Mitochondrial location of pyruvate carboxylase in Aspergillus niger

Pyruvate carboxylase has been found in the mitochondrial fraction of two strains of Aspergillus niger along with the marker enzymes of citrate synthase and cytochrome c oxidase. The location of pyruvate carboxylase in A. nidulans was, however, confirmed to be in the cytosolic fraction. The enzyme from the former sources was dependent upon the presence of acetyl-CoA for full activity; the enzyme from A. nidulans was unaffected by the presence or absence of acetyl-CoA.     

A multienzyme complex for CO2 fixation.

Acetyl-coenzyme A carboxylase from Euglena gracilis strain Z was isolated as a component of a multienzyme complex which includes phosphoenolpyruvate carboxylase and malate dehydrogenase. The multienzyme complex was shown to exist in crude extracts and was purified to a homogeneous protein with a molecular weight of 360,000 by gel filtration. The ratio of the activities of the constituent enzymes was acetyl-CoA carboxylase:phosphoenolpyruvate carboxylase:malate dehydrogenase, 1:25:500. The complex is proposed to operate in conjunction with malic enzyme, which is present in Euglena, to facilitate the formation of substrates, malonyl-CoA, and NADPH, for fatty acid biosynthesis. The interaction of the enzymes may represent a means of control of acetyl-CoA carboxylase activity in organisms which do not possess an enzyme subject to allosteric regulation. The acetyl-CoA carboxylase activity from Euglena is unaffected by citrate and isocitrate.     

Immunochemical studies with soluble and mitochondrial pyruvate carboxylase activities from rat tissues

1. Pyruvate carboxylase (EC 6.4.1.1), purified from rat liver mitochondria to a specific activity of 14 units/mg, was used for the preparation of antibodies in rabbits. 2. Tissue distribution studies showed that pyruvate carboxylase was present in all rat tissues that were tested, with considerable activities both in gluconeogenic tissues such as liver and kidney and in tissues with high rates of lipogenesis such as white adipose tissue, brown adipose tissue, adrenal gland and lactating mammary gland. 3. Immunochemical titration experiments with the specific antibodies showed no differences between the inactivation of pyruvate carboxylase from mitochondrial or soluble fractions of liver, kidney, mammary gland, brown adipose tissue or white adipose tissue. 4. The antibodies were relatively less effective in reactions against pyruvate carboxylase from sheep liver than against the enzyme from rat tissues. 5. Pyruvate carboxylase antibodies did not inactivate either propionyl-CoA carboxylase or acetyl-CoA carboxylase from rat liver. 6. It is concluded that pyruvate carboxylase in lipogenic tissues is similar antigenically to the enzyme in gluconeogenic tissues and that the soluble activities of pyruvate carboxylase detected in many rat tissues do not represent discrete enzymes but are the result of mitochondrial damage during tissue homogenization.     

Structural aspects of the dye-linked alcohol dehydrogenase of Rhodopseudomonas acidophila.

1. A dye-linked alcohol dehydrogenase was purified 60-fold from extracts of Rhodopseudomonas acidophila 10050 grown aerobically on ethanol. 2. The properties of this enzyme were identical with those of the alcohol dehydrogenase synthesized by this organism during growth on methanol anaerobically in the light, and they are judged to be the same enzyme. 3. The enzyme gave a single protein band, coincident with alcohol dehydrogenase activity, during electrophoresis on polyacrylamide gel. 4. The amino acid composition, ioselectric point, u.v. and visible absorption spectra of the enzyme were determined and compared with those of other similar enzymes. 5. The presence of 0.7--1.0 g-atom of non-haem, acidlabile iron/mol of enzyme was shown by atomic absorption spectrophotometry and colorimetric assay. The iron could not be dissociated from the enzyme by dialysis against chelating agents. 6. E.p.r. spectroscopy of the enzyme did not indicate any redox function for the iron during alcohol dehydrogenation, but showed a signal at g = 2.00 consistent with the presence of a protein-bound organic free radical. 8. Antisera were raised against alcohol (methanol) dehydrogenases purified from Rhodopseudomonas acidophila, Paracoccus denitrificans and Methylophilus methylotrophus. 9. The antiserum to the Rhodopseudomonas acidophila enzyme cross-reacted with neither of the two other antisera, nor with crude extracts of methanol-grown Hyphomicrobium X and Pseudomonas AM1, thus emphasizing its singular biochemical properties.     

The interaction between heme and protein in cytochrome c1

The hormonal regulation of two regulatory enzymes of fatty acid synthesis acetyl-CoA carboxylase (EC 6.4.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49), has been investigated in human diploid fibroblasts. There was a 35% increase in acetyl-CoA carboxylase activity, 72 h following addition of 10 microU/ml insulin to the culture medium. Addition of 1 microgram/ml of 3,3'5-triiodothyronine for 72 h resulted in an increase in acetyl-CoA carboxylase activity to 166% of the controls. The simultaneous addition of 1 microgram/ml triiodothyronine and 10 mU/ml insulin caused the enzyme activity to rise to 240% of the controls. A dose-dependent reduction in acetyl-CoA carboxylase activity was brought about by 1 X 10(-4) to 1 X 10(-3) M dibutyryl cyclic AMP. The earliest effect of dibutyryl cyclic AMP was observed within 24 h. Glucose-6-phosphate dehydrogenase followed qualitatively the same pattern of response, whereas the constitutive enzyme, lactate dehydrogenase (EC 1.1.1.27), did not show significant changes in these experiments. The data demonstrate common features of hormonal regulation of lipogenesis in human fibroblasts with liver and adipose tissue and substantiate the growing evidence that thyroid hormones are of major importance for the regulation of this process.     

Differential effect of clofibrate on acetyl-CoA carboxylase mRNA level in rat white and brown adipose tissue

Regulation of some lipogenic enzyme gene expression by clofibrate was studied in rat white and brown adipose tissue. In white adipose tissue the drug administration for 14 days to rats resulted in the increase in acetyl-CoA carboxylase, ATP-citrate lyase, and glucose 6-phosphate dehydrogenase mRNA levels. Opposing effect of clofibrate on the acetyl-CoA carboxylase, ATP-citrate lyase, and glucose 6-phosphate dehydrogenase mRNA levels was found in brown adipose tissue. These data indicate a tissue specificity of clofibrate action on lipogenic enzyme gene expression. The results presented in this paper provide further evidence that hypolipidaemia caused by the treatment with clofibrate cannot be related to the inhibition of fatty acid synthesis in white adipose tissue in rat.     

Lipogenesis at the suckling-weaning transition in liver and brown adipose tissue of the rat

The responses of rat hepatic and brown adipose tissue in vivo lipogenesis to premature (15 days) and normal (21 days) weaning have been correlated to changes in the activities of acetyl-CoA carboxylase and two NADPH-producing enzymes, malic enzyme and glucose-6-phosphate dehydrogenase. Both tissues show an induction of lipogenesis in response to weaning. In the liver, lipogenic flux is closely linked to the activity of acetyl-CoA carboxylase, but not necessarily that of malic enzyme or glucose-6-phosphate dehydrogenase, whereas no such dissociation between enzyme activity and flux rate occurs in brown adipose tissue. Thyroid hormones, implicated in many physiological changes around weaning, do not seem to play a primary role in the adaptation of lipogenesis to the dietary change at this time, although a permissive role in both tissues is possible.     

Nutritional and hormonal regulation of mRNA levels of lipogenic enzymes in primary cultures of rat hepatocytes.

The effects of nutrients and hormones on the mRNA levels of acetyl-CoA carboxylase, fatty acid synthase, malic enzyme, and glucose 6-phosphate dehydrogenase were examined in primary cultures of rat hepatocytes during the process of induction. The addition of both glucose and insulin to the culture medium markedly enhanced the lipogenic enzyme mRNA induction due to either of them, in 16 h. Fructose or glycerol proved to be an effective substitute for glucose, suggesting that glycolytic metabolites were involved in the mRNA induction. It is remarkable that mRNA induction of acetyl-CoA carboxylase was the most sensitive to glucose and also to insulin among the lipogenic enzymes. Polyunsaturated fatty acids markedly reduced the mRNA induction of lipogenic enzymes. Dexamethasone enhanced all the lipogenic enzyme mRNA induction by insulin. On the other hand, triiodothyronine addition greatly increased the mRNA concentrations of lipogenic enzymes, but dexamethasone decreased rather than increased the mRNA induction by triiodothyronine. The effects of insulin on the induction of the lipogenic enzyme mRNAs were similar, but those of triiodothyronine were not. Triiodothyronine markedly enhanced malic enzyme mRNA induction by insulin with dexamethasone, and tended to enhance the induction of the acetyl-CoA carboxylase and fatty acid synthase mRNAs, but not that of glucose 6-phosphate dehydrogenase mRNA. It appeared that insulin and triiodothyronine synergistically enhanced lipogenic enzyme mRNA induction by glucose, but the mechanisms were different.     

A 1H nuclear-magnetic-resonance study of the conformation and the molecular dynamics of the glycoprotein cow-colostrum trypsin inhibitor

1. One mitochondrial and one cytoplasmic malate dehydrogenase isoenzyme could be purified from acetate grown cells of the yeast Saccharomyces cerevisiae. 2. The purification procedure uses chromatography on dextran blue columns as an essential step for enrichment, and reverse ammonium sulfate chromatography on celite for isoenzyme separation. 3. The homogeneity of the preparations was established by gel electrophoreses in the presence of sodium dodecylsulfate and by a sedimentation run in the analytical ultracentrifuge. 4. Both enzymes are dimers with a molecular weight of 75 000 for the cytoplasmic and of 68 000 for the mitochondrial enzyme. 5. Amino acid analysis and peptide mapping showed that both enzymes are closely related, but genetically different (true isoenzymes). 6. The cytoplasmic enzyme shows electrophoretic splitting. This is most likely due to post-translational deamination in vivo. 7. Antibodies to both isoenzymes could be obtained in rabbits. The antisera to cytoplasmic malate dehydrogenase were specific for this enzyme. Antisera to mitochondrial malate dehydrogenase react with both isoenzymes. Neither type of antisera precipitated an inactive protein after the glucose-dependent inactivation of cytoplasmic malate dehydrogenase in vivo.     

Adaptation to a high protein, carbohydrate-free diet induces a marked reduction of fatty acid synthesis and lipogenic enzymes in rat adipose tissue that is rapidly reverted by a balanced diet

We have previously shown that in vivo lipogenesis is markedly reduced in liver, carcass, and in 4 different depots of adipose tissue of rats adapted to a high protein, carbohydrate-free (HP) diet. In the present work, we investigate the activity of enzymes involved in lipogenesis in the epididymal adipose tissue (EPI) of rats adapted to an HP diet before and 12 h after a balanced diet was introduced. Rats fed an HP diet for 15 days showed a 60% reduction of EPI fatty acid synthesis in vivo that was accompanied by 45%-55% decreases in the activities of pyruvate dehydrogenase complex, ATP-citrate lyase, acetyl-CoA carboxylase, glucose-6-phosphate dehydrogenase, and malic enzyme. Reversion to a balanced diet for 12 h resulted in a normalization of in vivo EPI lipogenesis, and in a restoration of acetyl-CoA carboxylase activity to levels that did not differ significantly from control values. The activities of ATP-citrate lyase and pyruvate dehydrogenase complex increased to about 75%-86% of control values, but the activities of glucose-6-phosphate dehydrogenase and malic enzyme remained unchanged 12 h after diet reversion. The data indicate that in rats, the adjustment of adipose tissue lipogenic activity is an important component of the metabolic adaptation to different nutritional conditions.     

Enzymatic activities of cytoplasmic and of microsomal acetyl-CoA carboxylase of rat epididymal adipose tissue; different regulatory effects of a short-term fasting and palmitoyl-CoA on these two enzymes

The existence of a microsomal acetyl-CoA carboxylase in the rat epididymal adipose tissue was demonstrated in vitro in the present study. Its specific activity was of the same order of magnitude as that of the cytoplasmic acetyl-CoA carboxylase. The effect of several experimental conditions on the enzymatic activities of both enzymes were tested; fasting for 24 hr strongly increased (2.5-4 times) the activity of the microsomal enzyme while the cytoplasmic enzyme remained unchanged. Palmitoyl-CoA (1 and 5 microM), an inhibitor of acetyl-CoA carboxylase, had a greater effect on the cytoplasmic (33 and 88% inhibition) than on the microsomal enzyme (0 and 37% inhibition).     

Diurnal variations of lipogenic enzymes, their substrate and effector levels, and lipogenesis from tritiated water in rat liver

The activities of glucose-6-phosphate dehydrogenase, malic enzyme, fatty acid synthetase and acetyl-CoA carboxylase (extracted with or without phosphatase inhibitor) in rat liver did not vary significantly during 24 h. The hepatic levels of glucose 6-phosphate and malate increased coordinately 3-6 h after the beginning (1900 h) of food intake and were high until morning, whereas the levels of acetyl-CoA and citrate peaked at 1900 h and then decreased. However, it is remarkable that the in vivo incorporation of 3H from tritiated water into fatty acids in liver increased with the level of malonyl-CoA after food intake. Comparing the substrate and effector levels with the Km and Ka values for the enzymes, the levels of acetyl-CoA, malonyl-CoA and citrate appear to limit the enzyme activities. It is suggested that, after food intake, the physiological activity of acetyl-CoA carboxylase was increased with the substrate increase and/or with the catalytic activation with citrate, and consequently, the fatty acid synthetase activity was also increased, whereas the enzyme activities measured under optimum conditions were not.     

Effects of dietary nutrients on substrate and effector levels of lipogenic enzymes, and lipogenesis from tritiated water in rat liver

When fasted rats were refed for 4 days with a carbohydrate and protein diet, a carbohydrate diet (without protein) or a protein diet (without carbohydrate), the effects of dietary nutrients on the fatty acid synthesis from injected tritiated water, the substrate and effector levels of lipogenic enzymes and the enzyme activities were compared in the livers. In the carbohydrate diet group, although acetyl-CoA carboxylase was much induced and citrate was much increased, the activity of acetyl-CoA carboxylase extracted with phosphatase inhibitor and activated with 0.5 mM citrate was low in comparison to the carbohydrate and protein diet group. The physiological activity of acetyl-CoA carboxylase seems to be low. In the protein diet group, the concentrations of glucose 6-phosphate, acetyl-CoA and malonyl-CoA were markedly higher than in the carbohydrate and protein group, whereas the concentrations of oxaloacetate and citrate were lower. The levels of hepatic cAMP and plasma glucagon were high. The activities of acetyl-CoA carboxylase and also fatty acid synthetase were low in the protein group. By feeding fat, the citrate level was not decreased as much as the lipogenic enzyme inductions. Comparing the substrate and effector levels with the Km and Ka values, the activities of acetyl-CoA carboxylase and fatty acid synthetase could be limited by the levels. The fatty acid synthesis from tritiated water corresponded more closely to the acetyl-CoA carboxylase activity (activated 0.5 mM citrate) than to other lipogenic enzyme activities. On the other hand, neither the activities of glucose-6-phosphate dehydrogenase and malic enzyme (even though markedly lowered by diet) nor the levels of their substrates appeared to limit fatty acid synthesis of any of the dietary groups. Thus, it is suggested that under the dietary nutrient manipulation, acetyl-CoA carboxylase activity would be the first candidate of the rate-limiting factor for fatty acid synthesis with the regulations of the enzyme quantity, the substrate and effector levels and the enzyme modification.     

Dissociation and characterization of enzymes from a multienzyme complex involved in CO2 fixation.

A multienzyme complex from Euglena, molecular weight about 360,000, containing phosphoenolpyruvate carboxylase, malate dehydrogenase, and acetyl-coenzyme A carboxylase has been dissociated into active constituent enzymes. The respective molecular weights are 183,000, 67,000, and 127,000. The malate dehydrogenase contained in the complex is electrophoretically distinct from other malate dehydrogenase isozymes found in Euglena. The K-m for HCO3minus of the free and complexed acetyl-CoA carboxylase is 4.2-5.4 mM, and the substrate dependency for acetyl-CoA describes a sigmoidal relationship. The HCO3minus K-m for the free phosphoenolpyruvate carboxylase is 7.3-5.4 mM while that for the same enzyme contained in the complex is 0.7-1.3 mM. Both the free and complexed forms ofphosphoenolpyruvate carboxylase have a K-m for phosphoenolpyruvate of 0.9-1.7 mM. The latter enzyme in both the complex and free forms is stimulated by NADH, acetyl-CoA, and ATP. In the free phosphoenolpyruvate carboxylase, the stimulation passes through a maximum depending on effector concentration. The effect of NADH is to increase V-max while K-m values remain unmodified.     

Changes in the enzyme pattern of the mammary gland of the lactating rat after hypophysectomy and weaning

1. The enzymes glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase, UDP-glucose pyrophosphorylase, phosphofructokinase, ATP-citrate lyase and acetyl-CoA carboxylase have been assayed in rat mammary glands in various stages of involution after hypophysectomy and weaning. 2. After hypophysectomy all seven enzymes decline in activity over a 12–16hr. period but the extent of the decline varies, with acetyl-CoA carboxylase becoming almost totally inactive, ATP-citrate lyase and phosphofructokinase showing a large decrease, and the remaining enzymes a less marked decline. 3. Within 24hr. of removing the litter a change in the pattern of enzyme activity is found very similar to that after hypophysectomy. 4. The significance of these results is discussed in relation to the endocrine control of mammary gland metabolism and the mechanisms of involution.     

Interactions between alpha-ketoisovalerate metabolism and the pathways of gluconeogenesis and urea synthesis in isolated hepatocytes

The mechanism of inhibition of pyruvate carboxylase, pyruvate dehydrogenase, and carbamyl phosphate synthetase induced by alpha-ketoisovalerate metabolism has been investigated in isolated rat hepatocytes incubated with lactate, pyruvate, ammonia, and ornithine as substrates. Half-maximum inhibitions of flux through each of these enzyme steps were obtained with 0.3 mM alpha-ketoisovalerate. The inhibition of pyruvate carboxylase flux by alpha-ketoisovalerate was largely reversed by oleate addition, but pyruvate dehydrogenase flux was inhibited further. Inhibition of flux through pyruvate carboxylase could be attributed mainly to the fall of its allosteric activator, acetyl-CoA, with some additional effect due to inhibition by methylmalonyl-CoA. Tissue acetyl-CoA levels decrease as a result of an inhibition of the active form of pyruvate dehydrogenase. Kinetic studies with the purified pig heart pyruvate dehydrogenase complex showed that methyl-malonyl-CoA, propionyl-CoA, and isobutyryl-CoA were inhibitory, the latter noncompetitive with CoASH with an apparent Ki of 90 microM. The observed inhibition of pyruvate dehydrogenase flux correlated with increases of the acetyl-CoA/CoASH and propionyl-CoA/CoASH ratios and isobutyryl-CoA levels, while increases of the mitochondrial NADH/NAD+ ratio explained differences between the effects of alpha-ketoisovalerate and propionate. Carbamyl phosphate synthetase I purified from rat liver was shown to be inhibited directly by methylmalonyl-CoA (apparent Ki of 5 mM). Inhibition of flux through carbamyl phosphate synthetase during alpha-ketoisovalerate metabolism could be attributed both to a direct inhibitory effect of methyl-malonyl-CoA and to a diminished activation by N-acetylglutamate. Direct effects of various acyl-CoA metabolites on these key enzymes may explain symptoms of hypoglycemia and hyperammonemia observed in patients with inherited disorders of organic acid metabolism.     

Cytochrome c oxidase from bakers' yeast. IV. Immunological evidence for the participation of a mitochondrially synthesized subunit in enzymatic activity.

In order to study the role of the individual subunits of yeast cytochrome c oxidase, rabbit antisera were prepared against Subunit II (a mitochondrially made polypeptide) and Subunit VI (a cytoplasmically made polypeptide). Antisera were also obtained against a mixture of the two mitochondrially made subunits (I PLUS II) and against mixtures of the following cytoplasmically made subunits: (IV PLUS VI); (V PLUS VII); and (IV PLUS V PLUS VI PLUS VII). Neither anti-II serum nor anti-VI serum cross-reacted with any of the other six subunits of cytochrome c oxidase as judged by a sensitive ring test or by double diffusion in agarose gels. Anti-II serum inhibited the oxidation of ferrocytochrome c by purified yeast cytochrome c oxidase or by freshly isolated as well as sonically fragmented yeast mitochondria. Anti-(V, VII) serum and anti-(IV, V, VI, VII) serum were also strongly inhibitory. Anti-VI serum and anti-(IV, VI) serum inhibited only weakly. If purified cytochrome c oxidase was inhibited with a saturating amount of anti-VI serum, anti-II serum elicited a further increment of inhibition, as would be expected if the inhibitory effects of these two antisera involved different antigenic sites on the holoenzyme. Each of the antisera precipitated all seven cytochrome c oxidase subunits from crude mitochondrial extracts. However, anti-VI and, particularly, anti-II were much less effective precipitants than antisera against Subunits IV to VII or antisera against the holoenzyme. These data suggest that the oxidation of ferrocytochrome c by cytochrome c oxidase required both mitochondrially as well as cytoplasmically made subunits.     

Rat mammary-gland acetyl-coenzyme A carboxylase. Interaction with milk fatty acids

1. Highly purified rat mammary-gland acetyl-CoA carboxylase was inhibited by milk obtained from rats 12h after their young were weaned. 2. All the inhibitory activity was found in the particulate fraction (R(105)) obtained on centrifuging the milk. It could be extracted from milk fraction R(105) with acetone and identified as a complex mixture of non-esterified fatty acids, present in high concentration (nearly 10mm) in the milk. 3. Inhibition of acetyl-CoA carboxylase was observed at low concentrations (0.2-20mum) of several of these fatty acids when fresh fully active enzyme was used. Enzyme that had been partly inactivated by aging, or by storing in the absence of citrate, was stimulated by low concentrations but inhibited by high concentrations of fatty acids. 4. Various experiments suggested that fatty acids produce irreversible inactivation of acetyl-CoA carboxylase. 5. The effects of palmitoyl-CoA on mammary-gland acetyl-CoA carboxylase were found to resemble those of fatty acids, except that palmitoyl-CoA was effective at lower concentration. 6. The effect of milk fraction R(105) was tested on six other enzymes previously shown to decline to various extents after weaning. Although several of these enzymes were affected by unfractionated milk fraction R(105), none was significantly inhibited by the acetone extract or by low concentrations of lauric acid. 7. The findings are consistent, both qualitatively and quantitatively, with a regulatory mechanism whereby milk fatty acids shut off fatty acid synthesis in the mammary gland after weaning by inhibiting acetyl-CoA carboxylase.     

The role of acetyl-CoA carboxylase phosphorylation in the control of mammary gland fatty acid synthesis during the starvation and re-feeding of lactating rats.

Activation of acetyl-CoA carboxylase during incubation of crude extracts of lactating rat mammary gland with Mg2+ and citrate can be blocked by NaF, suggesting that it represents a dephosphorylation of the enzyme. The greater extent of activation in extracts from 24 h-starved rats (200%) compared with fed controls (70%) implies that the decrease in acetyl-CoA carboxylase activity in response to 24 h starvation may involve increased phosphorylation of the enzyme. Acetyl-CoA carboxylase was purified from the mammary glands of lactating rats in the presence of protein phosphatase inhibitors by avidin-Sepharose chromatography. Starvation of the rats for 24 h increased the concentration of citrate giving half-maximal activation by 75%, and decreased the Vmax. of the purified enzyme by 73%. This was associated with an increase in the alkali-labile phosphate content from 3.3 +/- 0.2 to 4.5 +/- 0.4 mol/mol of enzyme subunit. Starvation of lactating rats for 6 h, or short-term insulin deficiency induced by streptozotocin injection, did not effect the kinetic parameters or the phosphate content of acetyl-CoA carboxylase purified from mammary glands. The effects of 24 h starvation on the kinetic parameters and phosphate content of the purified enzyme were completely reversed by re-feeding for only 2.5 h. This effect was blocked if the animals were injected with streptozotocin before re-feeding, suggesting that the increase in plasma insulin that occurs on re-feeding was responsible for the activation of the enzyme. The effects of re-feeding 24 h-starved rats on the kinetic parameters and phosphate content of acetyl-CoA carboxylase could be mimicked by treating enzyme purified from 24 h-starved rats with protein phosphatase-2A in vitro. Our results suggest that, in mammary glands of 24 h-starved lactating rats, insulin brings about a dephosphorylation of acetyl-CoA carboxylase in vivo, which may be at least partly responsible for the reactivation of mammary lipogenesis in response to re-feeding.     

Mechanism of increase in cytochrome c oxidase activity in sweet potato root tissue during aging of slices

The mechanism of an increase in cytochrome c oxidase [EC 1.9.3.1] activity during aging of sliced sweet potato root tissue was investigated with antibiotics and antibody to the purified enzyme. 1. The increase in cytochrome c oxidase activity was inhibited by chloramphenicol but not by cycloheximide. 2. Cytochrome c oxidase purified from wounded tissue was identical with that from intact tissue as judged by the subunit composition, sedimentation velocity, absorption spectrum, antigenicity, and activity per heme a. 3. An increase in the amount of cytochrome c oxidase protein took place during aging of slices. 4. Sweet potato cytochrome c oxidase consists of five subunits. When slices were aged in the presence of [3H]leucine, the three larger subunits (I, II, and III) of cytochrome c oxidase were labeled, while no radioactivity was incorporated into the other two subunits, IV and V. The results indicate that the increase in cytochrome c oxidase activity is due to an increase in the amount of the enzyme protein. We propose that excess amounts of subunits derived from the cytoplasm of the enzyme are present in intact tissue and are assembled with subunits of mitochondrial origin to form the holoenzyme after wounding of tissue.