Comparison of 3-hydroxybutyrate dehydrogenase from bovine heart and rat liver mitochondria

3-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with an absolute requirement of phosphatidylcholine for enzymatic activity. Purification of the enzyme to homogeneity from bovine heart mitochondria was described more than a decade ago [H. G. Bock and S. Fleischer (1975) J. Biol. Chem. 250, 5774-5781]. We have modified the purification procedure so that it is faster, the yield has been improved, and the specific activity is greater by approximately 50%. The updated procedure has also been applied to isolate the enzyme from rat liver mitochondria. Characteristics of the enzyme from bovine heart and rat liver mitochondria have been compared and found to be similar with respect to: (1) purification characteristics; (2) amino acid composition; (3) pH optimum for enzymatic activity; (4) kinetic characteristics; (5) molecular weight as determined by sedimentation equilibrium in guanidine hydrochloride; (6) peptide maps; (7) immunological cross-reactivity. These studies show that 3-hydroxybutyrate dehydrogenase from bovine heart and rat liver mitochondria, though similar, are not identical.     

Metabolic control of the expression of mitochondrial D-beta-hydroxybutyrate dehydrogenase, a ketone body converting enzyme

The effects of various metabolic conditions inducing an overproduction of ketone bodies in the rat were studied at different levels of D-beta-hydroxybutyrate dehydrogenase expression, i.e., enzymatic activity and protein content in purified mitochondria, and translational activity of isolated free cytosolic polysomes. The strongest variations were obtained in diabetes mellitus where the D-beta-hydroxybutyrate dehydrogenase expression is largely decreased. Insulin can reverse this strong effect. Modulation of liver enzyme activity and of enzyme content was observed under the other conditions tested, i.e., a decrease and an increase in starvation and hyperlipidemic conditions, respectively. A comparative study was carried out on the enzyme of extrahepatic tissues, i.e., heart, kidney and brain. The results indicate that the D-beta-hydroxybutyrate dehydrogenase function appears to be controlled, at least at the translational, post-translational and catalytic levels.     

Regulators of 3-hydroxybutyrate dehydrogenase activity in rat liver mitochondria

Effects of numerous organic acids on the 3-hydroxybutyrate dehydrogenase activity were studied in isolated rat liver mitochondria with nonspecific permeability. Amino acids, most of citric acid cycle intermediates, lactate, maleate, acetate, glycerol-3-phosphate, urea, palmitate, and phosphoenolpyruvate plus ADP were shown to modify the enzyme activity insignificantly. The inhibitory effect of pyruvate seems to be a result of the concomitant cytosolic lactate dehydrogenase activity, and the effect of oxaloacetate is that of the mitochondrial matrix malate dehydrogenase activity. Malonate proves to be a competitive inhibitor of the 3-hydroxybutyrate dehydrogenase activity, enzyme affinity for malonate being the same irrespective of the source or purification of the preparation.     

Modified oscillation behavior and decreased D-3-hydroxybutyrate dehydrogenase activity in diabetic rat liver mitochondria

One month after induction of diabetes in adult white rats with streptozotocin or 4–10 months after its induction by pancreatectomy (in every case glycemia was over 3 g/liter), the following alterations were observed in liver mitochondria: (a) a decrease of amplitude and an increase of the damping factor of volume oscillations induced by potassium ions and valinomycin; (b) a 50% decrease of d-3-hydroxybutyrate dehydrogenase (HBD) activity in mitochondria disrupted by repeated freeze-thawing; (c) a similar decrease in the rate of d-3-hydroxybutyrate oxidation by intact mitochondria; (d) a significant increase of cytochrome oxidase activity and cytochrome aa₃ content. Measurement of succinate dehydrogenase and NADH dehydrogenase activity, the cytochrome b, c₁, and c content, and the P:O ratio for mitochondria oxidizing d-3-hydroxybutyrate did not reveal significant differences between control and diabetic rat mitochondria. In the streptozotocin-injected rats, the variation of HBD activity and the modification of the mitochondrial oscillation pattern were time-dependent phenomena, both effects reaching their maximal expression about 1 month after the onset of diabetes. The variation of HBD activity followed a biphasic course, since it rose to above the control level during the first 2 weeks of diabetes, then fell progressively to about half the control value after the third week. Treatment of diabetic rats with NPH insulin (5 IU twice daily, for 3 days, reinforced by the same dose 45 min before sacrifice) restored the mitochondrial oscillation pattern, HBD activity, and rate of d-3-hydroxybutyrate oxidation by intact mitochondria to their normal values.     

Human liver long-chain 3-hydroxyacyl-coenzyme A dehydrogenase is a multifunctional membrane-bound beta-oxidation enzyme of mitochondria.

We have purified to homogeneity the long-chain specific 3-hydroxyacyl-CoA dehydrogenase from mitochondrial membranes of human infant liver. The enzyme is composed of non-identical subunits of 71 kDa and 47 kDa within a native structure of 230 kDa. The pure enzyme is active with 3-ketohexanoyl-CoA and gives maximum activity with 3-ketoacyl-CoA substrates of C10 to C16 acyl-chain length but is inactive with acetoacetyl-CoA. In addition to 3-hydroxyacyl-CoA dehydrogenase activity, the enzyme possesses 2-enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase activities which cannot be separated from the dehydrogenase. None of these enzymes show activity with C4 substrates but all are active with C6 and longer acyl-chain length substrates. They are thus distinct from any described previously. This human liver mitochondrial membrane-bound enzyme catalyses the conversion of medium- and long-chain 2-enoyl-CoA compounds to: 1) 3-ketoacyl-CoA in the presence of NAD alone and 2) to acetyl-CoA (plus the corresponding acyl-CoA derivatives) in the presence of NAD and CoASH. It is therefore a multifunctional enzyme, resembling the beta-oxidation enzyme of E. coli, but unique in its membrane location and substrate specificity. We propose that its existence explains the repeated failure to detect any intermediates of mitochondrial beta-oxidation.     

Distribution of 3-hydroxybutyrate dehydrogenase in primary lobules of rat liver.

In an attempt to establish the functional organization of the hepatic parenchymal unit, we used histo- and microchemical procedures to assess metabolic liver cell heterogeneity at the level of the primary lobule. Because of the close interrelation of glucogenesis and ketone body formation, and in view of the distinct regional differences of the in vivo activity of glucose-6-phosphatase (G6Pase), these techniques were used on livers from male rats to investigate the distribution of the ketogenic enzyme, 3-hydroxybutyrate dehydrogenase (3-HBDH), during the post-resorptive phase. A close reciprocity was found between the general increase in the activity of 3-HBDH and the decrease of the in vivo activity of G6Pase along the sinusoidal axis, and also with regard to enzyme gradients along sinusoids of different origin. The activity of the ketogenic enzyme was higher throughout septal than portal sinusoids, whereas the opposite applied to the glucogenic enzyme. Histo- and microchemical data support the concept of a lobular parenchymal unit composed of "primary lobules," and show also that hepatocyte function varies with cell location along the sinusoidal axis and with the origin of the sinusoids.     

Concerning the decreased D-3-hydroxybutyrate dehydrogenase activity in the liver and heart of hyperthyroid rats

Summary Whereas in rat liver mitochondria the hyperthyroid state causes an increase both in fatty acid unsaturation and in the Ea of D-3-hydroxybutyrate dehydrogenase and a decrease in phase transition temperature, in hyperthyroid rat heart mitochondria these changes are negligible. D-3-hydroxybutyrate dehydrogenase in both the liver and the heart mitochondria of hyperthyroid rats is reduced by about 35% [l2] but this reduction is not due to changes in membrane fluidity in either tissue. Hypothyroidism, on the other hand, affects BDH activity in neither heart nor liver.Abbreviations BDH D-3-hydroxybutyrate dehydrogenase - PTU 6n-propyl-2-thiouracil - T₃,3,3 5-L-triiodothyronine - Tm temperature phase transition - Ea apparent activation energy     

Diurnal changes in the 3-hydroxybutyrate dehydrogenase activity of rat liver mitochondria during chronic alcohol consumption and after cessation

The activity of 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) in the isolated rat liver mitochondria changes but slightly during 24 h. In animals which were fed 10% ethanol solution for 3.5 months the enzyme activity varies within the daily cycle: maximum--at 10 p. m., minimum--at 1-4 p. m. and at 4-7 a. m.; the average daily activity gets three times lower. The cessation of the alcohol consumption makes average daily activity of the enzyme only two times higher, but the character of daily changes in the activity is different: the maximum is observed at 4-7 p. m., the minimum at 4-7 a. m.     

Effects of 2-mercaptoacetate in isolated liver mitochondria in vitro. Competitive inhibition of 3-hydroxybutyrate dehydrogenase and depression of the beta-oxidation pathway.

The effects of 2-mercaptoacetate on the respiration rates induced by different substrates were studied in vitro in isolated liver mitochondria. With palmitoyl-L-carnitine or 2-oxoglutarate as the substrate, the ADP-stimulated respiration (State 3) was dose-dependently inhibited by 2-mercaptoacetate. with glutamate or succinate as the substrate. State-3 respiration was only slightly inhibited by 2-mercaptoacetate. In contrast, the oxidation rate of 3-hydroxybutyrate was competitively inhibited by 2-mercaptoacetate in both isolated mitochondria and submitochondrial particles. In uncoupled mitochondria and in mitochondria in which ATP- and GTP-dependent acyl-CoA biosynthesis was inhibited, the inhibitory effect of 2-mercaptoacetate on palmitoyl-L-carnitine oxidation was abolished; under the same conditions, however, inhibition of 3-hydroxybutyrate oxidation by 2-mercaptoacetate still persisted. These results led to the following conclusions: 2-mercaptoacetate itself enters the mitochondrial matrix, inhibits fatty acid oxidation through a mechanism requiring an energy-dependent activation of 2-mercaptoacetate and itself inhibits 3-hydroxybutyrate oxidation through a competitive inhibition of the membrane-bound 3-hydroxybutyrate dehydrogenase. This study also strongly suggests that the compound responsible for the inhibition of fatty acid oxidation is 2-mercaptoacetyl-CoA.     

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.     

Diurnal changes in succinate and D-3-hydroxybutyrate dehydrogenase activities of rat liver mitochondria after chronic alcohol consumption and withdrawal

1. The succinate dehydrogenase (SDH) and D-3-hydroxybutyrate dehydrogenase (HBDH) activities were measured over a 24-hr period in rat liver mitochondria after chronic alcohol ingestion and withdrawal. 2. The diurnal patterns of both the enzyme activities were shown to change after alcohol consumption, with 64-66% decrease in the daily mean levels. 3. The diurnal rhythms of the SDH and HBDH activities are partially restored 24-72 hr after alcohol withdrawal. 4. There was no correlation between changes in both the enzyme activities and the NAD+/NADH ratio of liver mitochondria from control, ethanol-fed and withdrawn rats over the day.     

Decreased rate of ketone-body oxidation and decreased activity of D-3-hydroxybutyrate dehydrogenase and succinyl-CoA:3-oxo-acid CoA-transferase in heart mitochondria of diabetic rats.

Heart mitochondria from chronically diabetic rats ('diabetic mitochondria'), in metabolic State 3, oxidized 3-hydroxybutyrate and acetoacetate at a relatively slow rate, as compared with mitochondria from normal rats ('normal mitochondria'). No significant differences were observed, however, with pyruvate or L-glutamate plus L-malate as substrates. Diabetic mitochondria also showed decreased 3-hydroxybutyrate dehydrogenase and succinyl-CoA: 3-oxoacid CoA-transferase activities, but cytochrome content and NADH-dehydrogenase, succinate dehydrogenase, cytochrome oxidase and acetoacetyl-CoA thiolase activities proved normal. The decrease of 3-hydroxybutyrate dehydrogenase activity was observed in diabetic mitochondria subjected to different disruption procedures, namely freeze-thawing, sonication or hypoosmotic treatment, between pH 7.5 and 8.5, at temperatures in the range 6-36 degrees C, and in the presence of L-cysteine. Determination of the kinetic parameters of the enzyme reaction in diabetic mitochondria revealed diminution of maximal velocity (Vmax) as its outstanding feature. The decrease in 3-hydroxybutyrate dehydrogenase in diabetic mitochondria was a slow-developing effect, which reached full expression 2-3 months after the onset of diabetes; 1 week after onset, no significant difference between enzyme activity in diabetic and normal mitochondria could be established. Insulin administration to chronically diabetic rats for 2 weeks resulted in limited recovery of enzyme activity. G.l.c. analysis of fatty acid composition and measurement of diphenylhexatriene fluorescence anisotropy failed to reveal significant differences between diabetic and normal mitochondria. The Arrhenius-plot characteristics for 3-hydroxybutyrate dehydrogenase in membranes of diabetic and normal mitochondria were similar. It is assumed that the variation of the assayed enzymes in diabetic mitochondria results from a slow adaptation to the metabolic conditions resulting from diabetes, rather than to insulin deficiency itself.     

Stimulation by 3-hydroxybutyrate of pyruvate carboxylation in mitochondria from rat liver

Isolated rat liver mitochondria incubated in the presence of 3-hydroxybutyrate display a markedly increased rate of pyruvate carboxylation as measured by malate and citrate production from pyruvate. The stimulation was demonstrable both with exogenously added pyruvate, even at saturating concentration, and with pyruvate intramitochondrially generated from alanine. The concentration of DL-3-hydroxybutyrate required for half-maximal stimulation amounted to about 1.5 mM. The intramitochondrial ATP/ADP ratio as well as the matrix acetyl-CoA level was found to remain unchanged by 3-hydroxybutyrate exposure, which, however, lowered the absolute intramitochondrial contents of the respective adenine nucleotides. The effects of 3-hydroxybutyrate were diminished by the concomitant addition of acetoacetate. Moreover, a direct relationship between mitochondrial reduction by proline and the rate of pyruvate carboxylation was observed. The results seem to indicate that the mitochondrial oxidation--reduction state might be involved in the expression of the 3-hydroxybutyrate effect. As to the physiological relevance of the findings, 3-hydroxybutyrate could be shown to activate pyruvate carboxylation in isolated hepatocytes.     

Evidence that the cytosolic activity of 3-hydroxybutyrate dehydrogenase in chicken liver is L-3-hydroxyacid dehydrogenase

Classical fractionation studies showed that chicken liver contains two enzymes which can oxidize DL-3-hydroxybutyrate. The cytosolic enzyme is specific for the L-(+) isomer and accounts for 60% of the total activity. The mitochondrial activity is specific for the D-(-) isomer and accounts for 40% of the total activity. Kinetic studies showed that L-gulonic acid is a competitive inhibitor of the enzyme. We conclude that the cytosolic enzyme is the previously described L-3-hydroxyacid dehydrogenase.     

Characterization of bdhA, encoding the enzyme D-3-hydroxybutyrate dehydrogenase, from Sinorhizobium sp. strain NGR234

A genomic library of Sinorhizobium sp. strain NGR234 was introduced into Escherichia coli LS5218, a strain with a constitutively active pathway for acetoacetate degradation, and clones that confer the ability to utilize D-3-hydroxybutyrate as a sole carbon source were isolated. Subcloning experiments identified a 2.3 kb EcoRI fragment that retained complementing ability, and an ORF that appeared orthologous with known bdhA genes was located within this fragment. The deduced NGR234 BdhA amino acid sequence revealed 91% identity to the Sinorhizobium meliloti BdhA. Site-directed insertion mutagenesis was performed by introduction of a OmegaSmSp cassette at a unique EcoRV site within the bdhA coding region. A NGR234 bdhA mutant, NGRPA2, was generated by homogenotization, utilizing the sacB gene-based lethal selection procedure. This mutant was devoid of D-3-hydroxybutyrate dehydrogenase activity, and was unable to grow on D-3-hydroxybutyrate as sole carbon source. NGRPA2 exhibited symbiotic defects on Leucaena but not on Vigna, Macroptilium or Tephrosia host plants. Furthermore, the D-3-hydroxybutyrate utilization phenotype of NGRPA2 was suppressed by presence of plasmid-encoded multiple copies of the S. meliloti acsA2 gene. The glpK-bdhA-xdhA gene organization and the bdhA-xdhA operon arrangement observed in S. meliloti are also conserved in NGR234.     

Molecular cloning and characterization of (R)-3-hydroxybutyrate dehydrogenase from human heart.

The complete amino acid sequence of human heart (R)-3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) has been deduced from the nucleotide sequence of cDNA clones. This mitochondrial enzyme has an absolute and specific requirement of phosphatidylcholine for enzymic activity (allosteric activator) and is an important prototype of lipid-requiring enzymes. Despite extensive studies, the primary sequence has not been available and is now reported. The mature form of the enzyme consists of 297 amino acids (predicted M(r) of 33,117), does not appear to contain any transmembrane helices, and is homologous with the family of short-chain alcohol dehydrogenases (SC-ADH) (Persson, B., Krook, M., and J?rnvall, H. (1991) Eur. J. Biochem. 200, 537-543) (30% residue identity with human 17 beta-hydroxysteroid dehydrogenase). The first two-thirds of the enzyme includes both putative coenzyme binding and active site conserved residues and exhibits a predicted secondary structure motif (alternating alpha-helices and beta-sheet) characteristic of SC-ADH. Bovine heart peptide sequences (174 residues in nine sequences determined by microsequencing) have extensive homology (89% identical residues) with the deduced human heart sequence. The C-terminal third (Asn-194 to Arg-297) shows little sequence homology with the SC-ADH and likely contains elements that determine the substrate specificity for the enzyme including the phospholipid (phosphatidylcholine) binding site(s). Northern blot analysis identifies a 1.3-kilobase mRNA encoding the enzyme in heart tissue.     

3-Hydroxybutyrate dehydrogenase in the liver mitochondria of rats and chickens

The activity of mitochondrial 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) in rat and chicken liver was found to be comparable with the activity of electron transport chain of rat liver mitochondria. This activity is absent in chicken liver mitochondria, which are devoid of the 3-hydroxybutyrate oxidase activity. Both types of mitochondria have nearly identical respiration parameters but respond differently to Mg2+. It was assumed that chicken liver mitochondria are characterized by a low rate of fatty acids oxidation due to the absence of 3-hydroxybutyrate dehydrogenase in these organelles.