssyed for mximl enzyme c stndrd error for t lest four determintions. tion is p sents en J. BACTERIOL. fectors were evluted t 1. mm. pthwys to the synthesis of citrte. Figure 1 indictes tht pproximtely one-third of the cetyl CoA molecules re derived from tricrboxylic cid cycle mlte. The clcultion bsed on these dt predicting 15% of the totl citrte lbel in the cette moiety ws closely pproched by experimentl dt for the [U-'4C]-. glutmte (Fig. 2). We therefore suggest tht ft Fig. 1 represents n ccurte profile of mino timed. Dt from the sprtte experiment do, however, offer evidence s to the tricrboxylic :ctivity s described in the text. Men ± cid cycle precursor of cetyl CoA. If mlte is the tricrboxylic cid cycle precursor for cetyl CoA nd the oxlocette nd mlte pools re resented in Fig. 4. The -h point repre- not in isotopic equilibrium (i.e., the specific rkzyme ctivity in growing cells entering dioctivity of oxlocette is greter thn ml- phse. No enzyme ctivity ws te), then the percent totl citrte lbel in Cl the sttiionry detectedi in the bcteril frction obtined in plus C2 should be less thn tht found with [Und concentrting the moebe. A rpid '4C]glutmte. Alterntively, if the tricrboxylic wshing increse in enzyme ctivity occurs in the first 4 cid cycle precursor for.cetyl CoA is the oxlh fter t'he onset of differentition (strvtion). ocette pool used in citrte synthesis, pproxi- tht the difference between grow- mtely 15% of the totl citrte lbel should To sceirtin ing mooebe nd 4-h strved cells ws not n pper in Cl plus C2, s in the [U-_4C]glutmte rtefct due to high levels of protese ctivity or experiment. The sprtte dt in Fig. 2, indi- in the moeb extrct, pproxi- cting only 4% of the totl citrte lbel in C1 n inhilbitor mtely e squl mounts of moebe nd 4-h cells plus C2, support the hypothesis tht mlte

7 VOL. 138, 1979 rther thn oxlocette is the mjor tricrboxylic cid cycle precursor for cetyl CoA. A citrte molecule with 4% totl counts in Cl plus C2 is expected if the oxlocette pool used for citrte synthesis is pproximtely fourfold greter in specific rdioctivity thn the mlte pool undergoing decrboxyltion to yield cetyl CoA. The pulse-lbeling with [2-14C]glutmte indicted tht reversl of the tricrboxylic cid cycle from -ketoglutrte to citrte is not source of lbel of C1 nd C2 of citrte in the [ U- 14C]glutmte pulse. If it is ssumed tht lbeled glutmte mixes with the cellulr glutmte pool, these results further indicte tht crbon from glutmte does not contribute to lipid synthesis t preculmintion. Finlly, there is nother possible pthwy from mlte to cetyl CoA not involving the mlic enzyme. Stem (19) hs found mlte clevge enzyme in E. coli which ctlyzes clevge rection of mlte yielding cetyl CoA nd glyoxylte. If this rection occurred in Dictyostelium, [2-'4C]glutmte. would produce lbeled cetyl CoA nd hence citrte lbeled in Cl (see reference 14 for detils). Since no rdioctivity ws detected in the cette moiety of citrte in this experiment (Tble 1), the mlte clevge enzyme of Stern (19) is not ctive in Dictyostelium under these conditions. The citrte dissection method used here should be of vlue in other systems where mino cid oxidtion hs been suggested s mjor energy source. Setlow nd Primus (17) clculted tht erly in Bcillus spore germintion mino cid oxidtion might supply ATP t level 2.5 times tht vilble from endogenous 3-P-glyceric cid reserves. Citrte clevge fter pulse-lbeling with mino cids could evlute this prediction. Mlic enzyme. Two enzymes re known to be involved in the decrboxyltion of tricrboxylic cid cycle intermedites to yield pyruvte or phosphoenolpyruvte:phosphoenolpyruvte crboxykinse nd mlic enzyme. We hve suggested tht mlte is the tricrboxylic cid cycle precursor of cetyl CoA nd tht mlic enzyme ctlyzes the first rection in this pthwy. Our evidence is bsed on three fcts. (i) The [U- '4C]sprtte lbeling experiment indictes tht the tricrboxylic cid cycle precursor of cetyl CoA is compound lower in specific rdioctivity thn oxlocette, nd thus phosphoenolpyruvte crboxykinse, which uses oxlocette s tricrboxylic cid cycle substrte, is not likely to be the enzyme involved. (ii) We were not ble to detect phosphoenolpyruvte crboxykinse ctivity in crude extrcts of culminting Dictyostelium (Kelleher, unpublished observtions). (iii) AMINO ACIDS AND MALIC ENZYME IN DICTYOSTELIUM 473 The regultory properties of these two enzymes in other systems (4, 7, 8, 24) suggest tht mlic enzyme is the more likely prticipnt in protein degrdtion in Dictyostelium. In generl, phosphoenolpyruvte crboxykinse is used by cells for gluconeogenesis, wheres mlic enzyme functions in mlte ctbolism (9, 14). The properties of mlic enzyme isolted from Dictyostelium re consistent with this hypothesis. The effect of vrious compounds on the rte of mlte decrboxyltion ws studied t.3 mm mlte, pproximtely the mlte concentrtion t culmintion (metbolite levels will be published elsewhere). The significnce of the inhibitory effects of oxlocette nd cetyl CoA (Tble 2) is uncler since the concentrtion of these metbolites in vivo is fr below 1. mm. However, severl effective modultors of enzyme ctivity re found in vivo t pproximtely 1. mm. These include glutmte, sprtte, nd succinte. Thus, these dt suggest tht some compounds whose complete oxidtion would require the prticiption of n ctive mlic enzyme my ctully serve s modultors of enzyme ctivity in vivo. The ctivity of mlic enzyme required t culmintion clculted from protein degrdtion profiles is.13 mmol of flux per min (Fig. 1), or 1.,umol of substrte decrboxylted/g (dry weight) of cells per min (22). Compring this vlue to the ctivity of mlic enzyme in preculminting cells of Dictyostelium, pproximtely 5. p.nol/g (dry weight) (Fig. 4), suggests tht cells contin n ctivity of mlic enzyme dequte to produce the flux hypothesized in Fig. 1. In ddition, the chnging pttern of enzyme ctivity in cell extrcts over the course of differentition (Fig. 4) follows the protein degrdtion profile in Dictyostelium (24). Tken together, these results suggest tht the kinetic behvior nd cellulr ctivity of mlic enzyme in Dictyostelium reflect its role in protein degrdtion. ACKNOWLEDGMENTS This investigtion ws supported by Public Helth Service postdoctorl fellowship AG565 to J. K. K. nd Public Helth Service grnts AG433, AG26, nd 551RR5711 from the Ntionl Institutes of Helth. LITERATURE CITED 1. Bruhmuller, M., nd B. E. Wright Glutmte oxidtion in the differentiting slime mold. Biochim. Biophys. Act 71: Clelnd, S. V., nd E. L. Coe Conversion of sprtic cid to glucose during culmintion in Dictyostelium discoideum. Biochim. Biophys. Act 192: Chng, T. W., nd A. L. Goldberg The metbolic ftes of mino cids nd the formtion of glutmine in skeletl muscle. J. Biol. Chem. 253: Diesterhft, M. D., nd E. Freese Role of pyru-

8 474 KELLEHER, KELLY, AND WRIGHT vte crboxylse, phosphoenolpyruvte crboxykinse, nd mlic enzyme during growth nd sporultion of Bcillus subtilis. J. Biol. Chem. 248: Goldstein, L, nd E. A. Newsholme The formtion of lnine from mino cids in diphrgm muscle of the rt. Biochem. J. 164: Hmes, B. D., nd J. M. Ashworth The metbolism of mcromolecules during the differentition of myxmoebe of the cellulr slime mould Dictyostelium discoideum contining different mounts of glycogen. Biochem. J. 142: Hsie, A. W., nd H. V. Rickenberg A mutnt of Escherichi coli deficient in phosphoenolpyruvte crboxykinse ctivity. Biochem. Biophys. Res. Commnun. 25: Jcobson, L. A., R. C. Brtholomus, nd I. C. Gunslus Repression of mlic enzyme by cette in Pseudomons. Biochem. Biophys. Res. Commun. 24: Krulwich, T. A., B. L. Shron, nd L. S. Perrin Nturl pucity of nplerotic enzymes: bsis for dependence of Arthrobcter pyridinolis on L-mlte for growth. J. Bcteriol. 127: Lngridge, W. H. R., P. Kominiecki, nd F. J. De- Tom Isoltion nd regultory properties of two glutmte dehydrogenses from the cellulr slime mold Dictyostelium discoideum. Arch. Biochem. Biophys. 178: Liddel, G. U., nd B. E. Wright The effect of glucose on respirtion of the differentiting slime mold. Dev. Biol. 3: Long, B. H., nd E. L. Coe Chnges in neutrl lipid constituents during differentition of the cellulr slime mold Dictyostelium discoideum. J. Biol. Chem. 249: Lowry,. H., nd J. V. Pssonneu A flexible system of enzymtic nlysis, p Acdemic Press Inc., New York. 14. Muri, T., M. Tokushige, J. Ngi, nd H. Ktsuki. J. BACTERIOL Studies on regultory functions of mlic enzymes. J. Biochem. (Tokyo) 71: Rosness, P. A., nd B. E. Wright In vivo chnges of cellulose, trehlose nd glycogen during differentition of Dictyostelium discoideum. Arch. Biochem. Biophys. 164: Schoner, W., nd W. Seubert Rdiochemicl ssy of cetyl CoA, p In H. U. Bergmeyer (ed.), Methods of enzymtic nlysis, vol. 4, 2nd English ed. Acdemic Press Inc., New York. 17. Setlow, P., nd G. Primus Protein metbolism during germintion of Bcillus megterium spores. J. Biol. Chem. 25: Srere, P. A The citrte enzymes: their structures, mechnisms, nd biologicl functions, Curr. Top.- Cell. Regul. 5: Stern, J. R Enzymtic ctivtion nd clevge of D- nd L-mlte. Biochim. Biophys. Act 69: Sussmn, M., J. Schindler, nd H. Kim Towrd biochemicl definition of the morphogenetic fields in Distyostelium discoideum, p In P. Cppuccinelli nd J. M. Ashworth (ed.), Development nd differentition in the cellulr slime moulds. Elsevier/North-Hollnd, New York. 21. Tristrm, G. R., nd R. H. Smith Amino cid composition of certin proteins, p In H. Neurth (ed.), The proteins, vol. 1. Acdemic Press Inc., New York. 22. Wlsh, J., nd B. E. Wright Kinetics of net RNA degrdtion during development in Dictyostelium discoideum. J. Gen. Microbiol. 18: Whet, R. W., nd S. J. Ajl Citritse, the citrtesplitting enzyme from Escherichi coli. J. Biol. Chem. 217: White, G. J., nd M. Sussmn Metbolism of mjor cell components during slime mold morphogenesis. Biochim. Biophys. Act 53: Zink, M. W Regultion of the "mlic" enzyme in Neurospor crss. Cn. J. Microbiol. 13: