THE OXIDATION OF TARTARIC ACID BY AN ENZYME SYSTEM OF MITOCHONDRIA*

BY ERNEST KUNt AND MARIO GARCIA HERNANDEZS

(From the Institute for Enzyme Research, University of Wisconsin, Madison, Wisconsin)

(Received for publication, May 25, 1955)

The r81e of certain hydroxy acids such as malic and lactic acids in ani- mal, plant, and microbial metabolism is generally recognized. It was shown recently that mammalian liver contains a specific enzyme which oxi- dizes another hydroxy acid, glycolic acid (1). A similar glycolic acid oxi-

dase was found in plant tissues (2). Relatively little is known, however, about the metabolism of a number of other hydroxy acids, such as tartaric, dihydroxyfumaric, hydroxypyruvic, and tartronic acids. Banga and Szent- Gyiirgyi (3) and Banga and Philippot (4) extracted dihydroxyfumaric acid oxidase from plants. The oxidation product was believed to be diketosuc- cinic acid, although its identity has not been established. Further infor- mation on certain enzymatic reactions of dihydroxyfumaric and diketo- succinic acids in plant tissues was recently obtained by Stafford, Magaldi, and Vennesland (5). The r&e of these acids in animal metabolism was hitherto only suggested but not proved by experimental work. As early

as 1912 Parnas and Baer (6) proposed a series of reactions, presumably occurring in muscle, which involved CO2 fixation into hydroxypyruvic acid to yield the keto form of dihydroxyfumaric acid. The experimental evi- dence for such a reaction was, however, missing. Stepanow and Kusin (7) observed glycogen synthesis in muscle from dihydroxyfumaric acid. This work was extended to hydroxypyruvic acid by Akabori, Uehara, and Muramatsu (8) who were primarily interested in sugar synthesis in muscle tissue.

The purpose of this paper is to describe an enzyme system which acts upon tartrate. The emphasis is on the qualitative and quantitative char- acterization of the sequence of enzymatic reactions, while the components of the enzyme system are the subject of further studies.

* Part of this work was presented before the annual meeting of the American Society of Biological Chemists, April 12, 1955, San Francisco, California. The work was supported in part by a grant from the United States Public Health Service and in part by a grant from Eli Lilly and Company (the latter issued to E. K. in 1953).

t Special Research Fellow, United States Public Health Service. $ During the tenure of a scholarship of the International Institute of Education.

From the National Polytechnic Institute of Mexico.

201

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202 TARTRATE OXIDATION

Results

Occurrence and Properties of Enzyme System-The tartrate oxidizing en- zyme system was found in the mitochondrial fraction of rat and beef tissue homogenates of all organs so far investigated. Conditions for enzymatic oxidation of tartrate are established when diphosphopyridine nucleotide (DPN) and Mg++ (or Mn++) are added to a suspension of sucrose mito- chondria with D( -)- or meso-tartrate as substrate. In intact mitochondria the cytochrome system is utilized as the terminal electron transfer mecha- nism to molecular oxygen. Highest rates of 02 uptake are obtained when the reaction is carried out at pH 8 to 8.3. The enzyme system was brought

TABLE I

Comparison of “Apparent Tartaric Acid Oxidase” Activity of Various Tissues

Tissue Activity, pl. 0~ per mg. protein per hr.

DC-)- meJo-

Rat liver Mw “ kidney Mw “ brain Mw. _. “ heart “

Beef heart Mw extract. Rat liver Mw extract.

10.0 4.6 16.4 9.6 6.2 3.1

20.0 11.4 74.0 106.0 19.2 30.8

The manometric test system contained in a volume of 3 ml. 30 pmoles of tartrate, 50 pmoles of Tris, pH 8.3, 15 pmoles of Mg++, and 1 mg. of DPN. With intact mito- chondria (Mw) 2 mg. of cytochrome c and in extracts 2 mg. of phenazine methonium sulfate were used as the carrier. Temperature, 37”; gas phase, air; center well, 0.1 ml. of 20 per cent KOH + filter paper. Time of reaction, 60 minutes.

into solution by either of two methods. Mitochondria prepared by the isotonic sucrose procedure of Schneider (9) were treated with cold (- 10”) acetone as described by Drysdale and Lardy (10). The resulting dry powder was stirred for 1 hour at 0” with 0.1 M tris(hydroxymet,hyl)amino- methane (Tris) buffer of pH 8.3 and centrifuged (6000 X g for 30 minutes). As a routine t’he extract of 1 gm. of acetone powder with 10 ml. of buffer, containing 5 to 10 mg. of protein per ml., was used in most experiments. The enzyme system was also made soluble by extraction of freshly prepared mitochondria according to a method described by Kun (11). The resulting extract was frozen and dried in vacua after high speed centrifugation. When mitochondrial extracts were tested for “tartaric acid oxidase” ac- tivity, besides DPN and Mg++ a carrier dye, phenazine met.honium sulfate, was required as a link to molecular oxygen.

Table I summarizes comparative data of “tartaric acid oxidase” activi-

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E. KUN AND M. GARCIA HERNANDEZ 203

ties of various tissues as well as of mitochondrial extracts as determined by the manometric procedure. The term “apparent tartaric acid oxidase” is merely used for convenience and indicates the sum total of several con- secutive reactions measured under conditions in which maximal O2 uptake is obtained. When intact mitochondria are in contact with the DC-) or meso form of tartaric acid, the D(-) form is oxidized at a greater rate. Aging of mitochondria, treatment with HzO, or extraction of the enzyme system from the particulate form into solution reverses the apparent affinity, and in each case meso-tartrate is oxidized preferentially. This difference between the structurally bound and the soluble system may be explained by selective permeability of mitochondria. n(+)-Tartrate is not oxidized by any of the preparations. The tartrate oxidizing enzyme system is fairly stable; its activity, which diminishes to 50 to 60 per cent in 1 to 2 days (in an ice bath), can be almost completely restored by the addition of Mg++.

Products of Tartrate Oxidation-Analyses of the deproteinized reaction mixtures resulted in the characterization of a number of keto acids, which were isolated as the 2,4-dinitrophenylhydrazine derivatives by means of a chromatographic procedure, described under “Methods” and reported in detail in another publication (12). The same products were formed by either intact mitochondria or extracts.

The identity of the keto acids was based upon the following criteria: (a) Rechromatography of a mixture of the synthetic pure compound to- gether with the derivative isolated from enzyme incubation did not result in separation into two components. (b) The catalytic reduction of the 2,4-dinitrophenylhydrazine derivatives of both samples yielded the same amino acids, which were identified by the chromatographic method as de- scribed by Levy and Chung (13).

The chromatographic separation of the 2,4-dinitrophenylhydrazine de- rivatives of the keto acids formed during the enzymatic oxidation of tartrate is shown in Fig. 1. Compound 1 is the 2,4-dinitrophenylhydrazone of oxaloglycolic acid (the keto form of dihydroxyfumaric acid). Both the synthetic and enzymatic products yield upon hydrogenolysis hydroxyas- partic acid as the major amino acid and small amounts of serine, the latter as the result of decarboxylation. Both enzymatically formed and synthetic products were decarboxylated by refluxing in water at 100’ for 1 hour to the bis-2,4-dinitrophenylhydrazine derivative of hydroxypyruvic acid. Compound 2 is the bis-2,4-dinitrophenylhydrazone (osazone) of diketosuccinic acid, which was also identified as diaminosuccinic acid. Compound 3 is the 2,4-dinitrophenylosazone of hydroxypyruvic acid, which upon reduction yielded a small amount of alanine and 2,3-diaminopro- pionic acid as the major product. Compound 4 separated into two com-

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204 TARTRATE OXIDATION

ponents which were identified as the isomers of the 2,4-dinitrophenylhy- drazone of glyoxylic acid. The two components, also obtained with pure synthetic 2,4-dinitrophenylhydrazone of glyoxylic acid, were eluted sepa- rately and converted to the corresponding amino acid, which was glycine. The cis and trans isomers of oxaloglycolic acid 2,4-dinitrophenylhydrazone did not occur, provided all chemical operations were kept below 50”.

The quantitative distribution and the rate of keto acid formation from tartrate are reported in Table II and Fig. 2. Oxygen consumption, CO, evolution, and glyoxylic acid production from both D(-)- and meso-tar- trate are increased by Mgt+, while a simultaneous diminution of oxalo-

.6 - \4 /

.8 -

v .

1.0 -

FIG. 1. The distribution of keto acid derivatives on a descending chromatogram. Relative values (RF) were determined by taking the alkaline decomposition product of 2,4-dinitrophenylhydrazine as RF = 1 (lowest line). Compound 1 is oxalogly- colic, Compound 2 diketosuccinic, Compound 3 hydroxypyruvic, Compounds 4 and’ 5 glyoxylic acid derivatives (see the text).

glycolate takes place. Hydroxypyruvate from meso-tartrate is doubled in the presence of M&+ (Experiment A). Further studies dealing with the stability of keto acids produced during the enzymatic oxidation of tartrate revealed that diketosuccinate is non-enzymatically decarboxylated to tar- tronate, which was identified as its oxidation product, i.e. mesoxalic acid. This was isolated as the 2,4-dinitrophenylhydrazone. As shown in Table III, ethylenediaminetetraacetate (Versene) retards this very rapid decar- boxylation. When Versene is present during the enzymatic oxidation of tartrate (Table II, Experiment B), oxaloglycolate and glyoxylate accumu- late, while CO2 evolution diminishes, as determined in other experiments. The metal ion-catalyzed decomposition of diketosuccinate thus markedly decreased the yield of total keto acids from tartrate. It was found in ex- periments with intact mitochondria (oxidation via the cytochrome system) that t’he main sequence of enzymatic reactions is the oxidation of tartrate

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E. KUN AND M. GARCIA HERNANDEZ 205

to oxaloglycolate, which then yields glyoxylate, while the oxidation to diketosuccinate and its decarboxylation to tartronate are considered as probably non-enzymatic side reactions. An additional interfering reaction

TABLE II

Distribution of a-Keto Acids Produced from Tartrate (in pmoles)

Keto acid No Mg++ + Mg++

Oxaloglycolic. .......... Diketosuccinic ......... Hydroxypyruvic ....... Glyoxylic ..............

Total keto acids. ..... 01 absorbed. ........ CO, produced. .......

Experiment A

xl-)- meso- ____~

0.196 0.378 0.018 0.026 0.006 0.010 0.037 0.072

n(-)- meso-

0.072 0.154 0.008 0.014 0.008 0.022 0.090 0.182

0.257 0.486 0.178 0.372 0.535 1.680 1.780 2.760 0.450 0.870 1.45 3.000

Experiment R

No Versent

0.550 0.026 0.008 0.109

0.693 1.300

0.684 0.028 0.018 0.278

1.008 1.300

Time of reaction, 15 minutes; temperature, 30”. In Experiment B no Mg++ was added; the substrate was meso-tartrate. Conditions as described in Table I. Each flask contained 0.5 ml. of extract of beef heart mitochondria (4 to 7 mg. of protein),

.400 RATE OF KETOACID FORMATION FROM DC-) TARTRATE

30 60 90 120 TIME IN MINUTES

FIG. 2. The enzyme system was the same as that described in Table II. Sub- strate, D(-)-tartrate; 15 @moles of MgSOd were added. Temperature, 30”.

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206 TARTRATE OXIDATION

which obscures the balance of products is the secondary oxidation of glyox- ylate by HzOz, as discussed elsewhere (1, 2). Analyses for simultaneous disappearance of tartrate showed that 1.4 to 1.6 pmoles of tartrate were used up while 1 pmole of O2 was absorbed. Unfortunately the analytical method for D( -)-tartrate by means of its complex formed with metavana- date (14) is not sufficiently accurate and sensitive to permit us to rely on it beyond an error of f20 per cent.

It is interesting, but as yet unexplained, that the stoichiometric mixture of Versene + MgH (final concentration KF2 M) caused complete inhibition of tartrate oxidation.

TABLE III

Decomposition of Diketosuccinate at pH 8.8

Time

In Tris

Amount recovered

In Tris + Versene (10-Z Y)

mh.

5 10 15 20 30

per cwt per cent

20 -35 43-50 l-5 X6-40 O.l- 0.5 2730 0 15 0 12

1 pmole of diketosuccinate was added at zero time, and the remaining keto acid was analyzed at the indicated times. The volume of reactants and the amount of Tris buffer were the same as in the enzymatic experiments. Temperature, 30”. No Mg++ was added.

Reversibility of Dehydrogenation of Tar&ate-Studies concerned with the reversibility of the reactions catalyzed by the tartrate oxidizing enzyme system were confined to the first step of dehydrogenation to oxaloglycolate. The reduction of oxaloglycolate was measured as follows: crystalline yeast alcohol dehydrogenase (130,000 units) (15) was incubated at 30” for 10 minutes with 2 mg. of DPN and 0.05 ml. of 95 per cent ethanol in the pres- ence of 20 pmoles of Tris buffer (pH 8.3) and 0.5 ml. of an aqueous extract of an acetone powder of beef heart mitochondria. After this preincuba- tion, which insured complete reduction of DPN, 0.5 ml. of a freshly pre- pared solution of oxaloglycolate containing 6 pmoles of the partly neu- tralized acid was added. The pH of the final reaction mixture was 7.0. The enzyme mixture was again incubated for 10 minutes, then depro- teinized with 2 ml. of 10 per cent HC104, and the filtrate analyzed for keto acids and tartrate. The result,s were compared with those of an identical enzyme reaction mixture which was deproteinized at zero time; i.e., just

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E. KUN AND M. GARCIA HERNANDEZ 207

at the moment oxaloglycolate was added. During 10 minutes incubation, 0.344 pmole oxaloglycolate was reduced. The rate of the reverse reaction is of the same order of magnitude as the oxidation of tartrate to oxalogly- colate (compare with Table II). Only traces of hydroxypyruvate (0.006 pmole) and no glyoxylate at all were found. Neither alcohol dehydrogen- ase nor the extract of mitochondria reduced glyoxylate to glycolate in the presence of DPNH. The color reaction of tartrate could not be quan- titatively evaluated in this range; however, the appearance of the red metavanadate complex (absorption maximum at 505 rnp) of one of the optically active enantiomorphs clearly indicated that tartrate was actually formed.

Methods

Chemical Preparations-Dihydroxyfumaric acid was made by a modified procedure of Fenton (16) as described by Nef (17). The method recently published by Hartree (18) is essentially the same as that of Nef and does not appear to offer any advantages over that of the older paper. Hydroxy- pyruvic acid was prepared from chloropyruvic acid as described by Sprin- son and Chargaff (19). Mesoxalic and diketosuccinic acids were obtained from the Aldrich Chemical Company, Inc., both as free acids and as the sodium salt. The properties of the acids agreed closely with those de- scribed by Fenton (20). Glyoxylic acid was obtained by the reduction of oxalic acid as described by Weinhouse and Friedmann (21). The 2,4- dinitrophenylhydrazine derivatives were prepared by dissolving the keto acids in an excess of the reagent (in 2 N HCl). After standing for 2 hours at room temperature, the mixture was kept at 4” overnight, and the crys- tals were removed by filtration, washed with 2 N HCl, and dried. Under such conditions the 2,4-dinitrophenylhydrazone of oxaloglycolic (from dihydroxyfumaric) and glyoxylic acids was obtained. Dihydroxyfumaric acid was quantitatively recovered as the 2,4-dinitrophenylhydrazone of the keto form from an aqueous solution. Diketosuccinic and hydroxypyruvic acids under these conditions yielded quantitatively the bis-2,4-dinitrophen- ylhydrazone (osazone).

Analytical Methods-Each derivative was purified by descending chro- matography on What’man No. 1 filter paper, which was previously impreg- nated with 0.1 M potassium phosphate buffer of pH 7.3 and dried. The solvent was ethanol (83 per cent) and Hz0 (17 per cent). The chamber was equilibrated with 95 per cent ethanol. For the quantitative analyses of the keto acid derivatives the 2,4-dinitrophenylhydrazones were eluted from the paper and determined spectrophotometrically (12).

The reduction of W,4-dinitrophenylhydraxones to amino acids was carried out, as follows: Approximately 3 to 5 mg. of the 2,4-dinitrophenylhydrazine

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208 TARTRATE OXIDATION

derivative were dissolved in 30 ml. of glacial acetic acid, 1 mg. of platinum oxide catalyst was added, and the mixture vigorously shaken in Hz atmos- phere for 3 to 6 hours at room temperature. After the uptake of Hz ceased, the solution was filtered and dried in vacua. The residue was taken up in a small volume (0.05 to 0.1 ml.) of glacial acetic acid and quantita- tively applied to a sheet of Whatman No. 1 filter paper and chromato- graphed (descending technique) (13). The amino acid was located by the ninhydrin reagent on a small strip of the total chromatogram, and the rest was purified by reextraction and further chromatography.

DISCUSSION

The results of experiments obtained with the tartrate oxidizing enzyme system suggest a definite pattern of consecutive enzymatic reactions. There is little doubt that the first step consists of the dehydrogenation of t,artrate to oxaloglycolate. This type of dehydrogenation reminds us of the reaction catalyzed by malic dehydrogenase. Malic dehydrogenase is specific for the naturally occurring L(-)-malate (22) and does not act on dihydroxyfumarate, while the “tartrate dehydrogenase” acts on D( - )- and meso-tartaric acids. Malic dehydrogenase can react with both di- and triphosphopyridine nucleotides (23) as coenzymes; on the other hand, tartaric acid dehydrogenation occurs only in the presence of DPN. Var- ious preparations of purified malic dehydrogenase of the type described by Straub (24) oxidize to a small and varying degree D(-)- and meso- tartrate; however, these preparations contain other enzymes (transami- nases) and cannot be considered suitable for experiments which are to de- cide whether or not malic dehydrogenase acts on both substrates or, alternatively, whether different dehydrogenases are involved.

The main pathway of the enzymatic degradation of oxaloglycolic acid leads to the formation of glyoxylic acid. The formation of diketosuccinate was experimentally demonstrated, but we do not consider this reaction to be on the main path to glyoxylate. It should be mentioned that besides Ranga, Szent-Gyorgyi, and Philippot (3, 4) who worked with plant ex- tracts, Swedin and Theorell (25, 26) and more recently Chance (27) also studied the enzymatic oxidation of dihydroxyfumaric acid by purified horseradish peroxidase. This reaction is catalyzed by a Mn++-activated peroxidase-peroxide complex (27) and occurs under different conditions (pH 4.7) from the oxidation of tartaric acid (pH 8.3). According to Chance (27) the oxidation of dihydroxyfumaric acid by the peroxidase-peroxide complex requires Mn++, while the formation of glyoxylate from tartrate goes equally well with Mg++, which was used exclusively in all our experi- ments.

While diketosuccinate decomposes non-enzymatically to CO2 and tar-

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E. KUN AND M, GARCIA HERNANDEZ 209

tronate, glyoxylate appears only in the presence of the enzyme system, DPN, and tartrate as substrate. Dihydroxyfumarate is enzymatically re- duced to tartrate (in the presence of continually regenerated DPNH), but it does not yield glyoxylate. This observation suggests that the precursor of glyoxylate is not the dienol but the keto form of dihydroxyfumarate, i.e., oxaloglycolate. This question could not be clarified by the identifica- tion of oxaloglycolate as its 2,4-dinitrophenylhydrazone, since both the synthetic dienol and the enzymatically formed product yielded the same derivative upon prolonged (24 hours) incubation with the carbonyl reagent. On the other hand, it is known that dihydroxyfumarate is a- relatively

I

$oo-

II: m coo- coo-

HtOH I

3 H:OH -6H

koo-

(+3 DPNj3 Hf:H M9

++

too- \

H

-co2cM/\2H yH20H 700- coo-

Y0 F0 - co2 H&OH

coo- F0 Mg++ coo-

coo-

m Y PL

I=MESO-TARTRATE IZ=HYDROXYPYRUVATE

IE=OXALOGLYCOLATE Y=DIKETOSUCCINATE

llI=GLYOXYLATE PI=l-ARTRONATE

FIG. 3

stable dienol (18) and thus may not be acted upon by an enzyme which is specific for the keto form. It is suggested that glyoxylate is formed from oxaloglycolate (which is the primary product of the dehydrogenation of tartrate) by an intramolecular transfer of H, followed by cleavage between CZ and CI.

Alternative possibilities of glyoxylate formation via the oxidative de carboxylation of hydroxypyruvate or tartronate were ruled out by experi- ments which showed that under given conditions (during tartrate oxida- tion) both added hydroxypyruvate and tartronate were inert and yielded no glyoxylate. Concomitantly with this major pathway of glyoxylate formation, a small but consistent decarboxylation of oxaloglycolate to hy- droxypyruvate occurs. It is not certain that this reaction is catalyzed by an enzyme. The summary of major reactions is schematically outlined in Fig. 3, which merely represents a qualitative pathway.

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210 TARTRATE OXIDATION

It is hardly possible at present to predict whether or not the sequence of reactions outlined in this paper represents a major pathway of metabo- lism. The ubiquitous appearance of the enzyme reactions may indicate this. On the other hand, an interesting possibility may be suggested, that transaminations of some of the keto acids formed from tartrate result in amino acids which, in small amounts, could serve as physiological regu- lators of cell metabolism, as antimetabolites. Such examples have been described; e.g., diaminopropionic acid is a constituent of the antibiotic viomycin (28), and hydroxyaspartic acid as well as diaminosuccinic acid is known to inhibit the growth of Escherichia coli (29). These possibilities in animal, plant, and microbial metabolism as well as the characterization of the enzyme components of the tartrate oxidizing enzyme system are now under investigation.

SUMMARY

1. Suspensions of mitochondria prepared from various tissues of rat and beef oxidize D(-)- and meso-tartrate. The enzyme system was brought into solution by extraction. The reaction required DPN; added Mg++ in- creased 02 uptake and CO2 evolution.

2. The products of the enzymatic oxidation were isolated and identified as (a) oxaloglycolate, which yields (b) glyoxylate. The formation of (c) diketosuccinate, (d) hydroxypyruvate, and (e) tartronate was also estab- lished.

3. The reduction of oxaloglycolate to tartrate was demonstrated. The sequence of reactions and their possible r61e in cell metabolism are dis- cussed.

We are greatly indebted to Professor H. A. Lardy for his continued interest. Our thanks are due to Dr. Helmut Beinert for a sample of malic dehydrogenase and diaphorase, and to Miss Patricia Broberg for a sample of crystalline alcohol dehydrogenase.

BIBLIOGRAPHY

1. Kun, E., Dechary, J. M., and Pitot, H. C., J. Biol. Chem., 210, 269 (1954). 2. Zelitch, I., and Ochoa, S., J. Riol. Chem., 201,707 (1953). 3. Banga, I., and Szent-Gyorgyi, A., Z. physiol. Chem., 266,58 (1938).

4. Banga, I., and Philippot, E., Z. physiol. Chem., 268,147 (1939). 5. Stafford, H. A., Magaldi, A., and Vennesland, B., Science, 120, 265 (1954). 6. Parnas, T., and Baer, T., Biochem. Z., 41,386 (1912). 7. Stepanow, A., and Kusin, A., Ber. them. Ges., 67,723 (1934). 8. Akabori, S., Uehara, K., and Muramatsu, J., Proc. Japan Acad., 28,41 (1952). 9. Schneider, W. C., in Umbreit, W. W., Burris, R. H., and Stauffer, J. F., Mano-

metric techniques and tissue metabolism, Minneapolis (1949). 10. Drysdale, G. R., and Lardy, H. A., J. Biol. Chem., 202, 119 (1953).

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E. KUN AND M. GARCIA HERNANDEZ 211

11. Kun, E., Proc. Sot. Exp. Biol. and Med., 77,441 (1951). 12. Kun, E., and Garcia Hernandez, M., Biochim. et biophys. acta, in press. 13. Levy, A. L., and Chung, D., Anal. Chem., 26,396 (1953). 14. Vaughn, R. H., Marsh, G. L., Stadtman, T. C., and Cantino, B. C., J. Bact., 62,

311 (1946). 15. Racker, E., J. Biol. Chem., 184,313 (1950). 16. Fenton, H. J. H., J. Chem. Sot., 71,375 (1897). 17. Nef, J. V., Ann. Chem., 367,290 (1907). 18. Hartree, E. F., Biochem. Preparations, 3,56 (1953). 19. Sprinson, D. B., and Chargaff, E., J. Biol. Chem., 164,417 (1946). 20. Fenton, H. J. H., J. Chem. Xoc., 73,71 (1898). 21. Weinhouse, S., and Friedmann, B., J. BioZ. Chem., 191, 707 (1951). 22. Green, D. E., Biochem. J., 30,2095 (1936). 23. Mehler, A. H., Kornberg, A., Grisolia, S., and Ochoa, S., Federation Proc., 6,

278 (1947). 24. Straub, B. F., 2. physiol. Chem., 276,63 (1942). 25. Swedin, B., and Theorell, H., Nature, 143,71 (1939). 26. Theorell, H., and Swedin, B., Naturwissenschaften, 27,95 (1939). 27. Chance, B., J. BioZ. Chem., 197,577 (1952). 28. Haskell, T. H., Fusari, S. A., Frohardt, R. P., and Bartz, Q. R., J. Am. Chem. Sot.,

74, 599 (1952). 29. Shive, W., and Macow, J., J. BioZ. Chem., 102,451 (1946).

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Ernest Kun and Mario Garcia HernandezMITOCHONDRIA

BY AN ENZYME SYSTEM OF THE OXIDATION OF TARTARIC ACID

1956, 218:201-211.J. Biol. Chem. 

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