Expression of R-3-hydroxybutyrate dehydrogenase, a ketone body converting enzyme in heart and liver mitochondria of ruminant and non-ruminant mammals.

1. The properties of rat liver and bovine heart R-3-hydroxybutyrate dehydrogenase (BDH) have been extensively studied in the past 20 years, but little is known concerning the biogenesis and the regulation of this dehydrogenase over different species. 2. In addition, controversial results were often reported concerning the activity, the level and the subcellular location of this enzyme in ruminants. 3. BDH activity found in liver and kidney mitochondria from ruminants (cow and sheep) is low, while it is much higher in rat. 4. However, the enzyme activity is detected in microsomes and in cytosol of liver and of kidney cells from ruminants. These activities are not correlated to ketonaemia level. 5. Although low BDH activity is detected in liver mitochondria from ruminants; the bovine liver BDH gene seems to be translated since BDH can be immunodetected by using an antiserum raised against bovine heart BDH. 6. Beside this, the good cross-reactivity between heart BDH and liver BDH suggests their high level of homology in ruminants.     

Fluorometric assays for succinyl-CoA and propionyl-CoA in mitochondrial extracts

Fluorometric assay procedures are described for the quantitative measurements of succinyl-CoA and propionyl-CoA down to concentrations of 0.1 μm in the reaction mixture. The enzymatic assay for succinyl-CoA couples the reaction of 3-ketoacid CoA transferase (succinyl-CoA transferase) to β-OH butyryl-CoA dehydrogenase. A simple purification procedure is described for the isolation of succinyl-CoA transferase from beef heart. Two enzyme assays for propionyl-CoA are described. In the first, CoA, acetyl-CoA and propionyl-CoA are assayed by sequential addition of α-ketoglutarate dehydrogenase, citrate synthase and phosphotransacetylase. The second assay for propionyl-CoA utilized propionyl-CoA carboxylase to convert propionyl-CoA to methylmalonyl-CoA in the presence of ATP and bicarbonate, and the ADP formed was assayed by coupling pyruvate kinase with lactate dehydrogenase. Illustrations are given for the application of these assay procedures to measurements of succinyl-CoA and propionyl-CoA in neutralized perchloric acid extracts prepared from rat heart and liver mitochondria incubated under a variety of conditions.     

Crystallization and some properties of glutamate dehydrogenase from rat liver

1. Glutamate dehydrogenase (L-glutamate:NAD(P) oxidoreductase, EC 1.4.1.3) from rat liver has been crystallized with a method carefully avoiding all denaturating agents. A 236-fold purification was achieved at a yield of 20%. The specific activity was 185 units/mg protein. The enzyme was homogeneous by analytical zone electrophoresis and sedimentation studies. The s0(20),w value was 13.2. 2. Sedimentation studies in the analytical ultracentrifuge and the behaviour of the enzyme in the disc-electrophoresis revealed that glutamate dehydrogenase from rat liver did not undergo a reversible association-dissociation reaction as reported of glutamate dehydrogenase of nearly all other mammalians. 3. Using antibodies prepared against crystalline bovine liver glutamate dehydrogenase, no immunological differences between the rat and the bovine liver enzyme could be observed.     

Rat liver mitochondria contain two immunologically distinct dihydrolipoamide dehydrogenases

We have raised antisera against dihydrolipoamide dehydrogenase. One antigen was isolated from purified bovine kidney pyruvate dehydrogenase complex (PDC). The other antigen was a commercial preparation of porcine heart dihydrolipoamide dehydrogenase (E3) which did not first involve purification of the alpha-keto acid dehydrogenase complex(es). Both antibody preparations cross-reacted with the E3 components of PDC, alpha-ketoglutarate dehydrogenase complex, and branched-chain keto acid dehydrogenase complex. This demonstrates the immunological identity of the E3 components. These sera totally precipitated E3 activity from the purified complexes, from purified preparations of E3, and from extracts of rat heart and kidney mitochondria. The two sera vary in their reaction with rat liver mitochondrial extracts: the anti PDC-E3 serum left residual E3 activity (approximately 50% of the original) that was precipitable by the anti-E3 anti-serum. This indicates that liver contains two immunologically distinct forms of E3. Metabolic assays measuring the differential effects of the two sera on the glycine decarboxylation reaction suggest that the form which is immunologically nonreactive with the anti-PDC-E3 serum could represent the E3 involved in the glycine cleavage system.     

The induction of mitochondrial L-3-glycerophosphate dehydrogenase by thyroid hormone

L-3-Glycerophosphate dehydrogenase was purified from porcine brain mitochondria by a shorter and simpler procedure than previously reported. Immunoblotting with antiserum to the porcine enzyme established that rat liver L-3-glycerophosphate dehydrogenase has the same Mr (76 000) by SDS-polyacrylamide gel electrophoresis. In liver mitochondria from normal and hyperthyroid rats, changes in L-3-glycerophosphate dehydrogenase activity were parallelled by changes in enzyme content assayed by immunoblotting. Similar changes were found in the amount of enzyme synthesised in vitro by reticulocyte lysate programmed with rat liver mRNA, suggesting that thyroid hormone causes specific induction of L-3-glycerophosphate dehydrogenase mRNA.     

Pyruvate dehydrogenase phosphate (PDHb) phosphatase in brain: activity, properties, and subcellular localization

The activity of pyruvate dehydrogenase phosphate (PDHb) phosphatase in rat brain mitochondria and homogenate was determined by measuring the rate of activation of purified, phosphorylated (i.e., inactive) pyruvate dehydrogenase complex (PDHC), which had been purified from bovine kidney and inactivated by phosphorylation with Mg . ATP. The PDHb phosphatase activity in purified mitochondria showed saturable kinetics with respect to its substrate, the phospho-PDHC. It had a pH optimum between 7.0 and 7.4, depended on Mg and Ca, and was inhibited by NaF and K-phosphate. These properties are consistent with those of the highly purified enzyme from beef heart. On subcellular fractionation, PDHb phosphatase copurified with mitochondrial marker enzymes (fumarase and PDHC) and separated from a cytosolic marker enzyme (lactate dehydrogenase) and a membrane marker enzyme (acetylcholinesterase), suggesting that it, like its substrate, is located in mitochondria. PDHb phosphatase had similar kinetic properties in purified mitochondria and in homogenate: dependence on Mg and Ca, independence of dichloroacetate, and inhibition by NaF and K-phosphate. These results are consistent with there being only one type of PDHb phosphatase in rat brain preparations. They support the validity of the measurements of the activity of this enzyme in brain homogenates.     

A protein inhibitor of the mitochondrial adenosine triphosphatase complex of rat liver. Purification and characterization.

A heat-stable protein has been purified from rat liver mitochondria which inhibits the ATP hydrolytic activity of both the soluble and membrane-bound mitochondrial F1-ATPase. The overall purification is about 2400-fold with the major purification step consisting of Sephadex "affinity" chromatography. The purified rat liver inhibitor is homogeneous as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 12,300. Amino acid analysis reveals a high content of glutamic acid, lysine, and arginine and the absence of cysteine, proline and methionine. Whether tested with the rat liver or bovine heart ATPase, the liver inhibitor is equally as potent and specific as the heart inhibitor preparation of Pullman and Monroy (Pullman, M.E., and Monroy, G.C. (1963) J. Biol. Chem. 238, 3762-3769). Although the results presented show that the rat liver ATPase inhibitor resembles closely the ATPase inhibitors from other tissues with respect to specific activity and reaction specificity, it is important to note that the rat liver inhibitor is almost 2000 daltons larger than the bovine heart inhibitor, about 5000 daltons larger than ATPase inhibitors of yeast, and contains significantly more lysine residues than both the bovine heart and yeast inhibitors.     

A simple procedure for isolating adenosine triphosphatase from mitochondria

A simple method for isolation of adenosine triphosphatase (EC 3.6.1.3) from mitochondria is described. The enzyme is released from mitochondrial Lubrol particles by drastic sonication and purified by gel filtration on Sepharose 6-B. The described procedure is effective in isolating adenosine triphosphatase from rat liver as it is from beef heart mitochondria. The enzyme isolated from beef heart has a specific activity of 120 μmol P/min per mg protein and enzyme isolated from rat liver has a specific activity of 70 μmol P/min per mg protein when measured as a release of inorganic phosphate.     

Binding of mitochondrial malate dehydrogenase to mitoplasts.

The binding of 14C-labelled bovine and porcine malate dehydrogenase (EC 1.1.1.37) to rat liver mitochondria and mitoplasts was examined. The bovine enzyme was found to associate nonspecifically with isolated mitochondria and sonicated mitoplasts. Scatchard plot analysis suggested a specific binding to mitoplasts of the order of 5 pmol malate dehydrogenase per milligram of mitoplast protein. Porcine malate dehydrogenase dimer but not monomer exhibited a similar binding. The results are discussed in relation to the mechanism of uptake of the enzyme by mitochondria after synthesis on cytosolic ribosomes.     

Cellular localization of α-ketoglutarate: Glyoxylate carboligase in rat tissues

α-Ketoglutarate : glyoxylate carboligase activity has been reported by other laboratories to be present in mitochondria and in the cytosol of mammalian tissues; the mitochondrial activity is associated with the α-ketoglutarate decarboxylase moiety of the α-ketoglutarate dehydrogenase complex. The cellular distribution of the carboligase has been re-examined here using marker enzymes of known localization in order to monitor the composition of subcellular fractions prepared by differential centrifugation. Carboligase activity paralleled the activity of the mitochondrial matrix enzyme citrate synthase in subcellular fractions prepared from rat liver, heart and brain as well as from rabbit liver. Whole rat liver mitochondria upon lysis released both carboligase and citrate synthase. The activity patterns of several other extramitochondrial marker enzymes differed significantly from that of carboligase in rat liver. In addition, the distribution pattern of carboligase was similar to that of α-ketoglutarate decarboxylase and of α-ketoglutarate dehydrogenase complex.The data indicate that α-ketoglutarate : gloxylate carboligase activity is located exclusively within the mitochondria of the rat and rabbit tissues investigated. There is no evidence for a cytosolic form of the enzyme. Thus the report from another laboratory that the molecular etiology of the human genetic disorder hyperoxaluria type I is a deficiency of cytosolic carboligase must be questioned.     

Evidence for mitochondrial localization of betaine aldehyde dehydrogenase in rat liver: purification, characterization, and comparison with human cytoplasmic E3 isozyme.

Betaine aldehyde dehydrogenase has been purified to homogeneity from rat liver mitochondria. The properties of betaine aldehyde dehydrogenase were similar to those of human cytoplasmic E3 isozyme in substrate specificity and kinetic constants for substrates. The primary structure of four tryptic peptides was also similar; only two substitutions, at most, per peptide were observed. Thus, betaine aldehyde dehydrogenase is not a specific enzyme, as formerly believed; activity with betaine aldehyde is a property of aldehyde dehydrogenase (EC 1.2.1.3), which has broad substrate specificity. Up to the present time the enzyme was thought to be cytoplasmic in mammals. This report establishes, for the first time, mitochondrial subcellular localization for aldehyde dehydrogenase, which dehydrogenates betaine aldehyde, and its colocalization with choline dehydrogenase. Betaine aldehyde dehydrogenation is an important function in the metabolism of choline to betaine, a major osmolyte. Betaine is also important in mammalian organisms as a major methyl group donor and nitrogen source. This is the first purification and characterization of mitochondrial betaine aldehyde dehydrogenase from any mammalian species.     

Immunochemical Characterization of Pyruvate Dehydrogenase Complex in Rat Brain

Pyruvate dehydrogenase complex (PDHC) in rat brain was studied immunochemically, using antibodies against the bovine kidney PDHC, by immunoblotting, immunoprecipitation, inhibition of enzyme activity, and enzyme-linked immunoabsorbent assay (ELISA). The immunoblots showed that the antibodies bound strongly to the alpha peptide of the pyruvate dehydrogenase (E1) component, and to the dihydrolipoyl transacetylase (E2) and the dihydrolipoyl dehydrogenase (E3) components of PDHC. A similar immunoblotting pattern was observed in all eight brain regions examined. On immunoblotting of the subcellular fractions, these PDHC peptides were observed in mitochondria and synaptosomes but not in the postmitochondrial supernatants. This agrees with other evidence that brain PDHC is localized in the mitochondria. These results, together with those from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitin, also showed that the alpha E1, beta E1, and E3 peptides of rat brain PDHC are very similar in sizes to those of the bovine kidney PDHC, being 42, 36, and 58 kD, respectively. The size of the E2 peptide, 66 kD, is different from that of bovine kidney E2, 73 kD. The relative abundance of PDHC protein in nonsynaptic mitochondria was compared by enzyme activity titration and ELISA. Both methods demonstrated that the amount of PDHC antigen in the mitochondria from cerebral cortex is greater than that in the olfactory bulb mitochondria. This is consistent with the results of the activity measurement. The ELISA also showed that the PDHCs in both mitochondrial populations are antigenically similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha-methylisocitrate. A selective inhibitor of TPN-linked isocitrate dehydrogenase from bovine heart and rat liver.

Alpha-Methylisocitrate (3-hydroxy-1,2,3-butanetricarboxylate) is a potent inhibitor, competitive with isocitrate (1-hydroxy-1,2,3-propanetricarboxylate), of the TPN-linked isocitrate dehydrogenase from bovine heart and rat liver; it does not inhibit the DPN-specific enzyme from these tissues. In the presence of magnesium ion, values of Kis for DL-alpha-methylisocitrate for purified bovine heart enzyme, rat liver cytosol, and rat liver mitochondrial extract were in the range of 0.1 muM to 0.3 muM. This compared to values of apparent Km for DL-isocitrate for the same tissue preparations of 14 muM to 20 muM. One of the DL isomer pairs of alpha-methylisocitrate was inactive; the observations suggest that it is threo-alpha-methylisocitrate which inhibits TPN-linked isocitrate dehydrogenase. A method of synthesis of DL-threo-alpha-methylisocitric lactone (2-methyl-5-oxo-2,3-furandicarboxylic acid) from dimethyl trans-epoxymethylsuccinate and dimethylmalonate is described.     

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.     

Characterization of the glucose 6-phosphate dehydrogenase activity in rat liver mitochondria.

Glucose 6-phosphate dehydrogenase activity in rat liver mitochondria can be released by detergent. The released activity is separated by chromatography into two peaks. One peak has the kinetic behaviour and mobility similar to the soluble sex-linked enzyme, whereas the other peak is similar to the microsomal hexose 6-phosphate dehydrogenase. There is no evidence for the existence of a new glucose 6-phosphate dehydrogenase activity in rat liver mitochondria.     

Isolation of inactive phenylalanine hydroxylase from autopsy specimens of human liver

An electrophoretically homogeneous protein has been isolated from human liver autoptats, using a procedure employed for the isolation of phenylalanine hydroxylase from rat liver. The procedure includes chromatography of liver extracts on phenyl-Sepharose and subsequent purification on DEAE-Toyopearl. The activity of phenylalanine hydroxylase in the autoptats was markedly decreased in comparison with that in bioptats. The isolated protein possessed no enzymatic activity. However, the subunit composition of the protein, the molecular masses of protein subunits (55 and 57 kD) and the amino acid composition were close to those of the human enzyme. Antibodies to the protein inhibited the phenylalanine hydroxylase activity in human liver bioptats and weakly inhibited the rat enzyme. The experimental results suggest that the structural organization of phenylalanine hydroxylase does not alter as a result of the loss of enzymatic activity in cadaverous human liver.     

Inhibition of mitochondrial phospholipase A2 by mono- and dilysocardiolipin

Phospholipase A2 extracted from the acetone powder of previously frozen rat liver mitochondria is strongly inhibited compared to the activity manifest before acetone powder preparation. Activity is substantially recovered upon partial purification of the enzyme by gel filtration chromatography. Inhibitor activity elutes in the void volume from the column and is obtained in the chloroform layer when void volume fractions are subjected to a Folch extraction. Structural studies support the inhibitor being monolysocardiolipin. Under the assay conditions employed, 1 molecule of the inhibitor per 5000 substrate molecules or 40 nM on a nominal concentration basis is I50 for the mitochondrial enzyme. The agent is similarly effective against pancreatic and snake venom phospholipases A2. Monolysocardiolipin and dilysocardiolipin prepared enzymatically from bovine heart cardiolipin are less potent than the material arising from rat liver cardiolipin by factors of 10- and 30-fold, respectively, yet are still highly potent compared to the other known inhibitors of this enzyme. Differences in acyl group composition, in the degree of acyl group oxidation, or in structural isomerism between the sn-1 and sn-2 positions of the lyso compounds may account for the difference in potency between the materials derived from rat liver and bovine heart.     

Purification of L-3-glycerophosphate dehydrogenase from rat liver mitochondria

A rapid three-step procedure is presented for the purification of flavin-linked L-3-glycerophosphate dehydrogenase (E.C. 1.1.99.5.) from rat liver mitochondria. Solubilization of the enzyme is achieved selectively by digitonin, at a detergent-to-protein ratio of 0.7 mg/mg (mitochondrial protein concentration 10 mg/ml). The procedure involves chromatography on hydroxymethyl-hexamethylenediamine-succinyl-hexamethylenediamin e Sepharose 4B, followed by anion exchange chromatography using a FPLC technique. Subunit molecular weight of the enzyme was found to be 77,000 when prepared in the presence of the protease inhibitor phenylmethylsulphonyl fluoride. The Kmapp value for glycerophosphate was not influenced by the purification, and the ability of the enzyme to be activated by Ca2+ was preserved as well.     

Calpain inhibition by peptide epoxides.

The protein activator of phosphorylated branched-chain 2-oxo acid dehydrogenase complex was purified greater than 1000-fold from extracts of rat liver mitochondria; the specific activity was greater than 1000 units/mg of protein (1 unit gives half-maximum re-activation of 10 munits of phosphorylated complex). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave two bands (Mr 47700 and 35300) indistinguishable from the alpha- and beta-subunits of the branched-chain dehydrogenase component of the complex. On gel filtration (Sephacryl S-300), apparent Mr was 190000. This and other evidence suggests that activator protein is free branched-chain dehydrogenase; this conclusion is provisional until identical amino acid composition of the subunits has been demonstrated. Activator protein (i.e. free branched-chain dehydrogenase) was inhibited (up to 30%) by NaF, whereas branched-chain complex was not inhibited. There was no convincing evidence for interconvertible active and inactive forms of activator protein in rat liver mitochondria. Activator protein was detected in mitochondria from liver (ox, rabbit and rat) and kidney (ox and rat), but not in rat heart or skeletal-muscle mitochondria. In rat liver mitochondrial extracts, branched-chain complex sedimented with the mitochondrial membranes, whereas activator protein remained in the supernatant. Activator protein re-activated phosphorylated (inactive) particulate complex from rat liver mitochondria, but it did not activate dephosphorylated complex. Liver and kidney, but not muscle, mitochondria apparently contain surplus free branched-chain dehydrogenase, which is bound by the complex with lower affinity than is the branched-chain dehydrogenase intrinsic to the complex. It is suggested that this functions as a buffering mechanism to maintain branched-chain complex activity in liver and kidney mitochondria.     

Cloning and expression of the bovine 11beta-hydroxysteroid dehydrogenase type-2

The bovine 11β-hydroxysteroid dehydrogenase type 2 enzyme (11β-HSD-2) cDNA was cloned from three overlapping PCR fragments using primers based on the human and ovine 11β-HSD-2 cDNA sequences. Both cDNA ends were obtained by a modified RACE (Rapid Amplification of cDNA Ends) method. The bovine 11β-HSD-2 cDNA is 1878 bp long, excluding the poly(A) tail. It consists of a 5′-untranslated region of 133 bp, an open reading frame of 1215 bp and a 3′-untranslated region of 530 bp. Bovine 11β-HSD-2 cDNA is highly homologous to that of the sheep (92%) and less related to the human (67%), rabbit (65%), rat (52%) and mouse (45%) cDNA. The predicted bovine 11β-HSD-2 protein contains 404 amino acid residues with a calculated mol wt of 43,985. It is homologous to the sheep (98%) and human (88%) protein, and less related to that of the rabbit (76%), rat (80%) and mouse (77%). The cloned 11β-HSD-2 cDNA was transfected into CHOP cells and the enzymatic characteristics determined. The enzyme functions primarily as an oxidase, uses NAD⁺ and is more active with corticosterone as a substrate than with cortisol or dexamethasone. It is expressed in high concentrations in kidney, adrenal and colon, and in small concentrations in liver, heart and lung. In conclusion, the 11β-HSD-2 enzyme of cattle is very similar to that of other species in its structure and enzymatic characteristics.