Mechanism of citrinin-induced dysfunction of mitochondria. IV—Effect on Ca2+ transport

The effect of citrinin on Ca²⁺ transport was studied in isolated kidney cortex and liver mitochondria, and baby hamster kidney cultured cells. The mycotoxin significantly inhibited the activity of 2-oxoglutarate and pyruvate dehydrogenases in both kidney cortex and liver mitochondria. Citrinin promoted a decrease in the velocity and in the total capacity of Ca²⁺ uptake, in both mitochondria. Apparently, citrinin acts by a mechanism similar to ruthenium red. In intact cultured cells, citrinin also had a preferential effect on mitochondrial Ca²⁺ fluxes. Citrinin promoted a marked decrease in the Ca²⁺ level in the mitochondrial matrix, whereas that of the extramitochondiral fraction became less affected. All the observed effects were dependent on the citrinin concentration.     

Studies on the metabolism of glycolyl-CoA

Glycolyl-CoA can be formed during the course of the beta-oxidation by rat liver mitochondria of 4-hydroxybutyrate. The existence of this beta-oxidation has been previously supported by the occurrence of 4-hydroxybutyrate and its beta-oxidation catabolites in urine from patients with 4-hydroxybutyric aciduria, an inborn error of gamma-aminobutyric acid metabolism due to the deficiency of succinic semialdehyde dehydrogenase. The characteristics of the mitochondrial beta-oxidation of 4-hydroxybutyrate were, in rat liver, compared with those of the mitochondrial beta-oxidation of butyrate. The inhibition by malonate of the oxidation of 4-hydroxybutyrate was about twofold weaker than that of oxidation of butyrate, whereas both oxidations were abolished by preincubating the mitochondria with 1 mM valproic acid, a known inhibitor of mitochondrial beta-oxidation. Mitochondria from rat kidney cortex were demonstrated to catalyse, as previously shown for hepatic mitochondria, the carnitine-dependent oxidation of 12-hydroxylauroyl-CoA-omega-Hydroxymonocarboxylyl-CoAs are thus concluded to be precursors of glycolyl-CoA also in rat kidney cortex. In addition, 3-hydroxypyruvate was found to be a precursor of glycolyl-CoA, since it was oxidized by bovine heart pyruvate dehydrogenase with a cofactor requirement similar to that of pyruvate oxidation. Glycolyl-CoA was a substrate of carnitine acetyltransferase (pigeon breast muscle). Pig heart citrate synthase was capable of catalyzing the condensation of glycolyl-CoA with oxaloacetate. The product of this reaction induced low NADH production rates dependent on the addition of porcine heart aconitase and isocitrate dehydrogenase.     

Mitochondrial pyruvate metabolism in liver and kidney during acidosis

Pyruvate transport and carboxylation have been determined in mitochondria from liver and kidney cortex isolated from Wistar rats with acidosis produced by three different treatments: fasting, exercise and ingestion of ammonium chloride. Fasting for 48 h or swimming for 2 h resulted in an increased rate of CO₂ fixation by mitochondria from both organs incubated with pyruvate. This increase was accompanied by a rise in the rate of pyruvate transport in all cases except in mitochondria derived from the kidney of the fasted animals. Acute acidosis produced by the ingestion of ammonium chloride resulted in increases in pyruvate transport and carboxylation in kidney mitochondria, but a drop in pyruvate carboxylation was observed in mitochondria from the liver. The results are discussed in terms of the differential regulation of the mitochondria steps for gluconeogenesis from three carbon precursors in liver and kidney, taking into consideration the hormonal status of the animals and the prevailing available substrates in each condition.     

Resolution of rat mitochondrial matrix proteins by two-dimensional polyacrylamide gel electrophoresis.

The mitochondrial matrix subfractions from rat liver, kidney cortex, brain, heart, and skeletal muscle were isolated and their protein components were resolved by two-dimensional polyacrylamide gel electrophoresis, revealing between 120 and 150 components for each matrix subfraction. Excellent resolution was obtained utilizing a pH 5 to 8 gradient in the first dimension and in 8 to 13% exponential acrylamide gradient in the second dimension, increasing the number of mitochondrial matrix proteins observed 3-fold over one-dimensional systems. Protein components tentatively identified by co-migration with pure enzymes and by known tissue distributions are carbamoyl-phosphate synthetase (EC 2.7.2.5), ornithine transcarbamylase (EC 2.1.3.3), glutamate dehydrogenase (EC 1.4.1.3), pyruvate carboxylase (EC 6.4.1.1), citrate synthase (EC 4.1.3.7), fumarase (EC 4.2.1.2), aconitase (EC 4.2.1.3), alpha-ketoglutarate dehydrogenase (EC 1.2.4.2), dihydrolipoyl transsuccinylase (EC 2.3.1.12), lipoamide dehydrogenase (EC 1.6.4.3), glutamate-aspartate aminotransferase (EC 2.6.1.1), and the two subunits of pyruvate dehydrogenase (EC 1.2.4.1). Protein components unambiguously identified by peptide mapping are citrate synthase, aconitase, and pyruvate carboxylase. The inner membrane subfraction from rat liver mitochondria was also resolved two dimensionally; the alpha and beta subunits of ATPase (F1) (EC 3.6.1.3) were identified by peptide mapping.     

The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by alpha-Cyano-4-hydroxycinnamate and related compounds.

1. Effects of alpha-cyano-4-hydroxycinnamate and alpha-cyanocinnamate on a number of enzymes involved in pyruvate metabolism have been investigated. Little or no inhibition was observed of any enzyme at concentrations that inhibit completely mitochondrial pyruvate transport. At much higher concentrations (1 mM) some inhibition of pyruvate carboxylase was apparent. 2. Alpha-Cyano-4-hydroxycinnamate (1-100 muM) specifically inhibited pyruvate oxidation by mitochondria isolated from rat heart, brain, kidney and from blowfly flight muscle; oxidation of other substrates in the presence or absence of ADP was not affected. Similar concentrations of the compound also inhibited the carboxylation of pyruvate by rat liver mitochondria and the activation by pyruvate of pyruvate dehydrogenase in fat-cell mitochondria. These findings imply that pyruvate dehydrogenase, pyruvate dehydrogenase kinase and pyruvate carboxylase are exposed to mitochondrial matrix concentrations of pyruvate rather than to cytoplasmic concentrations. 3. Studies with whole-cell preparations incubated in vitro indicate that alpha-cyano-4-hydroxycinnamate or alpha-cyanocinnamate (at concentrations below 200 muM) can be used to specifically inhibit mitochondrial pyruvate transport within cells and thus alter the metabolic emphasis of the preparation. In epididymal fat-pads, fatty acid synthesis from glucose and fructose, but not from acetate, was markedly inhibited. No changes in tissue ATP concentrations were observed. The effects on fatty acid synthesis were reversible. In kidney-cortex slices, gluconeogenesis from pyruvate and lactate but not from succinate was inhibited. In the rat heart perfused with medium containing glucose and insulin, addition of alpha-cyanocinnamate (200 muM) greatly increased the output and tissue concentrations of lactate plus pyruvate but decreased the lactate/pyruvate ratio. 4. The inhibition by cyanocinnamate derivatives of pyruvate transport across the cell membrane of human erythrocytes requires much higher concentrations of the derivatives than the inhibition of transport across the mitochondrial membrane. Alpha-Cyano-4-hydroxycinnamate appears to enter erythrocytes on the cell-membrane pyruvate carrier. Entry is not observed in the presence of albumin, which may explain the small effects when these compounds are injected into whole animals.     

Effects of ischaemia on content of metabolites in rat liver and kidney in vivo

1. The time-course of changes in content of intermediates of glycolysis in rat liver and kidney cortex after severance of blood supply was investigated. 2. The decline in content of ATP was more rapid in kidney (1.7-0.5mumol/g in 30s) than in liver (2.7-1.6mumol/g in 60s). In both tissues AMP and P(i) accumulated. 3. Net formation of lactate was 1.7mumol/g during the second minute of ischaemia in liver from well-fed rats, 1.1mumol/g in liver from 48h-starved rats, and about 1.0mumol/g during the first 30s of ischaemia in kidney. Net formation of alpha-glycerophosphate was rapid, especially in liver. 4. In kidney the concentration of beta-hydroxybutyrate rose, but that of alpha-oxoglutarate and acetoacetate decreased. 5. In both organs the concentrations of fructose diphosphate and triose phosphates increased during ischaemia and those of other phosphorylated C(3) intermediates decreased. 6. The concentration of the hexose 6-phosphates rose rapidly during the first minute of ischaemia in liver, but decreased during renal ischaemia. 7. In kidney the content of glutamine fell after 2min of ischaemia, and that of ammonia and glutamate rose. 8. The redox states of the cytoplasmic and mitochondrial NAD couple in kidney cortex were similar to those in liver. 9. The regulatory role of glycogen phosphorylase, pyruvate kinase and phosphofructokinase is discussed in relation to the observed changes in the concentrations of the glycolytic intermediates.     

A synthetic peptide-directed antibody as a probe of the phosphorylation site of pyruvate dehydrogenase

A synthetic peptide, corresponding to the 14-amino acid tryptic fragment containing phosphorylation sites one and two of bovine mitochondrial pyruvate dehydrogenase, was coupled to Limulus polyphemus hemocyanin and used to produce rabbit polyclonal antibodies. A positive signal was observed when Western blots of bovine, porcine, or yeast mitochondrial pyruvate dehydrogenase complexes were probed with the antibodies but not with blots of bacterial, cellular slime mold, plant mitochondrial, or plant plastid pyruvate dehydrogenase complexes. The antibodies also gave a positive signal when used to probe blots of the bovine kidney branched chain 2-oxo acid dehydrogenase complex. The ATP-dependent phosphorylation/inactivation of rat liver mitochondrial pyruvate dehydrogenase complex could be inhibited by prior incubation with the anti-peptide antibodies.     

Mitochondrial respiration in heart, liver, and kidney of copper-deficient rats

Morphological observations in some tissues indicate that dietary copper deficiency results in structural damage to mitochondria. The purpose of this study was to determine whether mitochondrial function is impaired as well. Male, weanling Sprague-Dawley rats were fed diets deficient or sufficient in copper for 4 weeks. Mitochondria were isolated from heart, liver, kidney cortex, and kidney medulla. P/O ratio, state 3 and state 4 respiration rates (oxygen consumed in the presence and absence of ADP, respectively), and acceptor control index (ratio of state 3:state 4) were determined using succinate or pyruvate/malate as substrate. State 3 respiration rate in mitochondria from copper-deficient hearts and livers was lower than in mitochondria from copper-sufficient hearts. Copper deficiency reduced the state 4 respiration rate only in cardiac mitochondria. Neither respiration rate was affected by copper deficiency in mitochondria from kidney medulla or cortex. P/O ratio was not significantly affected by copper deficiency in any tissue examined. Acceptor control index was reduced only in liver mitochondria. The observed decreases in respiration rates are consistent with decreased cytochrome c oxidase activity, shown by others to occur in mitochondria isolated from hearts and livers of copper-deficient rats.     

Phosphorylation of rat kidney pyruvate kinase type L by cyclic 3',5'-AMP-dependent protein kinase.

Pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) type L was partly purified from rat kidney. During the last two purification steps, the incorporation of [32P]phosphate into protein on incubation with [32P]ATP and cyclic 3',5'-AMP-dependent protein kinase was found to parallel the pyruvate kinase activity. After phosphorylation of the enzyme, a major radioactive band with a molecular weight of 57 000 was found on polyacrylamide gel electrophoresis [32P]Phosphorylserine was isolated from the kidney pyruvate kinase. Immunological identity was found between the liver and kidney pyruvate kinases type L. By autoradiography of high-voltage electropherograms after partial acid hydrolysis of the phosphorylated rat liver and kidney pyruvate kinases type L, identical results were obtained. The affinity for phosphoenolpyruvate was found to be decreased by phosphorylation of the enzyme with a change in the apparent Km from 0.15 mM to 0.35 mM. After incubation of the phosphorylated kidney pyruvate kinase with phosphatase the phosphoenolpyruvate saturation curve was found to be identical to that for the unphosphorylated enzyme. Thus, the activity of the rat kidney pyruvate kinase type L is with all probability regulated by a reversible phosphorylation-dephosphorylation reaction, thereby indicating that hormonal regulation of gluconeogenesis via cyclic AMP may be of importance in the renal cortex.     

Inhibitory effect of gentamicin on gluconeogenesis from pyruvate, propionate, and lactate in isolated rabbit kidney-cortex tubules

The effect of gentamicin on glucose production in isolated rabbit renal tubules was studied with lactate, propionate, malate, 2-oxoglutarate, and succinate as substrates. This antibiotic at 5 mM concentration inhibited gluconeogenesis from lactate by about 60% and that from either pyruvate or propionate by about 30%. In contrast, it did not alter the rate of glucose formation from other substrates studied. The rate of gluconeogenesis was higher at 1 mM propionate than at increasing concentrations of this substrate and was stimulated in the presence of 1 mM carnitine. However, the addition of carnitine did not affect the degree of inhibition of glucose formation by gentamicin. Since the mitochondrial free coenzyme A level was significantly lower in the presence of 10 than 1 mM propionate and increased on the addition of carnitine to the reaction medium, the inhibitory effect of propionate concentrations above 1 mM on gluconeogenesis in rabbit renal tubules may be due to a depletion of the free mitochondrial coenzyme A level, resulting in an inhibition of the mitochondrial coenzyme A-dependent reactions. In intact rabbit kidney cortex mitochondria incubated in State 4 as well as in Triton X-100-treated mitochondria, 5 mM gentamicin inhibited by about 30-40% the incorporation of 14CO2 into both pyruvate and propionate. The results indicate that the inhibitory effect of gentamicin on glucose formation in isolated kidney tubules incubated with lactate, pyruvate, or propionate is likely due to a decrease of the rate of carboxylation reactions.     

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.     

Substrate-dependent differential regulation of mitochondrial bioenergetics in the heart and kidney cortex and outer medulla

The kinetics and efficiency of mitochondrial oxidative phosphorylation (OxPhos) can depend on the choice of respiratory substrates. Furthermore, potential differences in this substrate dependency among different tissues are not well-understood. Here, we determined the effects of different substrates on the kinetics and efficiency of OxPhos in isolated mitochondria from the heart and kidney cortex and outer medulla (OM) of Sprague-Dawley rats. The substrates were pyruvate+malate, glutamate+malate, palmitoyl-carnitine+malate, alpha-ketoglutarate+malate, and succinate±rotenone at saturating concentrations. The kinetics of OxPhos were interrogated by measuring mitochondrial bioenergetics under different ADP perturbations. Results show that the kinetics and efficiency of OxPhos are highly dependent on the substrates used, and this dependency is distinctly different between heart and kidney. Heart mitochondria showed higher respiratory rates and OxPhos efficiencies for all substrates in comparison to kidney mitochondria. Cortex mitochondria respiratory rates were higher than OM mitochondria, but OM mitochondria OxPhos efficiencies were higher than cortex mitochondria. State 3 respiration was low in heart mitochondria with succinate but increased significantly in the presence of rotenone, unlike kidney mitochondria. Similar differences were observed in mitochondrial membrane potential. Differences in H₂O₂ emission in the presence of succinate±rotenone were observed in heart mitochondria and to a lesser extent in OM mitochondria, but not in cortex mitochondria. Bioenergetics and H₂O₂ emission data with succinate±rotenone indicate that oxaloacetate accumulation and reverse electron transfer may play a more prominent regulatory role in heart mitochondria than kidney mitochondria. These studies provide novel quantitative data demonstrating that the choice of respiratory substrates affects mitochondrial responses in a tissue-specific manner.     

Isolation and characterization of mitochondrial alanine aminotransferase from porcine tissue.

Mitochondrial alanine aminotransferase L-alanine:2-oxoglutarate aminotransferase, EC 2.6.1.2) has been isolated in homogeneous form from both porcine liver and kidney cortex, but in low yield. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate or 8 M urea gave a single band. An isoelectric point of 8.5 +/- 0.5 and a molecular weight of 75--80 000 were obtained. The enzyme is specific for L-alanine and is inhibited by D-alanine, aminooxyacetate and cyclosterine. The Km for pyruvate and glutamate is 0.4 mM and 32 mM, respectively. These values are similar to those determined for the cytoplasmic enzyme; however, at high concentrations, both compounds strongly inhibit the mitochondrial enzyme, an inhibition not observed with cytosolic alanine aminotransferase. These characteristics and the fact that the mitochondrial alanine aminotransferase was inactivated by procedures effective in the preparation of the cytosolic enzyme, clearly differentiate the two proteins and further support different roles for the two alanine aminotransferases in vivo.     

PPAR-gamma-independent inhibitory effect of rosiglitazone on glucose synthesis in primary cultured rabbit kidney-cortex tubules

Therapeutic effect of rosiglitazone has been reported to result from an improvement of insulin sensitivity and inhibition of glucose synthesis. As the latter process occurs in both liver and kidney cortex the aim of this study was to elucidate the rosiglitazone action on glucose formation in both tissues. Primary cultured cells of both liver and kidney cortex grown in defined medium were use throughout. To identify the mechanism responsible for drug-induced changes, intracellular gluconeogenic intermediates and enzyme activities were determined. In contrast to hepatocytes, the administration of a 10 micromol/L concentration of rosiglitazone to renal tubules resulted in about a 70% decrease in the rate of gluconeogenesis, accompanied by an approximately 75% decrease in alanine utilization and a 35% increase in lactate synthesis. The effect of rosiglitazone was not abolished by GW9662, the PPAR-gamma irreversible antagonist, indicating that this action is not dependent on PPAR-gamma activation. In view of rosiglitazone-induced changes in gluconeogenic intermediates and a diminished incorporation of 14CO2 into pyruvate, it is likely that the drug causes a decline in flux through pyruvate carboxylase and (or) phosphoenolpyruvate carboxykinase. It is likely that the hypoglycemic action of rosiglitazone is PPAR-gamma independent and results mainly from its inhibitory effects on renal gluconeogenesis.     

Effects of pyruvate and other metabolites on cyclic GMP levels in incubations of rat hepatocytes and kidney cortex

Pyruvate increased cyclic GMP levels in rat hepatocytes. The effects were observed without or with 1-methyl-3-isobutylxanthine. Lactate, acetate, oxaloacetate, alpha-ketoglutarate, succinate, acetoacetate and beta-hydroxybutyrate also increased cyclic GMP levels. Some compounds increased cyclic GMP in kidney cortex slices. The effects were dependent upon Ca2+ in the medium. Cyclic AMP was increased 30-50% by some of these substances with 2.6 mM Ca2+. Rotenone, oligomycin, antimycin, dinitrophenol, KCN, and arsenate decreased GTP and ATP, basal cyclic GMP and the pyruvate effect, but did not alter cyclic AMP. Although fluoroacetate alone had no effect on cyclic nucleotides, GTP, or ATP, it potentiated the pyruvate effect on cyclic GMP. Adenosine and guanosine increased cyclic GMP and GTP to a similar extent of 30-50%. Aminooxyacetate, cycloserine, pentenoic acid and mepacrine decreased the pyruvate effect while cycloserine or mepacrine alone increased cyclic GMP. Citrate and mepacrine inhibited soluble and particulate guanylate cyclase from rat liver while cycloserine and acetoacetate increased guanylate cyclase activity. None of the other compounds altered guanylate cyclase activity. These results indicate that various metabolites and inhibitors can alter cyclic GMP accumulation in hepatocytes and renal cortex slices. Several mechanisms may be involved in these effects.     

Immunohistochemical localization of pyruvate kinase isoenzymes in chicken tissues.

A method for the localization of pyruvate kinase isoenzymes type L, M2 and M1 in tissue sections is described. Mono-specific antibodies directed against isoenzymes of pyruvate kinase from chicken and the peroxidase antiperoxidase method were used. The following preferential localizations of the isoenzymes in chicken tissues were observed: Pyruvate kinase M1 was found in skeletal muscle. The white muscle fibers were more intensely stained than the red. Some dark muscles (e.g., anterior latissimus dorsi) and the heart muscle showed no reaction with antiserum against pyruvate kinase M1. Pyruvate kinase type L was found in the hepatocytes and in kidney cortex. Pyruvate kinase type M2 was seen in the distal tubules of kidney, in hepatocytes and sinusoidal cells in liver, in lung, adipose tissue, and in the spleen mainly in the bursa dependent areas. Pyruvate kinase type M2 was detected in high concentrations in the granulation tissue of regenerating liver after partial hepatectomy. Liver sections of a hen bearing a pancreatic tumor showed an unusually high content of pyruvate kinase type M2 in some hepatocytes, which were each clustered to spots in the liver parenchyma. Thus, contrary to previous reports, the tissue distribution of isoenzymes in chicken is similar to that of other vertebrates.     

Possible interrelationship between gluconeogenesis and ketogenesis in the liver

Effeects of various ketogenic substrates on gluconeogenesis from lactate were examined. D,L-3-Hydroxybutyrate (5 mM) stimulated gluconeogenesis by 41%, the effect being the same as that of 5 mM acetate (49%). No stimulating effect of acetoacetate was observed; conversely, acetoacetate (up to 40 mM) partially or completely abolished the observed stimulating effects of acetate, oleate, and 3-hydroxybutyrate. The results suggest that, in intact liver cells, pyruvate is transported into mitochondria in exchange for acetoacetate and that an interrelationship between gluconeogenesis and ketogenesis at the level of mitochondrial pyruvate carrier may exist in the liver.     

Effect of long-chain fatty acyl-CoA on mitochondrial and cytosolic ATP/ADP ratios in the intact liver cell.

The effect of long-chain acyl-CoA on subcellular adenine nucleotide systems was studied in the intact liver cell. Long-chain acyl-CoA content was varied by varying the nutritional state (fed and starved states) or by addition of oleate. Starvation led to an increase in the mitochondrial and a decrease in the cytosolic ATP/ADP ratio in liver both in vivo and in the isolated perfused organ as compared with the fed state. The changes were reversed on re-feeding glucose in liver in vivo or on infusion of substrates (glucose, glycerol) in the perfused liver, respectively. Similar changes in mitochondrial and cytosolic ATP/ADP ratios occurred on addition of oleate, but, importantly, not with a short-chain fatty acid such as octanoate. It is concluded that long-chain acyl-CoA exerts an inhibitory effect on mitochondrial adenine nucleotide translocation in the intact cell, as was previously postulated in the literature from data obtained with isolated mitochondria. The physiological relevance with respect to pyruvate metabolism, i.e. regulation of pyruvate carboxylase and pyruvate dehydrogenase by the mitochondrial ATP/ADP ratio, is discussed.     

The activities and intracellular distribution of nicotinamide-adenine dinucleotide phosphate-malate dehydrogenase, phosphoenolpyruvate carboxykinase and pyruvate carboxylase in rat, guinea-pig and rabbit tissues.

1. Measurements are presented of the activity and intracellular distribution of phosphoenolypruvate carboxykinase, pyruvate carboxylase and NADP-malate dehydrogenase in rat, guinea-pig and rabbit liver and kidney cortex, together with previously obtained measurements of these enzymes in adipose tissue. 2. In all three tissues pyruvate carboxylase activity was greatest in the rat and lowest in the rabbit. 3. Guinea pig and rabbit were very similar to each other with respect to the extramitochondrial-mitochondrial distribution of phosphoenolpyruvate carboxykinase in all three tissues. 4. NADP-malate dehydrogenase was present in all three tissues in the rat, present in kidney cortex and adipose tissue in the guinea pig and absent from all tissues examines in the rabbit.