PYRUVIC CARBOXYLASE OF ACETOBACTER SUBOXYDANS*

BY TSOO E. KING AND VERNON H. CHELDELIN

(From the Deparfment of Chemisfry and the Science Research Institute, Oregon State College, Corvallis, Oregon)

(Received for publication, November 30, 1953)

Biochemical decarboxylation of pyruvate can in general occur in two ways. Simple cr-decarboxylation has been recorded in yeast and plants (1). In animals and most bacteria, pyruvate is generally either consumed through an oxidative pathway whereby acetaldehyde is not an intermedi- ate (2) or converted to acetylmethylcarbinol (acetoin) by the carboligase system (3, 4).

Acetaldehyde has been reported as a product of pyruvate fermentation by a few bacteria, particularly species of Pseudomonas and Acetobacter (5). In Acetobacter suboxydans, pyruvate is oxidized to acetate with a consump- tion of 1 atom of oxygen per molecule of the substrate (6,7). Through the use of cell-free extracts it has now been shown that pyruvate is first decar- boxylated to acetaldehyde and that this compound is in turn oxidized to acetate. A. stboxydans is thus one of a relatively few bacteria with a yeast type decarboxylation. In this paper, the behavior of this pyruvic car- boxylase and its separation from other oxidative enzymes are reported in detail.

EXPERIMENTAL

The crude cell-free extracts were prepared by grinding the lyophilized or fresh cells with alumina as reported previously (7). Removal of pro- tein from appropriate reaction mixtures was accomplished with either tri- chloroacetic acid or a zinc sulfate-sodium hydroxide mixture. Chemical analyses were then made on the deproteinized filtrates. Acetate was de- termined as total volatile acids by steam distillation (8). Acetaldehyde was quantitatively determined by the calorimetric methods of Barker and Summerson (9), except that an aliquot of protein-free extract was treated twice with copper sulfate and calcium hydroxide instead of once as origi- nally described. In a control experiment with a 4: 1 mixture of pyruvic

* A preliminary report, was presented before the Nineteenth International Con- gress of Physiology, Montreal, Canada, September, 1953. Published with the ap- proval of the Monographs Publications Committee, Oregon State College, Research paper No. 234, School of Science, Department of Chemistry. This work was sup- ported by the Nutrition Foundation, Inc., the Rockefeller Foundation, and the Division of Research Grants and Fellowships, National Institutes of Health, United States Public Health Service.

821

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822 PYRUVIC CARBOXYLASE OF A. SUBOXYDANS

acid and acetaldehyde, the pyruvic acid contributed less than 0.5 per cent of the color.

The dinitrophenylhydrazone derivatives were prepared by passing an air stream through the deproteinized enzyme reaction mixture or the ap- proximate corresponding concentration of an authentic aldehyde solution into a 2,4-dinitrophenylhydrazine reagent. The precipitated product was recrystallized immediately from aqueous alcohol three times. A pyruvic acid solution tested under the same conditions did not give any precipi- tate. Acetaldehyde solutions were standardized by titration with bisulfite (10). Acetoin was determined by t,he Happold and Spencer modification (11) of the Neuberg and Strauss method (12) and also by the creatine- sodium carbonate reaction (13).

Protein was determined by the usual Kjeldahl method or by the biuret reaction. Thiamine was determined by microbiological and thiochrome methods (14, 15). Manometric determinations were performed at 29” in a Warburg apparatus, with corrections for carbon dioxide retention in the medium.

Materials-These were procured as follows: Sodium pyruvate was a commercial sample (Schwarz), recrystallized from

alcohol; it was also prepared from pyruvic acid according to Price and Levintow (16).

a-Ketoglutaric and oxalacetic acids were commercial samples (Krishell Laboratories). The purity of the oxalacetate was 96 f 5 per cent as judged by decarboxylation with Alz(S04)a (17).

Diphosphopyridine nucleotide (DPN) was a commercial sample (Pabst), of about 80 per cent purity.

Cocarboxylase was a commercial sample (Delta). The bound thiamine assay indicated a purity of 80 per cent.

Phenyl azlactone was furnished by Dr. E. G. Bubl. This was converted to phenylpyruvic acid by hydrolysis (18); m.p. 150-152” (literature value, 150-154” (18)).

a-Ketobutyric acid was prepared and furnished by Dr. B. E. Christensen, by an unpublished method; m.p. 30” (uncorrected) (literature value, 32” (19)). For further evidence on the identity of this compound, see the text.

Sodium or-ketoisovalerate, Lu-ketoisocaproate, and oc-keto-/3-methylvalerate were furnished and prepared by Dr. Alton Meister, by deamination of the corresponding amino acids (20).

RESULTS AND DISCUSSION

Dissimilation. of Pyruvic Acid in Crude Cell-Free Extracts

The oxidation of pyruvate to acetate could be demonstrated in cell-free extracts. As shown in Table I, under aerobic conditions 1 mole of CO2

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T. E. KING AND V. H. CHELDELIN 823

was formed per mole of pyruvate. However, both the oxygen consumed and the volatile acids formed were slightly lower than the stoichiometric values based on the simple conversion of pyruvate to acetate. This dis- crepancy was found to be due to the formation of acetoin. The details of the latter reaction will be reported elsewhere.

In the oxidation of pyruvate a short lag period was always observed in oxygen consumption, but not in COZ formation. On the other hand, there was no lag period in the oxidation of acetaldehyde. This raised the ques- tion whether the enzyme followed the scheme of oxidative decarboxylation common to most bacteria. The oxidation of pyruvic acid was therefore

TABLE I

Oxidation of Pyruvic Acid in Cell-Free Extracts of A. suboxydans

Oxygen consumed.

COz formed. ..................... Volatile acid formed .................. Acetoin formed. ...................... Acetaldehyde recovered ................

Aerobic Anaerobic

system I System II System I system II

microatoms microaloms

45 0 GM PM PM )IM 52 50 52 50

- Each flask contained 50 PM of pyruvic acid and 0.067 M phosphate buffer; pH 6.0;

total volume, 3.0 ml. In addition, System I contained 0.3 MM of DPN and 10 mg. (in terms of protein content) of the crude cell-free extract, and System II contained 2.8 mg.( in terms of protein) of Fraction A.

tested in the presence of dimedon. With this reagent, carbon dioxide formation proceeded nearly normally, whereas oxygen consumption was greatly impaired, as shown in Table II. Dimedon also inhibited the oxi- dation of exogenous acetaldehyde to an ext,ent almost paralleling the in- hibition of pyruvate oxidation. This observation strengthened the sug- gestion of a simple a! decarboxylation prior t,o oxidation in A. suboxydans. Accordingly, the reaction was tested under anaerobic conditions. The theoretical yield of COz was obtained (Table I), and the intermediate acetaldehyde was recovered as its 2,4-dinitrophenylhydrazone; m.p. 165” (uncorrected). The melting point of an authentic sample was 164”, and the mixed melting point was 164”. The small amount of acetic acid formed was probably due to some residual electron transfer coenzymes in the crude enzyme extract. The yield of acetoin was slightly higher than that under aerobic conditions.

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824 PYRUVIC CARBOXYLASE OF A. SUBOXYDANS

Separation and Properties of Pyruvic Carboxylase

Separation of Pyruvic Carboxylase from Crude Extract-Since the oxida- tion of pyruvate to acetate in A. suboxydans occurred in stepwise fashion, an attempt was made to separate the carboxylase from the oxidative en- zymes. After a few unsuccessful experiments, this was accomplished by a simple acetone precipitation.

25 ml. of cold acetone were added slowly to 25 ml. of the crude cell-free extract at O-5”. The precipitate thus formed was suspended in 15 ml. of water. The insoluble matter was separated by centrifugation. The clear supernatant liquid was dialyzed against running distilled water at 5”. With some samples a second acetone precipitation was required in order

TABLE II

Efect of Dimedon on Oxidation of Pyruvate and Acetaldehyde by Crude Cell-Free Extract of A. suboxydans

Dimedon

w.

0 7.4

22.2

Pyruvate Acetaldehyde

CO2 formation 02 consumption 02 consumption

PM nhwtoms microato?lls

62 54 56 60 36 33 51 12 10

Each flask contained 60 .UL~M of substrate, 20 PM of MgCL, 0.3 PM of DPN, 2 PM of cocarboxylase, and 10 mg. of enzyme (in terms of protein content); 0.067 M phosphate buffer, pH 7.0. Total volume, 3.0 ml. Atmosphere, air.

to remove all oxygen-reacting enzymes. The supernatant solution was designated Fraction A.

As shown in Table I, Fraction A (System II) produced the theoretical amount of COZ from pyruvate under both anaerobic and aerobic conditions. No oxygen uptake could be demonstrated even in the presence of added DPN (System I). The acetaldehyde formed was determined colorimetri- tally (9) and identified as its 2,4-dinitrophenylhydrazone. Added acetal- dehyde was not oxidized by Fraction A.

No attempt was made to purify Fraction A further. However, it was precipitable at an acetone concentration of 30 per cent. The precipitate, which was called Fraction B, was used for all the experiments described below. This fraction occasionally (three out of fourteen experiments) ex- hibited a marginal dependence upon MgK and cocarboxylase.

Optimal pH-The optimal pH was found to be 6.0, as shown in Fig. 1. No special effects were produced by the individual buffers; phosphate, suc- cinate, and citrate at about pH 6 gave practically the same rate of CO,

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T. E. KING AND V. H. CHELDELIN 825

formation throughout the course of the experiment. As expected, the re- action was independent of phosphate, as in the case of the simple carboxyl- ases from other organisms.

It- SUCCINATE (0) 4

It PHOSPHATE W)

-------CITRATE (*)-A

3.6 4.1 5.1 6.1 7.1

PH FIG. 1

I.

I.

f

1.

0

0

4r

.2 -

O-

.8 -

.6- 0

Vwmuu 2 1.48 )J MOLS / MINUTE

KS -7.4 X Id MOLS/ LITER

5 IO I5

&(X102M)

FIG. 2

FIQ. 1. Optimal pH for pyruvic cerboxylase activity in A. suboxydans. The system contained 200 PM of buffer, 50 PM of pyruvic acid, 10 pM of MgCL, 1 pM of co- carboxylase,and 2 mg. (in terms of protein content) of Fraction B; total volume, 3.0 ml.

FIG. 2. The Michaelis constant of A. suboxydans pyruvic carboxylase. The system contained 10 PM of MgCle, 1 PM of cocarboxylase, 0.067 M phosphate buffer, 1.0 mg. (in terms of protein content) of Fraction B, and the indicated amounts of pyruvic acid; pH 6.0; volume, 3.0 ml. V is expressed as micromoles per minute (aver- age of the first 4 minutes), except at the concentration of 6.68 X 10m4 M (l/s = 1500) when the average of the first 2 minutes was taken. s = substrate concentration.

Thiamine Content of Holoenzyme-The thiamine content of Fraction B, which usually did not respond to added cocarboxylase, was found to be about 0.05 per cent (based on protein content) by both microbiological and thiochrome methods. A provisional “molecular weight” of this frac- tion would thus be about 650,000, assuming an equimolar combination of cocarboxylase to protein. Since Fraction B was impure, the actual molecular weight is probably much lower; thus, it may be calculated that the apoenzyme in Fig. 3 possesses at least twice the specific activity of Fraction B (Fig. 1). The turnover rate may be seen from Fig. 3 and

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826 PYRUVIC CARBOXYLASE OF A. SUBOXYDANS

from the V,,,. value obtained in Fig. 2 to approximate 150 (moles of COZ formed per minute per 100,000 gm. of enzyme).

Stability-The cocarboxylase activity in the crude extract as well as in Fractions A and B was not impaired by heating at 55” for 5 minutes, in- cubation at 37” for 10 hours, or storage at - 10” for several weeks. How- ever, heating for 3 minutes at 80” destroyed all the activity. Lyophiliza- tion had no effect on the potency, although the lyophilized crude extract did not completely redissolve.

Michaelis Constant of Enzyme-Substrate Complex-This was determined over a substrate concentration range from 6.68 X W4 to 6.68 X W3 M.

The reaction rate remained constant for only 2 to 3 minutes at pyruvate

LIMITING COMPONENT

- COCARBOXYLASE I

07 I I I 0 2.5 5 IO

COCARBOXYLASE (X lO-4 M) MO& (X 10-3M)

FIG. 3. Reconstructed pyruvic carboxylase of A. suboxydans. The system con- tained 50 PM of pyruvic acid, 0.067 M phosphate buffer, and 1.0 mg. (in terms of pro- tein content) of Fraction C; pH 6.0; total volume, 3.0 ml.

concentrations below 1.67 X W3 M, and hence the rate during the first 2 minutes was used in computation. With the method of Lineweaver and Burk (21), K, was found to be approximately 7.4 X 1W4 mole per liter and V,,,. to be 1.48 j.bM per minute (see Fig. 2). The Michaelis constant of this enzyme is thus much smaller than that of the yeast enzyme (approxi- mately 3 X 1c2 (3)).

InhiBitors-It has been shown that yeast pyruvic carboxylase contains sulfhydryl groups. Sulfhydryl reagents were therefore tested against the carboxylase of A. suboxyduns. Cu ++, Ag+, Hg*, and p-chloromercuri- benzoic acid, as shown in Table III, were very powerful inhibitors when tested with Fraction B. Iodosobenzoic acid, which is considered as a selective oxidizing agent for sulfhydryl, was not as potent as the heavy metals. Acetaldehyde was a strong inhibitor of pyruvate breakdown. This may be responsible for the rapid retardation of the rate of decomposi- tion mentioned above. Formaldehyde showed much less inhibition.

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T. E. KING AND V. H. CBELDELIN 827

Resolution of Pyruvic Carboxylase

Attempts to separate the prosthetic group from carboxylase in Fractions A and B by prolonged dialysis at pH 6.0, or by other methods previously employed (22, 23)) were unsuccessful. However, the following modified procedure effectively resolved the enzyme.

31 ml. of saturated (NH&SO, solution, which had been adjusted to pH 8.4 with ammonium hydroxide, were slowly added to 100 ml. of the diluted Fraction A (protein concentration at about 1 mg. per ml.) at 0”. The mixture was stored at that temperature for 30 minutes. The precip- itate was centrifuged and discarded.

TABLE III

Inhibition of Pyruvic Carboxylase of A. suboxydans

Inhibitor

cusoc “

AgNOa: : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : Hg(NO&

“ . . . . p-Chloromercuribenzoic acid.

“ C‘ ..,............. Iodoaobenzoic acid. Acetaldehyde.

L‘ . . . . . . . . . . . Formaldehyde.

“ .

mole per 1. ger cent

0.002 95 0.0002 80 0.0002 90 0.002 100 0.0002 95 0.002 100 0.0002 95 0.002 50 0.002 10 0.02 65 0.008 5 0.08 50

Inhibition of COn production

The conditions were the same as in Fig. 1, except that 0.067 M phosphate buffer was used; pH 6.0.

To 120 ml. of the clear supernatant solution were slowly added 60 ml. of a saturated (NH&S04 solution at pH 8.4. After standing for 30 minutes at O”, the precipitate was separated by centrifugation and dissolved in 100 ml. of water. 40 ml. of cold acetone were carefully added to the aqueous solution at O-5”. The precipitate was collected by centrifugation and redissolved in about 20 ml. of water. It was dialyzed in a rotating, “tilt over” cellophane bag against 0.01 M phosphate buffer at pH 8.4 for 3 hours at 0”. This was called Fraction C. The preparation could be stored in the frozen condition or lyophilized without loss of activity.

Recmastructed Pyruvic Carboxylase-Fraction C possessed no decarbox- ylating power. The activity was, however, restored by the addition of Mg++ and cocarboxylase. The effect of various concentrations of these factors is shown in Fig. 3. The optimal requirements of both (about 7.5

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828 PYRUVIC CARBOXYLASE OF A. SUBOXYDANS

X W3 M MgCl, and 8 X 1O-4 M cocarboxylase) were much higher in the present system than in yeast pyruvic apocarboxylase (23).

Action of Bivabnt Cations Other Than MgH-Many Mg++-requiring en- zymes can use Mntt- and other bivalent cations. This was also true of the present enzyme. MgH could be replaced by Co++, Mn++, Zn++, Cd++, Ca++, and Fe++. Under the conditions tested in Fig. 3 (4 to 5 X NY3 M),

Co* and Mntc- were slightly more effective than Mg++. The activity of Ca* and Fe++ was much lower than that of Mg+t, whereas Ba* was in- active.

TABLE IV

Comparison of Substrate Specificity of A. suboxydans and Yeast Pyruvic Carboxylase

Yea& Acid added

Kobayasi (24) lGreen et al. (23)

Pyruvic. . a-Ketobutyric. . cu-Ketoglutaric cu-Ketoisovaleric. or-Ketoisocaproic Oxalacetic Phenylpyruvic

.

1.00 0.75 0.00 0.00 0.00

t 0.00

1.00 0.80 0.04 0.26

0.54 0.00

1.00

0.01 0.88 0.05 0.32

* Recalculated from the data of Green el al. (23) and Kobayasi (24). A direct quantitative comparison is not possible because of differences in test conditions. The numbers represent relative rates of decarboxylation compared to the rate with pyruvic acid.

t See Fig. 4.

Substrate SpeciJcity

Yeast pyruvic carboxylase can decompose a number of keto acids other than pyruvate (23, 24). In contrast, the enzyme from A. suboxydans did not attack ar-ketoglutarate, phenyl pyruvate, a-ketoisovalerate, cu-ketoiso- caproate, or a-keto-P-methyl valerate. As shown in Table IV, the present preparation was apparently more specific in its activity than the yeast preparations of Green et al. (23), which were judged to be highly purified.

Fraction B decarboxylated Lu-ketobutyrate to give 1 mole of COz per mole of the substrate. The initial rate was 75 per cent of that of pyruvate decarboxylation. A boiled sample of Fraction B did not attack ol-keto- butyric acid. The product was identified as propionaldehyde by its 2,4- dinitrophenylhydrazone, m.p. 156O.l The melting point of an authentic

1 One sample of the 2,4-dinitrophenylhydrazone of the enzymatic decarboxylation product melted at 12%131”, even after three recrystallizationa from alcohol. How-

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T. E. KING AND V. H. CHELDELIN 829

sample was 154”, and the mixed melting point was 156”. The 2,4-dinitro- phenylhydrazone of the starting material melted at 202-204”.

Both Green et al. (23) and Kobayasi (24) have reported significant de- carboxylation of oxalacetate by yeast ‘pyruvic carboxylase. Oxalacetate is also decomposed by A. suboxydans carboxylase, as is seen in Fig. 4. The total yield of COZ was about two-thirds that from pyruvate breakdown over the first 15 minutes (Curve B). Much of this yield, but not all, could be expected from the non-enzymatic decarboxylation of oxalace- tate in Curve C, followed by further breakdown of the pyruvate pro- duced. However, the CO2 produced in Curve B should hardly be ex- pected to exceed twice that in Curve C, even in view of the removal of

-0 3 6 9 I2 I5 MINUTES

FIG. 4. Decarboxylation of oxalacetate by pyruvic carboxylase in A. suboxudans. The conditions were the same as in Table III.

pyruvate and possible further enhancement of the non-enzymatic de- carboxylation. This is particularly evident during the first few minutes. It therefore appears that some decarboxylation of oxalacetate per se oc- curred with the present pyruvate enzyme. Whether this was an (Y or /3 decarboxylation (1) cannot be told from the data at hand.

Although the present study does not provide proof of the normal path- way of pyruvate oxidation in A. suboxyduns, it strongly suggests that in the intact cells the oxidation proceeds through a preliminary decarboxylation to form free acetaldehyde as an intermediate. This behavior is unusual among bacteria; the principal examples are Acetobacter species and related pseudomonads, e.g. Pseudomonas lindneri (5, 25). In A. suboxydans, this

ever, the X content (Dumas method) agreed with the calculated value of 2,4- dinitrophenylhydrazone of propionaldehyde (found, 23.50 percent; calculated, 23.53). The reason for the lower melting point is unknown.

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830 PYRUVIC CARBOXYLASE OF A. SUBOXYDANS

may likely be associated with the failure to form acetyl phosphate or acetyl CoA, or to otherwise metabolize acetate.

SUMMARY

Pyruvate oxidation in cell-free extracts of Acetobacter suboxydans has been shown to consist of a preliminary decarboxylation step, followed by oxidation. The decarboxylation product is free acetaldehyde. Acetoin is also formed in small amounts.

Pyruvic carboxylase has been separated from the oxidative enzymes. The turnover rate of the purest fractions prepared was above 150 (moles of CO2 per minute per 100,000 gm. of enzyme). Such preparations readily catalyzed the breakdown of cu-ketobutyrate and were slightly active upon oxalacetate. However, they had no effect upon ol-ketoglutarate, oc-keto- isovalerate, or-ketoisocaproate, oc-keto-p-methyl valerate, or phenyl pyru- vate.

The enzyme has been successfully resolved to reveal a dependence upon cocarboxylase and Mg* or other divalent ions.

The technical assistance of Marion MacDonald and Gwendolyn C. Ca- hill is gratefully acknowledged, as well as the kindness of Dr. C. A. Storvick and her associates in performing the thiamine determinations.

BIBLIOGRAPHY

1. Vennesland, B., in Sumner, J. B., and Myrback, K., The enzymes, New York, 2, 183 (1951).

2. Green, D. E., Science, 116, 661 (1952). 3. Ochos, S., Physiol. Rev., 31, 56 (1951). 4. Dolin, M. I., and Gunsalus, I. C., J. &et., 62, 199 (1951). 5. Kobel, M., and Neuberg, Cl., in Klein, G., Handbuch der Pflanzenanalyse, Vienna,

4, 1253 (1933). 6. King, T. E., and Cheldelin, V. H., J. Biol. Chem., 198,127 (1952). 7. King, T. E., and Cheldelin, V. H., Biochim. et biophys. acta, in press. 8. Friedemann, T. E., J. Biol. Chem., 123, 161 (1938). 9. Barker, S. B., and Summerson, W. H., J. Biol. Chem., 138,535 (1941).

10. Donnally, L. H., IncZ. and Eng. Chem., Anal. Ed., 6, 91 (1933). 11. Happold, F. C., and Spencer, C. P., Biochim. et biophys. acta, 8, 18 (1952). 12. Neuberg, C., and Strauss, E., Arch. Biochem., ‘7, 211 (1945). 13. Westerfeld, W. W., J. Biol. Chem., 161,495 (1945). 14. Cheldelin, V. H., Bennett, M. J., and Kornberg, H. A., J. Biol. Chem., 166, 779

(1946). 15. Friedemann, T. E., and Kmieciak, T. C., J. Lab. and Clin. Med., 28, 1262 (1943). 16. Price, V. E., and Levintow, L., Biochem. Preparations, 2, 22 (1952). 17. Umbreit, W. W., Burris, R. H., and Stauffer, J. F., Manometric techniques and

tissue metabolism, Minneapolis, 175 (1949). 18. Herbst, R. M., and Shemin, D., Org. Syntheses, ~011. 2, 519 (1943). 19. van der Sleen, M. G., Rec. tram. chim. Pays-Bus, 21, 209 (1902).

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20. Meister, A., J. Biol. Chem., 197, 309 (1952). 21. Lineweaver, H., and Burk, D., J. Am. Chem. Sot., 66,658 (1934). 22. Kubowitz, F., and Ltittgens, W., Biochem. Z., 307,170 (1941). 23. Green, D. E., Herbert, D., and Subrahmanyan, V., J. Biol. Chem., 138,327 (1941). 24. Kobayasi, S., J. Biochem., Japan, 33, 301 (1941). 25. De Moss, R. D., J. Cell. and Comp. Physiol., 41, suppl. 1, 207 (1953).

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Tsoo E. King and Vernon H. CheldelinACETOBACTER SUBOXYDANSPYRUVIC CARBOXYLASE OF

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