TRANSAMINASES IN SMOOTH BRUCELLA ABORTUS, STRAIN 19

TRANSAMINASES IN SMOOTH BRUCELLA ABORTUS, STRAIN 19 BY ROBERT A. ALTENBERN AND RILEY D. HOUSEWRIGHT (From the Chemical Corps Biological Laboratories, Camp Detrick, Frederick, Maryland) (Received for publication, February 13, 1953) Goodlow et al. (1) clearly demonstrated that the establishment of nonsmooth variants in originally smooth virulent cultures of Bruce&x species grown in Gerhardt-Wilson synthetic medium (2) is concurrent with the appearance of alanine in the medium. Previous work in this laboratory (3) has shown that a considerable amount of alanine is produced by a glutamate to pyruvate transamination reaction by strain 19 of Brucella abortus. The data reported in this paper show that in sonic extracts of this organism there is a variety of transaminases. Evidence has been obtained for the presence of transaminases which catalyze amino transfer from leucine, isoleucine, norleucine, or phenylalanine to pyruvate without proceeding through glutamic acid. Although glutamic acid participates in all transaminases that have been studied extensively (4), the work of Rowsell (5) and the present paper offer evidence that there are trans- aminases which do not involve glutamate. In addition, sonic extracts of this organism contain transaminases which transfer amino groups to cr-ketoglutarate from the four amino acids mentioned above. EXPERIMENTAL A smooth variant of Brucella abortus, strain 19, was employed. Methods for maintenance of stock cultures and for preparation of cell suspensions have been described previously (3). Cell suspensions were disintegrated by sonic oscillation in a Raytheon sonic oscillator for 90 minutes at low temperature (about 5 ). The resulting preparation was centrifuged for 1 hour at 8000 r.p.m. in a Servall high speed centrifuge at refrigerator temperature (4 ). The clear supernatant fluid was decanted and dialyzed in the cold for 48 to 7.2 hours against 0.1 M phosphate buffer adjusted to ph 7.4. This fluid extract consistently contained from 2 to 3 mg. of nitrogen per ml. and was devoid of detectable amounts of free amino acids. The dialyzed extract was used immediately or stored in a frozen state until needed. All ammonium sulfate precipitation procedures were carried out in the refrigerator. Residual salt was removed from redissolved precipitates by dialysis in the cold against 0.1 M phosphate buffer at ph 7.4. Substrates were dissolved in 0.1 M phosphate buffer 159

160 TRANSAMINASES IN BRUCELLA and adjusted to ph 7.4 if necessary. Determination of transaminase activity was performed by incubating enzyme solution plus substrate solution at 37 anaerobically in Thunberg tubes. At successive times, samples were removed and the tubes were evacuated again. These samples were analyzed for amino acids by the quantitative method of Housewright and Thorne (6). Qualitative and quantitative determinations of keto acids were performed by the methods of Magasanik and Umbarger (7). Results Many chromatograms of crude or undialyzed sonic extracts of this organism have shown that the free amino acids appearing in largest amount are the leucines, phenylalanine, alanine, glycine, glutamic acid, and perhaps some serine and aspartic acid. Many of these amino acids were found to participate in transamination reactions catalyzed by enzymes present in sonic extract. As in other organisms, enzymes transaminating to cr-ketoglutarate from leucine, isoleucine, norleucine, and phenylalanine were readily demonstrable, although glycine and serine were inactive. Incubation of the various leucines and phenylalanine plus pyruvic acid with sonic extracts indicated that there was considerable transfer of amino nitrogen to pyruvate to produce alanine. An analysis of reaction rates of pertinent transaminases established that the nitrogen is not transferred through glutamate. It was possible to show that glutamic acid was formed slightly more rapidly by aspartic-glutamic transamination than by leucine-glutamic transamination. Glutamic-alanine and aspartic-glutamic transaminases had been detected in earlier work (3). Therefore, if alanine were synthesized from leucine plus pyruvate by two steps proceeding through trace amounts of glutamate or cy-ketoglutarate, aspartic acid plus pyruvate should give rise to alanine as rapidly as does leucine. However, aspartate does not give rise to alanine, as is shown in Table I. Furthermore, at no time did detectable amounts of glutamic acid or any amino acid other than substrate constituents appear on any of the chromatograms. These data both eliminate aspartic-alanine transaminase as a possible enzyme and establish leucine to pyruvate transamination as a separate and probably single step. Only after prolonged incubation is alanine synthesized from aspartate and pyruvate. Since free amino acids appear in slight amount in sonic extract controls after long incubation, the formation of alanine from aspartate plus pyruvate seems not to be significant. Probably, P-decarboxylation of aspartate is not involved, since no alanine arises from aspartate alone. There is, likewise, no alanine synthesis from n-aspartate and pyruvate. Sonic extracts can metabolize carbohydrates by the Krebs cycle (8) and could produce intermediates,

R. A. ALTENBERN AND R. D. HOUSEWRIGHT 161 a-ketoglutarate in this case, which would facilitate a two-step amino transfer from aspartate to pyruvate proceeding through glutamate after lengthy incubation. TABLE Production of Alanine by Transamination by Sonic Extracts of Smooth B. abortus Strain 19 I Substrate Amino acid found Total amino acid after 2 hrs. 1 L hrs. 6 hrs. 4 hrs. L-Leucine (50 piu) + Na pyruvate (100 PM) Leucine Alanine PM 44.7 3.0 PM PM 49.5 51.4 5.4 7.4 nn-isoleucine (50 PM) + Isoleucine 43 43.6 48 38.7 Na pyruvate (100 PM) Alanine 2.1 3.7 4.7 5.2 nn-norleucine (50 PM) + Norleucine 41.2 51 48.6 Na pyruvate (100 PM) Alanine 2.9 5.4 6.2 nn-phenylalanine (50 PM) + Phenylalanine 44.7 54.0 48.6 51 Na pyruvate (100 MM) Alanine 2.0 2.3 4.5 4.4 n-aspartic acid (50 PM) + Aspartate 26.8 26.4 21.5 5.2 or-ketoglutaric acid (50 m) Glutamate 16.4 21.2 24.0 31.2 L-Leucine (50 flm) + Leucine 29.8 28.6 23.2 17.4 ol-ket oglutaric acid (50 MM) Glutamate 14.5 20.7 21.8 24.8 n-glutamic acid (50 PM) + Glutamate 43.0 48.6 44 32.7 Na pyruvate (100 PM) Alanine 5.3 7.4 10.2 17.6 n-aspartic acid (50 PM) f Na pyruvate (100 PM) Aspartate Alanine 42.0 47.0 48.5 0 0 0 GM 40.3 9.7 31.7 36.0 0 1 ml. of dialyzed sonic extract plus 1 ml. of substrate solution incubated anaerobically at 37 in Thunberg tubes at ph 7.4. A rough ammonium sulfate fractionation of sonic extract yielded supporting evidence for the presence of leucine-alanine transaminase. The fractions precipitated by 10, 20, 30, 40, and 50 per cent (weight by volume) ammonium sulfate were centrifuged, dissolved in buffer, and dialyzed to remove residual salt. The fractions were analyzed for relative amounts of four transaminases. By determining rates of reactions, it is possible to draw curves of the precipitating characteristics of the different transaminases. Arbitrarily, a unit of any transaminase was designated as that amount of enzyme which will catalyze the transfer of 10 mpm of amino

162 TRANSAMINASES IN BRUCELLA nitrogen per hour when the rate of transfer is linear. The data appear in Fig. 1. Leucine-glutamic transaminase is precipitated by 20 per cent ammonium sulfate. Both leucine-alanine and glutamic-alanine transaminases exhibit maximal precipitation at 30 per cent ammonium sulfate, while aspartic-glutamic transaminase is precipitated over a broad range with a maximum at 40 per cent ammonium sulfate. Repeated fractionation of sonic extract with ammonium sulfate has yielded preparations 8 60- E h g 50- LEUCINE-GLUTAMIC LEUCINE - ALAhQNE % ASPARTIC-GLUTAMM 2 >40- F Go- 8 e 20-8 I- z E Y io- W/V (NH4 In SO4 CONCENTRATION FIG. 1. Transaminase activity of precipitates obtained from ammonium sulfate fractionation of sonic extract of smooth i?. abortus, strain 19. 1 ml. amounts of redissolved precipitates added to 1 ml. amounts of substrate solutions and incubated anaerobicaffy at 37 at ph 7.4. Sampled and analyzed at 2,4, and 6 hours of incubation. Substrates, L-leucine (50 NM) + Na pyruvate (100 PM), n-leucine (50 PM) f or-ketoglutaric acid (50 PM), n-glutamic acid (50 PM) i- Na pyruvate (100 MM), n-aspartic acid (50 pm) f wketoglutaric acid (50 PM). All tubes cont,ained 6.4 y of pyridoxal phosphate. which catalyze leucine-alanine transamination directly, but which require pyridoxal phosphate before glutamic-alanine transamination occurs. Sonic extract was precipitated first with 30 per cent and then with 35 per cent ammonium sulfate. The supernatant fluid was freed of salt by dialysis after each step. The final salt-free supernatant fluid from 35 per cent precipitation was tested for the two transaminases cited above, with and without pyridoxa1 phosphate. These data are presented in Table II. Although the incubation time was great and the enzyme concentrations apparently small, it is clear that the transaminases may be separated on this basis.

R. A. ALTENBERN AND R. D. HOUSEWRIGHT 163 It has been reported that removal of nucleic acid and inert protein from bacterial extracts1 and culture filtrates (9) by the addition of protamine greatly facilitates enzyme separation by ammonium sulfate fractionation. Preliminary clearing of sonic extract by addition of protamine sulfate to a final concentration of 2 mg. per ml. sharpened ammonium sulfate fractionation. The precipitate formed by addition of protamine sulfate (1 hour in the refrigerator) was removed by centrifugation and discarded. The supernatant fluid was fractionated with ammonium sulfate in the cold and the 20,25,30,35, and 40 per cent (weight by volume) precipitates TABLE II Transaminase Activity of Precipitate Obtained from Sonic Extract between 30 and 36 Per Cent (Weight by Volume) Ammonium Sulfate L-Leucine (100 NM.) + Na pyruvate (ZOO PM) Substrate Leucine Alanine PM 117 6.4 L-Leucine (100 prvr) + Leucine 109 Ns pyruvate (200 PM) + 1 y pyridoxal phosphate Alanine 6.8 L-Glutamic acid (100 PM) -I- Na pyruvate (200 PM) L-Glutamic acid (100 pm) -I- Na pyruvate (200 PM) + 1 y pyridoxal phosphate Total amino acid found Glutamate Alanine Glutamate Alanine Precipitate obtained between 30 and 35 per cent ammonium sulfate redissolved in cold 0.1 M phosphate buffer at ph 7.4. 2 ml. aliquots of this solution were transferred to each Thunberg tube. 2 ml. of substrate solution were then added and the Thunberg tubes incubated anaerobically for 18 hours at 37. were collected and redissolved in buffer as before. After dialysis, the amounts of leucine-alanine and glutamine-alanine transaminases were determined by analysis of rates, as previously described. The ratios of amounts of these enzymes in each fraction were found to vary regularly and provided another basis for separation of the two transaminases. Table III shows the data consistently obtained in such experiments. A partially resolved preparation was obtained by thrice precipitating the fraction removed from sonic extracts between 20 and 35 per cent (weight by volume) ammonium sulfate. Dialysis followed each precipitation and resolution of the precipitate. Further, extensive reprecipitations resulted in rather great inactivation. Pyridoxal phosphate saturation curves were conducted on the partially resolved enzyme solution with i Wilson, P. W., personal communication (1952). 114 0 96 5.6

164 TRANSAMINASES IN BRUCELLA both leucine-pyruvate and glutamic-pyruvate substrates. The results appear in Fig. 2. Since the coenzyme concentration remaining in the preparation was not determined, no effort was made to calculate the Michaelis constant for each enzyme. However, it is apparent that the curves vary in shape and that the maximal velocities are quite different. From inspection of the curves, it is probable that the Michaelis constant for leutine-alanine transaminase is less than that for glutamic-alanine transaminase, supporting the data in Table II. Analysis of leucine to pyruvate transaminating system for a-keto acids by the method of Magasanik and Umbarger (7) revealed that at zero time pyruvic acid was the only keto acid present. During incubation, another TABLE Protamine and Ammonium Sulfate Fractionation of Sonic Extract for Separation of Leucine-Alanine and Glutamic-Alanine Transaminases Per cent ammonium sulfate (weight by volume) I III Rate of leucine-alanine transaminase Rate of glutamic-alanine transaminase 20 0.73 25 0.71 30 0.66 35 0.57 40 0.38 The Thunberg tubes contained 1 ml. of solution of precipitate from ammonium sulfate fractionation, 1 ml. of substrate solution containing either 50 PM of L-leucine and 100 PM of Na pyruvate or 50 pm of L-glutamic acid and 100 pm of Na pyruvate and 0.2 ml. of coenzyme solution containing 5.5 y of pyridoxal phosphate. Incubated anaerobically at 37 and sampled and analyzed at 2,4, and 6 hours of incubation; ph 7.4. keto acid of high RF in butanol-acetic acid-water solvent appeared. This acid seemed to increase in concentration as the time of incubation increased. By comparison with a pure sample of sodium a-ketoisocaproate, this component was found to be identical chromatographically with cy-ketoisocaproic acid. No other keto acids appeared during incubation. Quantitative analysis for the alanine synthesized and the Lu-ketoisocaproate released established that rates of production of these two components were parallel (Table IV). Although it is generally accepted that the well known transamination reactions involve glutamic acid or cu-ketoglutaric acid (4), it is becoming apparent that transaminases occur which may catalyze amino transfer without the participation of cr-ketoglutarate or glutamate. Earlier workers (10) claimed that transamination occurred between aspartic acid and alanine when enzyme preparations were fortified with several activators.

R. A. ALTENBERN AND R. D. HOUSEWRIGHT 165 Later it was shown that aspartic-alanine transaminase is an artifact and that the two activators are pyridoxal phosphate and glutamic acid (11). However, in the work presented here, there was no evidence which would implicate glutamic acid as a necessary component in transfer of amino 160- = 140 ii GLUTAMIC-ALANINE TRANSAMINASE x AX= 147.5 pg/ml/hr. LEUCINE-ALANINE TRANSAMINASE VMAX= ~VMAX= 76.8-0 76.8 gg/ml/hr.!3 I( w i,t G 40-,,,,, K J 20 0 I 2 3 4 5 pg PYRIDOXAL PHOSPHATE PER 2.2 ml. FIG. 2. Pyridoxal phosphate saturation curves for leucine-alanine and glutamicalanine transaminases in a partly resolved preparation from sonic extract of smooth B. abortus, strain 19. 1 ml. of enzyme solution + 1 ml. of substrate solution + 0.2 ml. of pyridoxal phosphate solution incubated anaerobically at 37 at ph 7.4. Sampled at 2, 4, and 6 hours of incubation. Substrate solutions, L-leucine (50 PM) + Na pyruvate (100 PM), n-glutamic acid (50 PM) k Na pyruvate (100 PM). TABLE Production of Alanine and cu-ketoisocaproate during Leucine-Alanine Transamination by Sonic Extract of Smooth B. abortus Strain 19 IV Incubation I Total alanine I Total a-ketoisocaproate 2 3.6 5.0 4 5.1 6.1 7 6.8 6.9 1 ml. of dialyzed sonic extract plus 1 ml. of substrate solution incubated anaerobically in Thunberg tubes at 37 ; ph 7.4. Total substrate, L-leucine 50 PM, Na pyruvate 100 MM.

166 TRANSAMINASES IN BRUCELLA groups from leucine to pyruvate. There was no requirement for either glutamate or a-ketoglutarate, nor were these two compounds ever detected in the complete system required for leucine-alanine transamination. The results of precipitation studies show that neither leucine-glutamic nor glutamic-alanine transaminase is precipitated in a constant ratio to leucine-alanine transaminase. Ammonium sulfate fractionation of sonic extract before protamine is apparently not very selective and served only to separate those enzymes with gross solubility differences, such as leucineglutamic and leucine-alanine transaminases (Fig. 1). However, treatment of sonic extract with protamine prior to ammonium sulfate fractionation, together with the decrease in ammonium sulfate concentration increments from 10 per cent to 5 per cent (Table III), gave good evidence that leucinealanine and glutamic-alanine transaminases can be separated by solubility differences. Although data obtained from coenzyme saturation studies are not entirely clear, these results do show that pyridoxal phosphate is the coenzyme for leucine-alanine transaminase. The data contained in this paper effectively demonstrate that leucine-alanine transamination is an enzymatic reaction separable from other transaminases and that the possible net transfer of amino group from leucine to pyruvate proceeding through glutamic acid has been eliminated. Transamination reactions involving transfer of amino group to pyruvate from isoleucine, norleucine, or phenylalanine have not been investigated as extensively as the leucinealanine system, but the data in Table I strongly indicate that these three transaminations also occur directly and do not pass through glutamic acid. SUMMARY Sonic extracts of smooth Brucella abortus strain 19 contain enzymes which catalyze amino transfer from leucine, isoleucine, norleucine, or phenylalanine to cr-ketoglutarate, producing glutamic acid. Data are presented to demonstrate that sonic extracts also possess transaminases which effect transfer of amino groups to pyruvate from leucine, isoleucine, norleucine, or phenylalanine. Leucine-alanine transaminase has been extensively investigated, and the results obtained indicate that this transamination occurs as a single step and that glutamic acid does not participate in this reaction. Pyridoxal phosphate is the coenzyme for leucinealanine transaminase. The authors wish to express their gratitude for generous gifts of calcium pyridoxal phosphate from Dr. W. W. Umbreit and of pure sodium ar-ketoisocaproate from Dr. Alton Meister.

R. A. ALTENBERN AND R. D. HOUSEWRIGHT 167 BIBLIOGRAPHY 1. Goodlow, R. J., Mika, L. A., and Braun, W., J. Bact., 60,291 (1950). 2. Gerhardt, P., and Wilson, J. B., J. Bact., 66, 17 (1948). 3. Altenbern, R. A., and Housewright, R. D., J. Bad., 62, 97 (1951). 4. Gale, E. F., in Werkman, C. H., and Wilson, P. W., Bacterial physiology, New York, 453 (1951). 5. Rowsell, E. V., Nature, 168, 194 (1951). 6. Housewright, R. D., and Thorne, C. B., J. Bact., 60,89 (1950). 7. Magasanik, B., and Umbarger, H. E., J. Am. Chem. Sot., 72, 2308 (1959). 8. Altenbern, R. A., and Housewright, R. D., Arch. Biochem. and Biophys., 36,345 (1952). 9. Christensen, L. R., J. Gen. Physiol., 30,465 (1947). 10. Braunstein, A. E., and Kritzmann, M. G., Biokhimiya, 8, 1 (1943). 11. O Kane, D. E., and Gunsalus, I. C., J. Biol. Chem., 170,433 (1947).

TRANSAMINASES IN SMOOTH BRUCELLA ABORTUS, STRAIN 19 Robert A. Altenbern and Riley D. Housewright J. Biol. Chem. 1953, 204:159-167. Access the most updated version of this article at http://www.jbc.org/content/204/1/159.citation Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/204/1/159.citation.full.h tml#ref-list-1