Degradation of Pyruvate by Micrococcus Lactilyticus

MICCORnMICK, N. G. (University of W1'ashington, Seattle), E. J. ORDAL, AND H. R. WHITELEY. Degradation of p)yruvate by Micrococcus lactilyticus. II. Studies of cofactors in the formate-exchange reaction. J. Bacteriol. 83:899-906. 1962. Enzyme preparations from ilicrococcus lactilyticus2 are rendered inactive with respect to formate exchange by treatment with charcoal or Dowex-5O, by dialysis, or by fractionation with ammonium sulfate. The activity may be completely restored by a "kochsaft" lprelparation (13ES) obtained from .ll. tactilyticus and partially restored by similar B3ES lpreparations from Escherichia coli and Clostridium butyricum. Diphosphothiamine is required for formate exchange but full activity cannot be restored by known cofactors. Brief exposure to increased temperatures, air, extremes of pH, and absorption with charcoal and Dow-ex-50 decrease the cofactor activity of B<ES preplarations. The addition of BES lprelparations from E. coli and Streptococcus faecalis causes a shift in the degradation of pyruvate by extracts of .lI. tactilyticus from the phosl)horoclastic cleavage (to acetyl phosphate and formate) to the dismutation of pyruvate (to lactate, acetate, and carbon dioxide). C. cylindrosporunim as found to mediate the formate-exchange reaction; the activity of crude extracts was stimulated by 11. tactilyticus and C. butyricwmiii BES lprelparations. Ul. tactilyticuts BES also increased the formate-exchange activitv of extracts of E. coli. The determination of cofactor requirements for the exchange of formate into p)yruvate by differ'Present address: I)ept. of Bacteriology, Univer;sty of Wisconsin, Madison, Wis. 2 Veillonella alcalescens Prevot, in Bergey's manual of determinative bacteriology, 7th ed., 1957. Classified as .Jlicrococciis lactilyticuls by Foubert and Douglas (1948). ent bacteria has been complicated by the fact that the enzymes involved are labile and have not been purified. Therefore, most investigations have been made with crude extracts which have been depleted of cofactors and then examined for activity after addition of known coenzymes, natural products (e.g. yeast extract), and heatinactivated crude extracts ("kochsaft"). Studies with Escherichia coli (Strecker, 1951; Novelli, Gest, and Krampitz, 1954; Asnis, Fritz, and Glick, 1956) and Streptococcuis faecalis (Wood and O'Kane, 1960) showed that natural products or "kochsafts" could be replaced only partially, or not at all, by mixtures of known cofactors although certain cofactors stimulated exchange. Thus in E. coli, coenzyme A (CoA) and diphosphothiamine (DPT) are required (Chantrenne and Lipmann, 1950; Strecker, 1951), and tetrahydrofolate is stimulatory (Chin, Krampitz, and Novelli, 1957), wvhereas in S.faecalis exchange is enhanced by tetrahydrofolate (Wood and O'Kane, 1960). The exchange of formate into p)yruvate by extracts of Jlicrococcus lactilyticus requires an alkaline pH (Novelli, 1955), a high phosphate concentration, and divalent cations, and is greatly stimulated by certain reducing agents and a hydrogen atmoslhere (AIcCormick, Ordal, and Whiteley, 1962). If extracts are dialyzed, fractionatecl, or treated with charcoal or certain resins, exchange is abolished. Full activity may be achieved only by addition of the supernatant fraction from a heat-inactivated crude extract, suggesting a requirement for additional components. This paper describes some of the lproperties of the Ml. lactilyticus "kochsaft" p)reparation, the effect of this lpreparation andI known cofactors on the formate-exchange activity of .If. lactilyticus, the effect of the "kochsaft" preparation on the formate-exchange activity of other organisms, and the effect of "kochsaft" preparations from other bacteria on the exchange reaction mediated by .M1. lactilyticuis. Brief reports of some of these results have al)899 on O cber 5, 2017 by gest http/jb.asm .rg/ D ow nladed fom MCCORMICK, ORDAL, AND WHITELEY peared previously (McCormick et al., 1959a, b, 1960). MATERIALS AND METHODS Mlaintenance and growth of cultures. The growth of .11. lactilyticus, strain 221, and the preparation of extracts and fractions have been described previously (McCormick et al., 1962). Peptococcus aerogenes was cultured in glutamate medium (Whiteley, Osborn, and Huenneckens, 1959), and E. coli was grown in a medium consisting of tryptone, 1.5%; yeast extract, 0.5%; K2 HPO4, 0.05%; and glucose, 1% (pH 7.2). C. butyricum, kindly supplied by R. S. Wolfe, was cultured in 0.5% tryptone, 0.5% yeast extract, 0.2% K2HPO4, and 1% glucose at pH 7.2. S. faecalis, strain 10 Cl, obtained through the courtesy of I. C. Gunsalus, was cultured in a medium consisting of tryptone, 1%; yeast extract, 1%; K2HPO4, 0.5%; and glucose, 0.5% (pH 7.2). To obtain large amounts of the above organisms, 18-liter quantities of medium were inoculated with 1700 ml of a culture of 8 to 10 hr and harvested after 16 to 18 hr incubation at 37 C. C. cylindrosporum, obtained from H. A. Barker, was cultivated in a medium consisting of yeast extract, 0.05%; K2HPO4, 0.03%; MgSO4-7H20, 0.005 %; CaSO4, 0.25% (saturated solution); FeSO4 * 7H20, 0.0002%; Na2S 9H20, 0.005%; and uric acid, 0.25%; adjusted to pH 7.4. A total of 20 liters of medium was inoculated with 1 liter of an overnight culture and harvested after 10 to 12 hr of incubation at 37 C. Chemicals. All organic and inorganic chemicals were the best grade available commercially. The preparation of tetrahydrofolate, N5,N10,methylene tetrahydrofolate, N5 ,N'0, -methenyl tetrahydrofolate, and N'0-formyl tetrahydrofolate has been described previously (Whiteley, 1960). E. L. Stokstad kindly provided N5-formyl tetrahydrofolate, C. Mathews supplied dehydrofolate, and R. Crane supplied coenzyme Q. Mlethods. Procedures used for the assay of formate exchange have been detailed elsewhere ('McCormick et al., 1962). Lipoic acid was measured manometrically by the S. faecalis pyruvic oxidase method described by Gunsalus and Razzell (1957). We wish to thank I. C. Gunsalus for the assay of certain extract preparations for lipoic acid. CoA content of extracts was determined by the arsenolysis of acetyl phosphate (Stadtman, 1955). Formiiate-activlting enzyme with a specific activity!of 800 xN-as )repared from extracts of P. aerogectcs an(l assaye(l as described by Whiteley et al. (1959). The determination of lactate and volatile acids lhas been described (McCormick et al., 1962). Depletion of i1. lactilyticius extracts of "cofactor activity." Dialy-sis of aimm)iionitum stulfate fractions (McCormick et ld. 1 959a, b) for 4 hr, or of crude extracts for 4 to 9 lhi, resulted in preparations having negligible activity. Treatment of extra1('cts w-itlh (harcoal or Dowex50 also yielded prel)arations (keficienlt in cofactor activity. Charcoal was l)rel)are(l in the following manner. Norit A wats stsl)en(led in 3 N HCl and left standing overnight, washed with water to a constant pH of bl)out 5.0, washed once in 0.01 M phosphate buffer (14i 8.5), and(I washed again with water. To 100 miig of the al)ove charcoal suspension were added 5 ml of crude extract containing 40 mg of protein per mifl; the mnixture was stirred for 15 miil in aln ice bath and centrifuged at 25,000 X g for 15 min in a Servall centrifuge. Dowex-50, 20 to 50 mlesh, w-as prepared by washing the resinl several timiies with 4 N HCl, followed by w-ashlilng 3 to 4 tinmes with water, then with 2 N NaOH several times, and finally washed exhaustively with water unIltil the pH of the resin suspension w\s 7.5. One volume of dry resin was added to tw0(o volumiles of extract, the mixture was allowe(d to stan(l in anl ice bath for 15 min with occasionial stirrinig, anld the extract was removed. Dowex-1, 20 to 50 mesh, was prepared by washing several times with 1 N HCl followed b1 exhlla,ustive washing, with water to a constant pH of abbout 6.0. I)owex-1 was added to extracts in the samiie im-anner as described above for D)owex-50. Elution of cofactor activity fr70n charcoal. Charcoal was sel)arate(d fromii the extract by centrifugation, susl)edlc(ld in 20 voltunes of 50% aqueous acetone whicih was 0.1 N with respect to NH40H, allowved to stnlnd in anl ice bath for 15 min, centrifuged, andol the sulperiatant evaporated to dryness in v-acuo. The residue was dissolved in a small (uti.untity of wvater, recentrifuged, and the supl)ernltant utsed as a source of cofactor. Preparation of 'kochisafts". 11. lactilyticus "kochsaft" (abbreviated BIES for boiled extract supernatant) was )repl)ared as follows. Crude extract was placed inl a simiall filter flask and 900 [VOL. 83 on O cber 5, 2017 by gest http/jb.asm .rg/ D ow nladed fom COFACTORS IN FORMATE-EXCHANGE REACTION alternately evacuated and filled with hydrogen several times to insure anaerobic conditions. The flask was immersed in a boiling water bath, swirled rapidly for 2 to 3 min until the temperature was approximately 65C., then cooled rapidly in an ice bath, and the heat-treated extract was centrifuged to yield a clear dark-amber supernatant (BES). BES prelparations from the other organisms were obtained in the same manner. Animal tissues were homogenized with water in a Potter-Elvehjem glass homogenizer in the cold, centrifuged, the supernatant fraction heated at 65 C for 3 min, and centrifuge(d again to remove denatured protein. Chromatography of BES preparation. Quantities (0.5 to 1.0 ml) of BES and other cofactor preparations were chromatographed on Whatman a 1 or # 3 paper using a n-butanol, pyridine, and water (1:1:1) solvent. Areas of the chromatograms were cut out, eluted with water, and the eluates were concentrated in vacuo. RESULTS AND DISCUSSION Loss of activity and reactivation by BES. Dialysis, fractionation with ammonium sulfate, and treatment of crude extracts with charcoal or Dowex-50 resin resulted in a complete loss of formate-exchange activity (Table 1). This activity was fully restored by a BES l)reparation. In contrast to the action of Dowex-50, treatment of crude extracts with Dowex-1 resulted in only a slight decrease in activity even though, as discussed below, the latter treatment removed CoA almost comnpletely. It will be noted (Table 1), that exchange activity was confined to the tower ammonium sulfate fraction. If assays were performedl in the presence of BES, fractionation did not cause a loss in total formate-exehange activity of an extract; however, only slight increases in specific activity have been achieved. The ability of crude extracts to carry out the exchange of formate into a-ketobutyrate and a-ketoglutarate (MIcCormick et al., 1962) was also lost upon dialysis or treatment w-ith charcoal. The addition of 13ES partially restored formate exchange into a-ketobutyrate but not into a-