Activities of hexokinase, phosphofructokinase, 3-oxo acid coenzyme A-transferase and aceto-acetyl-coenzyme A thiolase in nervous tissue from vertebrates and invertebrates

1. The maximum activities of hexokinase and phosphofructokinase in nervous tissue from 18 different animals from different phyla range from 5.1 to 17.6 and from 24.0mumol/min per g fresh wt. respectively. In any one tissue the activities of these two enzymes are, in general, very similar. The rate of glucose utilization by the brain in vivo is much lower than the activities of hexokinase or phosphofructokinase. It is suggested that the high activities of these enzymes indicate a capacity for glycolysis which may be used by the brain during hypoxia or during conditions of extreme neuronal activity. 2. The activities of 3-oxo acid CoA-transferase and acetoacetyl-CoA thiolase in the nervous tissues range from 1.1 to 15.3 and from 0.7 to 4.5mumol/min per g fresh wt. respectively. Unfortunately the activities of these enzymes cannot be used to estimate maximal flux through the ketone-body-utilization pathway, since they may catalyse reactions that are close to equilibrium. Nonetheless, the presence of these enzymes in nervous tissue from a large variety of animals suggests that the importance of ketone bodies as a fuel for nervous tissue may be widespread in the animal kingdom.     

Comparative studies on 3-oxo acid coenzyme A transferase from various rat tissues

1. Tissue activities, intracellular distribution as well as selected kinetic and molecular properties of succinyl-CoA-3-oxo acid CoA transferase (EC 2.8.3.5), which is an initiator of ketone body usage, were examined in rat kidney, heart, brain, skeletal muscle and liver. 2. The activities of the transferase in these tissues are similar to reported values and are somewhat affected by the homogenization medium. Higher recoveries of activity are obtained when a phosphate buffer is used during the homogenization; Tris solutions containing sucrose and mannitol lead to only slightly lower recoveries, but can be used in studies to determine the subcellular localization of the transferase activity. 3. A close correlation was observed between the relative activities of citrate synthase (a mitochondrial marker enzyme) and CoA transferase in the cytoplasmic, particulate and mitochondrial fractions from the five tissues. 4. The K(m) values for acetoacetate (measured in two different ways), the ratio of V(max.) values for the two enzyme-catalysed half-reactions, and succinate product inhibition are quite similar for the enzyme from each tissue. 5. The enzymes are also similar in molecular weight (with an approx. mol.wt. of 100000 as determined by gel filtration). All show an active band in isoelectric-focusing studies with pI 7.6, except for the enzyme from heart (pI 6.8). 6. The results demonstrate a mitochondrial origin for CoA transferase in these rat tissues and support the proposition that CoA transferase is a ketolytic enzyme, i.e. an enzyme uniquely involved in the complete oxidation of ketone bodies. The structural and functional similarities of these transferases suggest that factors other than differences in K(m) values account for differences in the utilization of ketone bodies by various tissues.     

Formation of acetoacetate from 3-hydroxy-3-methylglutarate by rat liver and isolation of a mitochondrial coenzyme A-transferase activity involved

1. Formation of acetoacetate from 3-hydroxy-3-methylglutarate was observed in the perfused rat liver. Production of 3.5mumol of acetoacetate/h per g of tissue was obtained. 2. Formation of acetoacetate was catalysed mainly by the mitochondrial fraction of the homogenized liver, at a rate of 62nmol/h per mg of protein. 3. Experiments with hydroxy-[3-(14)C]methylglutarate demonstrated that the acetoacetate formed was derived mainly from this compound. 4. A mitochondrial transferase activity catalysing the transfer of a CoA molecule from succinyl-CoA (3-carboxypropionyl-CoA) to hydroxymethylglutarate was shown. The K(m) value for hydroxymethylglutarate was 5x10(-3)m.     

Induction of succinyl-coenzyme A:3-oxoacid coenzyme A-transferase during differentiation of 3T3-L1 cells

Differentiation of confluent 3T3-L1 preadipocytes to adipocytes in the presence of dexamethasone and 1-methyl-3-isobutylxanthine for 7 days resulted in a 4-fold increase in the incorporation of acetoacetate-carbon into fatty acids and in the activity of 3-oxoacid CoA-transferase, which catalyzes the first committed step in the conversion of acetoacetate to acetoacetyl-CoA. The increase in enzyme activity was due to an increase in the cellular content of the enzyme, as determined by immunoprecipitation of 3-oxoacid CoA-transferase from 3T3-L1 preadipocytes and adipocytes with rabbit antiserum specific for the rat brain enzyme. The 4-fold increase in enzyme activity was accompanied by a 2.7-fold increase in the average relative rate of synthesis of 3-oxoacid CoA-transferase (between Days 4 and 7). Additionally, the half-life of the enzyme increased 1.9-fold relative to the half-life of total protein, indicating that changes in both synthesis and degradation of 3-oxoacid CoA-transferase are responsible for alterations in its activity. Previous studies on the turnover of other enzymes that are induced during differentiation of 3T3-L1 cells have assigned changes in enzyme synthesis as the primary or sole mechanism for changes in enzyme activity. This report provides the first documentation that both enzyme synthesis and degradation play a role in regulating the enzyme activity of an enzyme during differentiation of 3T3-L1 cells.     

Resolution and reconstitution of bovine kidney branched-chain 2-oxo acid dehydrogenase complex.

Branched-chain 2-oxo acid dehydrogenase complex was resolved into component E1 and E2-kinase subcomplex by gel filtration in the presence of 1 M-NaC1. Essentially all the original activity of the complex can be regained after reconstitution of the component enzymes, reassociation being a rapid process. The specific activities of E1 and E2 were 25.1 and 19.0 units/mg respectively. Non-phosphorylated active E1 has an approx. 6-fold higher affinity for E2 than does phosphorylated E1. The components of the branched-chain 2-oxo acid dehydrogenase complex do not crossreact with the respective components from the pyruvate dehydrogenase complex. The significance of these results and of the tight association of the kinase with E2 are discussed.     

Purification and characterization of different "acid" beta-galactosidases from sheep kidney.

1. Two "acid" forms, Am and Al, of beta-galactosidase from sheep kidney have been isolated and purified 349- and 154-fold, respectively, with a recovery of about 8%. 2. Their mol. wts were about 450,000 and 230,000, respectively. Am seems to be a dimer of Al. The aggregation is stimulated by NaCl. 3. The "acid" beta-galactosidase has a pH optimum between 4.0 and 5.0 for both forms. They are located in the lysosomes. The optimal temperature is 37 degrees C and 40 degrees C for Al and Am forms, respectively. 4. Three peaks were detected by isoelectric focusing. After sialidase treatment, these peaks were obtained at higher pH values. 5. The activation energy values were 10.75 and 11.72 kcal/mol for Am and Al, respectively. 6. A variety of chemicals were tested as possible activators or inhibitors. The enzyme is strongly inhibited by gamma-D-galactonolactone, and the kinetic evidence suggests a competitive inhibition in all cases.     

Steady-state kinetics of 3-mercaptopyruvate sulfurtransferase from bovine kidney

Mammalian 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2), purified to apparent homogeneity by a new procedure, was studied by steady-state kinetic methods. The enzyme-catalyzed transfer of a sulfur atom from 3-mercaptopyruvate either to 2-mercaptoethanol or to a second molecule of 3-mercaptopyruvate was found to proceed by a sequential formal mechanism. An overall mechanism incorporating both of these transfers was shown to be capable of generating all of the initial velocity and product inhibition behavior observed.     

The regulation of branched-chain 2-oxo acid dehydrogenase of liver, kidney and heart by phosphorylation.

1. Incubation of mitochondria from heart, liver and kidney with [32P]phosphate allowed 32P incorporation into two intramitochondrial proteins, the decarboxylase alpha-subunit of the pyruvate dehydrogenase complex (mol.wt 42000) and a protein of mol.wt. 48000. 2. This latter protein incorporated 32P more slowly than did pyruvate dehydrogenase, was not precipitated by antibody to pyruvate dehydrogenase and showed behaviour distinct from that of pyruvate dehydrogenase towards high-speed centrifugation and pyruvate dehydrogenase phosphate phosphatase. 3. 32P incorporation into the protein was greatly diminished by the presence of 0.1 mM-4-methyl-2-oxopentanoate, but enhanced by pyruvate (1 mM), hypo-osmotic treatment of mitochondria and, under some conditions, by uncoupler. 4. The activity of branched-chain 2-oxo acid dehydrogenase was assayed in parallel experiments. Under appropriate conditions the enzyme was inhibited when 32P incorporation was increased and activated when incorporation was decreased. The data suggest that the 48000-mol.wt. phosphorylated protein is identical with the decarboxylase subunit of branched-chain 2-oxo acid dehydrogenase and that this enzyme may be controlled by a phosphorylation-dephosphorylation cycle akin to that for pyruvate dehydrogenase. 5. Strict correlation between activity and 32P incorporation was not observed, and a scheme for the regulation of the enzyme is proposed to account for these discrepancies.     

Purification and kinetics of sheep kidney cortex glucose-6-phosphate dehydrogenase

Glucose-6-phosphate dehydrogenase (G-6-PD) is one of the important enzymes, which is responsible for the production of NADPH and ribose-5-phosphate. NADPH is used for the biosynthetic reactions and protection of the cells from free radicals. We have investigated some properties and kinetic mechanism of the sheep kidney cortex G-6-PD. This enzyme has been purified 1,384-fold with a yield of 16.96% and had a specific activity of 27.69 U/mg protein. The purification procedure consists of 2', 5'-ADP-Sepharose 4B affinity chromatography after ultracentrifugation. The sheep kidney cortex G-6-PD was found to operate according to a Ping Pong Bi Bi mechanism. The kinetic parameters from sheep K(m) values for G-6-P and NADP(+) and V(m) were determined to be 0.041+/-0.0043 mM, 0.0147+/-0.001 mM and 28.23+/-0.86 microMol min(-1) mg protein(-1), respectively. The pH optimum was 7.4 and the optimum temperature was 45 degrees C. In our previous study we have found that lamb kidney cortex G-6-PD enzyme obeys 'Ordered Bi Bi' mechanism. We suggest that kinetic mechanism altered due to the aging since sheep G-6-PD uses a 'ping pong' mechanism.     

Phosphorylation of branched-chain 2-oxo acid dehydrogenase complex in isolated adipocytes. Effects of 2-oxo acids.

Isolated adipocytes from rat epididymal fat-pads were incubated with [32P]Pi, and intracellular phosphoproteins were then analysed by SDS/polyacrylamide-gel electrophoresis and autoradiography. A phosphorylated polypeptide of apparent Mr 46,000 was identified as the alpha-subunit of branched-chain 2-oxo acid dehydrogenase complex by immunoprecipitation using antiserum raised against the homogeneous E1 component of branched-chain 2-oxo acid dehydrogenase complex. Immunoprecipitation of this phosphoprotein is blocked in a competitive manner by purified branched-chain 2-oxo acid dehydrogenase complex. Peptide mapping of the isolated phosphoprotein indicates that two sites on the polypeptide are phosphorylated in the intact cells. Addition of branched-chain 2-oxo acids to the incubation medium causes diminution in the extent of labelling of both phosphorylation sites on the alpha-subunit, an effect presumably mediated via their known inhibitory action on branched-chain 2-oxo acid dehydrogenase kinase. These observations provide direct evidence for phosphorylation of branched-chain 2-oxo acid dehydrogenase complex in intact cells.     

Ketone body and fatty acid metabolism in sheep tissues. 3-Hydroxybutyrate dehydrogenase, a cytoplasmic enzyme in sheep liver and kidney

1. 3-Hydroxybutyrate dehydrogenase (EC 1.1.1.30) activities in sheep kidney cortex, rumen epithelium, skeletal muscle, brain, heart and liver were 177, 41, 38, 33, 27 and 17μmol/h per g of tissue respectively, and in rat liver and kidney cortex the values were 1150 and 170 respectively. 2. In sheep liver and kidney cortex the 3-hydroxybutyrate dehydrogenase was located predominantly in the cytosol fractions. In contrast, the enzyme was found in the mitochondria in rat liver and kidney cortex. 3. Laurate, myristate, palmitate and stearate were not oxidized by sheep liver mitochondria, whereas the l-carnitine esters were oxidized at appreciable rates. The free acids were readily oxidized by rat liver mitochondria. 4. During oxidation of palmitoyl-l-carnitine by sheep liver mitochondria, acetoacetate production accounted for 63% of the oxygen uptake. No 3-hydroxybutyrate was formed, even after 10min anaerobic incubation, except when sheep liver cytosol was added. With rat liver mitochondria, half of the preformed acetoacetate was converted into 3-hydroxybutyrate after anaerobic incubation. 5. Measurement of ketone bodies by using specific enzymic methods (Williamson, Mellanby & Krebs, 1962) showed that blood of normal sheep and cattle has a high [3-hydroxybutyrate]/[acetoacetate] ratio, in contrast with that of non-ruminants (rats and pigeons). This ratio in the blood of lambs was similar to that of non-ruminants. The ratio in sheep blood decreased on starvation and rose again on re-feeding. 6. The physiological implications of the low activity of 3-hydroxybutyrate dehydrogenase in sheep liver and the fact that it is found in the cytoplasm in sheep liver and kidney cortex are discussed.     

Cloning and expression of Clostridium acetobutylicum ATCC 824 acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase in Escherichia coli.

Coenzyme A (CoA)-transferase (acetoacetyl-CoA:acetate/butyrate:CoA-transferase [butyrate-acetoacetate CoA-transferase] [EC 2.8.3.9]) of Clostridium acetobutylicum ATCC 824 is an important enzyme in the metabolic shift between the acid-producing and solvent-forming states of this organism. The purification and properties of the enzyme have recently been described (D. P. Weisenborn, F. B. Rudolph, and E. T. Papoutsakis, Appl. Environ. Microbiol. 55:323-329, 1989). The genes encoding the two subunits of this enzyme have been cloned by using synthetic oligodeoxynucleotide probes designed from amino-terminal sequencing data from each subunit of the CoA-transferase. A bacteriophage lambda EMBL3 library of C. acetobutylicum DNA was prepared and screened by using these probes. Subsequent subcloning experiments established the position of the structural genes for CoA-transferase. Complementation of Escherichia coli ato mutants with the recombinant plasmid pCoAT4 (pUC19 carrying a 1.8-kilobase insert of C. acetobutylicum DNA encoding CoA-transferase activity) enabled the transformants to grow on butyrate as a sole carbon source. Despite the ability of CoA-transferase to complement the ato defect in E. coli mutants, Southern blot and Western blot (immunoblot) analyses showed that neither the C. acetobutylicum genes encoding CoA-transferase nor the enzyme itself shared any apparent homology with its E. coli counterpart. Polypeptides of Mr of the purified CoA-transferase subunits were observed by Western blot and maxicell analysis of whole-cell extracts of E. coli harboring pCoAT4. The proximity and orientation of the genes suggest that the genes encoding the two subunits of CoA-transferase may form an operon similar to that found in E. coli.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reversible ATP-induced inactivation of branched-chain 2-oxo acid dehydrogenase.

1. Antiserum was raised against purified Wistar-rat liver UDP-glucuronyltransferase. 2. UDP-glucuronyltransferase activities towards 4-nitrophenol, bilirubin, 1-naphthol and morphine were co-immunoprecipitated from solubilized Wistar-rat liver preparations. 3. UDP-glucuronyltransferase activities towards 1-naphthol, 2-aminophenol and 4-nitrophenol were precipitated from solubilized Gunn-rat liver preparations by this antiserum. 4. UDP-glucuronyltransferase activities towards 1-naphthol, 4-nitrophenol and bilirubin, from Wistar-rat liver, were slightly inhibited by antiserum, whereas 1-naphthol UDP-glucuronyltransferase activity from Gunn-rat livers was greatly inhibited. 5. Measurable Wistar-rat liver glucuronyltransferase activities in washed immunoprecipitates indicate that the enzyme(s) were not merely inhibited by antiserum. 6. Immunoglobulin G purified from this antiserum immunoprecipitated transferase activities towards 4-nitrophenol, bilirubin and 1-naphthol. 7. The washed immunoprecipitates from both rat strains, containing UDP-glucuronyltransferase activity, appear to be similar when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 8. Radial-immunodiffusion studies suggest that a smaller amount of UDP-glucuronyltransferase protein is present in Gunn-rat liver than in Wistar-rat liver. 9. The significance of these results in relation to the genetic deficiency in the Gunn rat is discussed.     

Binding of coenzyme and substrate and coenzyme analogues to 6-phosphogluconate dehydrogenase from sheep liver. An X-ray study at 0.6 nm resolution.

The analogues of the coenzyme NADP+, nicotinamide--8-bromo-adenine dinucleotide phosphate (Nbr8ADP+) and 3-iodopyridine--adenine dinucleotide phosphate (io3PdADP+), were prepared. Nbr8ADP+ was found to be active in the hydrogen transfer adn io3PdADP+ is a coenzyme competitive inhibitor for 6-phosphogluconate dehydrogenase. The binding of NADP+, NADPH and NADPH together with 6-phosphogluconate as well as that of both analogues to crystals of the enzyme 6-phosphogluconate dehydrogenase has been investigated at 0.6-nm resolution using difference electron density maps. The molecules bind in a similar position in a cleft in the enzyme subunit distant from the dimer interface. The orientation of the coenzyme in the site has been determined from the io3PdADP+ -NADP+ difference density. The ternary complex difference density extends beyond that of the nicotinamide moiety of the coenzyme and tentatively indicates substrate binding. No clear identification of the bromine atom of Nbr8ADP+ can be made. However, the analogue is bound more deeply in the cleft than is NADP+. The NADPH density is the most clearly defined and has thus been used to fit a molecular model using an interactive graphics system, checking for preferred geometry. A possible conformation is presented which is significantly different from that of NAD+ in the lactate dehydrogenase ternary complex.     

The kinetics of rabbit muscle pyruvate kinase. Initial-velocity, substrate- and product-inhibition and isotopic-exchange studies of the reverse reaction.

1. An assay, based on the transfer of label from [gamma-32P]ATP to [32P]phosphoenolpyruvate, suitable for a steady-state kinetic analysis of pyruvate kinase in the reverse direction (i.e. phosphoenolpyruvate synthesis), is described. 2. This assay was used in a kinetic investigation of the rabbit muscle enzyme including initial-rate and product-inhibition experiments, at a pH of 7.4 and constant concentrations of total K+ and free Mg2+. 3. These studies indicate that there is a random release of ADP and phosphoenolpyruvate from the enzyme and that there is a competitive substrate inhibition by ATP. Some of the results were suggestive that the rapid-equilibrium assumption, generally used for this enzyme was not valid. 4. Techniques were developed to measure the rate of isotopic exchange between all the substrate-product pairs. 5. By using these techniques the rates of isotopic exchange at chemical equilibrium were measured. The results indicate that this enzyme does not catalyse a truly rapid-equilibrium random mechanism, although in the forward reaction all initial-rate data obtained to date are consistent with this assumption.     

Medium-chain acyl coenzyme A dehydrogenase from pig kidney has intrinsic enoyl coenzyme A hydratase activity

The flavoprotein medium-chain acyl coenzyme A (acyl-CoA) dehydrogenase from pig kidney exhibits an intrinsic hydratase activity toward crotonyl-CoA yielding L-3-hydroxybutyryl-CoA. The maximal turnover number of about 0.5 min-1 is 500-1000-fold slower than the dehydrogenation of butyryl-CoA using electron-transferring flavoprotein as terminal acceptor. trans-2-Octenoyl- and trans-2-hexadecenoyl-CoA are not hydrated significantly. Hydration is not due to contamination with the short-chain enoyl-CoA hydratase crotonase. Several lines of evidence suggest that hydration and dehydrogenation reactions probably utilize the same active site. These two activities are coordinately inhibited by 2-octynoyl-CoA and (methylenecyclopropyl)acetyl-CoA [whose targets are the protein and flavin adenine dinucleotide (FAD) moieties of the dehydrogenase, respectively]. The hydration of crotonyl-CoA is severely inhibited by octanoyl-CoA, a good substrate of the dehydrogenase. The apoenzyme is inactive as a hydratase but recovers activity on the addition of FAD. Compared with the hydratase activity of the native enzyme, the 8-fluoro-FAD enzyme exhibits a roughly 2-fold increased activity, whereas the 5-deaza-FAD dehydrogenase is only 20% as active. A mechanism for this unanticipated secondary activity of the acyl-CoA dehydrogenase is suggested.