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ABSTRACT: We report characterization of a novel member of the short chain dehydrogenase/reductase superfamily. The 1513-base pair cDNA encodes a 319-amino acid protein. The corresponding gene spans over 26 kilobase pairs on chromosome 2 and contains five exons. The recombinant protein produced using the baculovirus system is localized in the microsomal fraction of Sf9 cells and is an integral membrane protein with cytosolic orientation of its catalytic domain. The enzyme exhibits an oxidoreductase activity toward hydroxysteroids with NAD(+) and NADH as the preferred cofactors. The enzyme is most efficient as a 3alpha-hydroxysteroid dehydrogenase, converting 3alpha-tetrahydroprogesterone (allopregnanolone) to dihydroprogesterone and 3alpha-androstanediol to dihydrotestosterone with similar catalytic efficiency (V(max) values of 13-14 nmol/min/mg microsomal protein and K(m) values of 5-7 microm). Despite approximately 44-47% sequence identity with retinol/3alpha-hydroxysterol dehydrogenases, the enzyme is not active toward retinols. The corresponding message is abundant in human trachea and is present at lower levels in the spinal cord, bone marrow, brain, heart, colon, testis, placenta, lung, and lymph node. Thus, the new short chain dehydrogenase represents a novel type of microsomal NAD(+)-dependent 3alpha-hydroxysteroid dehydrogenase with unique catalytic properties and tissue distribution.
Journal of Biological Chemistry 07/2001; 276(25):22278-86. · 4.77 Impact Factor
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ABSTRACT: This manuscript reports further characterization of the recently discovered human short-chain alcohol dehydrogenase, proposed to oxidize 3alpha-androstanediol to dihydrotestosterone in testis and prostate (M. G. Biswas and D. W. Russell, 1997, J. Biol. Chem. 272, 15959-15966). Enzyme expressed using the Baculovirus System localized in the microsomal fraction and catalyzed oxidation and reduction of the functional groups on steroids at carbons 3 and 17. Autoradiography assays revealed that the enzyme was most efficient as a 3alpha-hydroxysteroid oxidoreductase. High affinity of the enzyme for NADH (Km of 0.18 microM), lack of stereospecificity in the reductive direction, and poor efficiency for 3beta- versus 3alpha-hydroxyl oxidation could account for the observed transient accumulation of 3beta-stereoisomers in the oxidative reaction. Consistent with the 65% sequence identity with RoDH dehydrogenases, the enzyme oxidized all-trans-retinol with the Km value of 3.2 microM and Vmax value of 1.2 nmol/min per milligram microsomes. 13-cis-Retinol and all-trans-retinol bound to the cellular retinol-binding protein were not substrates. Neurosteroid allopregnanolone was a better substrate than all-trans-retinol with the Km and Vmax values of 0.24 microM and 14.7 nmol/min per milligram microsomes. Northern blot analysis revealed that the corresponding mRNA was present in adult human brain (caudate nucleus, amygdala, hippocampus, substantia nigra, thalamus) and spinal cord in addition to other tissues. The message was also detected in fetal lung, liver, and brain. Antibodies against the enzyme recognized a protein of approximately 35 kDa in the particulate fraction of human tissues. This study presents new information about enzymatic properties, substrate specificity, and tissue distribution of this enzyme, and provides a better insight into its possible physiological function(s).
Archives of Biochemistry and Biophysics 03/2001; 386(1):1-10. · 2.93 Impact Factor
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ABSTRACT: We have previously characterized the first human NAD(+)-dependent short chain dehydrogenase capable of oxidizing all-trans-retinol and androgens, and found only in the liver and skin. In a search for related human enzymes, we identified a partial open reading frame, which exhibited >60% sequence identity to human RoDH-4. The full-length cDNA for this enzyme was determined in our laboratory by 5'-RACE PCR and was found to be identical to the recently reported novel type of oxidative human 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD). Analysis of the genomic structure revealed that the gene for RoDH-like 3alpha-HSD has four translated exons and, possibly, a fifth exon that codes for the 5'-untranslated region. The gene for RoDH-4 appears to have only four exons. The positions of exon-intron boundaries and the sizes of the protein coding regions are identical in 3alpha-HSD and RoDH-4. Moreover, both genes are mapped to chromosome 12q13, and are located in a close proximity to each other. Both genes appear to have satellite pseudogenes. Thus, RoDH-4 and 3alpha-HSD genes share similar structural organization and cluster on human chromosome 12, near the gene for 11-cis retinol dehydrogenase.
Chemico-Biological Interactions 01/2001; 130-132(1-3):457-67. · 2.46 Impact Factor
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Methods in Enzymology 02/2000; 324:207-18. · 2.04 Impact Factor
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ABSTRACT: All-trans retinoic acid (atRA) is a powerful morphogen synthesized in a variety of tissues. Oxidation of all-trans retinol to all-trans retinal determines the overall rate of atRA biosynthesis. This reaction is catalyzed by multiple dehydrogenases in vitro. In the cells, most all-trans retinol is bound to cellular retinol binding protein (CRBP). Whether retinoic acid is produced from the free or CRBP-bound retinol in vivo is not known. The current study investigated whether human medium-chain alcohol/retinol dehydrogenases (ADH) can oxidize the CRBP-bound retinol. The results of this study suggest that retinol bound to CRBP cannot be channeled to the active site of ADH. Thus, the contribution of ADH isozymes to retinoic acid biosynthesis will depend on the amount of free retinol in each cell. Physiological levels of ethanol will substantially inhibit the oxidation of free retinol by human ADHs: class I, alpha alpha and beta 2 beta 2; class II, pi pi; and class IV, sigma sigma.
Biochemical and Biophysical Research Communications 09/1998; 249(1):191-6. · 2.48 Impact Factor
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ABSTRACT: We report the cDNA sequence and catalytic properties of a new member of the short chain dehydrogenase/reductase superfamily. The 1134-base pair cDNA isolated from the human liver cDNA library encodes a 317-amino acid protein, retinol dehydrogenase 4 (RoDH-4), which exhibits the strongest similarity with rat all-trans-retinol dehydrogenases RoDH-1, RoDH-2, and RoDH-3, and mouse cis-retinol/androgen dehydrogenase (</=73% identity). The mRNA for RoDH-4 is abundant in adult liver, where it is translated into RoDH-4 protein, which is associated with microsomal membranes, as evidenced by Western blot analysis. Significant amounts of RoDH-4 message are detected in fetal liver and lung. Recombinant RoDH-4, expressed in microsomes of Sf9 insect cells using BacoluGold Baculovirus system, oxidizes all-trans-retinol and 13-cis-retinol to corresponding aldehydes and oxidizes the 3alpha-hydroxysteroids androstane-diol and androsterone to dihydrotestosterone and androstanedione, respectively. NAD+ and NADH are the preferred cofactors, with apparent Km values 250-1500 times lower than those for NADP+ and NADPH. All-trans-retinol and 13-cis-retinol inhibit RoDH-4 catalyzed oxidation of androsterone with apparent Ki values of 5.8 and 3.5 microM, respectively. All-trans-retinol bound to cellular retinol-binding protein (type I) exhibits a similar Ki value of 3.6 microM. Unliganded cellular retinol-binding protein has no effect on RoDH activity. Citral and acyclic isoprenoids also act as inhibitors of RoDH-4 activity. Ethanol is not inhibitory. Thus, we have identified and characterized a sterol/retinol-oxidizing short chain dehydrogenase/reductase that prefers NAD+ and recognizes all-trans-retinol as substrate. RoDH-4 can potentially contribute to the biosynthesis of two powerful modulators of gene expression: retinoic acid from retinol and dihydrotestosterone from 3alpha-androstane-diol.
Journal of Biological Chemistry 07/1998; 273(31):19778-85. · 4.77 Impact Factor
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ABSTRACT: A human liver carboxylesterase (hCE-2) that catalyzes the hydrolysis of the benzoyl group of cocaine and the acetyl groups of 4-methylumbelliferyl acetate, heroin, and 6-monoacetylmorphine was purified from human liver. The purified enzyme exhibited a single band on SDS-polyacrylamide gel electrophoresis with a subunit mass of approximately 60 kDa. The native enzyme was monomeric. The isoelectric point of hCE-2 was approximately 4.9. Treatment with endoglycosidase H caused an increase in electrophoretic mobility indicating that the liver carboxylesterase was a glycoprotein of the high mannose type. The complete cDNA nucleotide sequence was determined. The authenticity of the cDNA was confirmed by a perfect sequence match of 78 amino acids derived from the hCE-2 purified from human liver. The mature 533-amino acid enzyme encoded by this cDNA shared highest sequence identity with the rabbit liver carboxylesterase form 2 (73%) and the hamster liver carboxylesterase AT51p (67%). Carboxylesterases with high sequence identity to hCE-2 have not been reported in mouse and rat liver. hCE-2 exhibited different drug ester substrate specificity from the human liver carboxylesterase called hCE-1, which hydrolyzes the methyl ester of cocaine. hCE-2 had higher catalytic efficiencies for hydrolysis of 4-methylumbelliferyl acetate, heroin, and 6-monoacetylmorphine and greater inhibition by eserine than hCE-1. hCE-2 may play an important role in the degradation of cocaine and heroin in human tissues.
Journal of Biological Chemistry 07/1997; 272(23):14769-75. · 4.77 Impact Factor
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ABSTRACT: This study was undertaken to identify the cytosolic 40-kDa zinc-containing alcohol dehydrogenases that oxidize all-trans-retinol and steroid alcohols in fetal tissues. Degenerate oligonucleotide primers were used to amplify by polymerase chain reaction 500-base pair fragments of alcohol dehydrogenase cDNAs from chick embryo limb buds and heart. cDNA fragments that encode an unknown putative alcohol dehydrogenase as well as the class III alcohol dehydrogenase were identified. The new cDNA hybridized with two messages of approximately 2 and 3 kilobase pairs in the adult chicken liver but not in the adult heart, muscle, testis, or brain. The corresponding complete cDNA clones with a total length of 1390 base pairs were isolated from a chicken liver lambdagt11 cDNA library. The open reading frame encoded a 375-amino acid polypeptide that exhibited 67 and 68% sequence identity with chicken class I and III alcohol dehydrogenases, respectively, and had lower identity with mammalian class II (55-58%) and IV (62%) isozymes. Expression of the new cDNA in Escherichia coli yielded an active alcohol dehydrogenase (ADH-F) with subunit molecular mass of approximately 40 kDa. The specific activity of the recombinant enzyme, calculated from active site titration of NADH binding, was 3.4 min-1 for ethanol at pH 7.4 and 25 degrees C. ADH-F was stereospecific for the 3beta,5alpha- versus 3beta,5beta-hydroxysteroids. The Km value for ethanol at pH 7.4 was 17 mM compared with 56 microM for all-trans-retinol and 31 microM for epiandrosterone. Antiserum against ADH-F recognized corresponding protein in the chicken liver homogenate. We suggest that ADH-F represents a new class of alcohol dehydrogenase, class VII, based on its primary structure and catalytic properties.
Journal of Biological Chemistry 04/1997; 272(11):7494-500. · 4.77 Impact Factor
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Advances in experimental medicine and biology 02/1997; 414:321-9. · 1.09 Impact Factor
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ABSTRACT: Recent evidence from this laboratory indicates that at least two isoenzymic forms of pyruvate dehydrogenase kinase (PDK1 and PDK2) may be involved in the regulation of enzymatic activity of mammalian pyruvate dehydrogenase complex by phosphorylation (Popov, K.M., Kedishvili, N.Y., Zhao, Y., Gudi, R., and Harris, R.A. (1994) J. Biol. Chem. 269, 29720-29724). The present study was undertaken to further explore the diversity of the pyruvate dehydrogenase kinase gene family. Here we report the deduced amino acid sequences of three isoenzymic forms of PDK found in humans. In terms of their primary structures, two isoenzymes identified in humans correspond to rat PDK1 and PDK2, whereas a third gene (PDK3) encodes for a new isoenzyme that shares 68% and 67% of amino acid identities with PDK1 and PDK2, respectively. PDK3 cDNA expressed in Eschierichia coli directs the synthesis of a polypeptide with a molecular mass of approximately 45,000 Da that possesses catalytic activity toward kinase-depleted pyruvate dehydrogenase. PDK3 appears to have the highest specific activity among the three isoenzymes tested as recombinant proteins. Tissue distribution of all three isoenzymes of human PDK was characterized by Northern blot analysis. The highest amount of PDK2 mRNA was found in heart and skeletal muscle, the lowest amount in placenta and lung. Brain, kidney, pancreas, and liver expressed an intermediate amount of PDK2 (brain > kidney = pancreas > liver). The tissue distribution of PDK1 mRNA differs markedly from PDK2. The message for PDK1 was expressed predominantly in heart with only modest levels of expression in other tissues (skeletal muscle > liver > pancreas > brain > placenta = lung > kidney). In contrast to PDk1 and PDK2, which are expressed in all tissues tested, the message for PDK3 was found almost exclusively in heart and skeletal muscle, indicating that PDK3 may serve specialized functions characteristic of muscle tissues. In all tissues tested thus far, the level of expression of PDK2 mRNA was essentially higher than that of PDK1 and PDK3, consistent with the idea that PDK2 is a major isoenzyme responsible for regulation of pyruvate dehydrogenase in human tissues.
Journal of Biological Chemistry 12/1995; 270(48):28989-94. · 4.77 Impact Factor
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ABSTRACT: Cimetidine, an H2-receptor antagonist, is one of the most commonly prescribed drugs in the world. It has been reported to increase blood alcohol concentrations in drinking individuals. To determine if this increase could be due to inhibition of alcohol dehydrogenase activity, the effect of the drug on ethanol oxidation by gastric sigma sigma alcohol dehydrogenase and liver beta 2 beta 2, pi pi, and chi chi alcohol dehydrogenase isoenzymes was observed. Cimetidine inhibited all isoenzymes studied except chi chi; the chi chi isoenzyme showed no inhibition up to 5 mM cimetidine. Inhibition of the alcohol dehydrogenase isoenzymes by the H2-receptor antagonists nizatidine, ranitidine, and famotidine was negligible. Docking simulations with the beta 2.NAD+.4-iodopyrazole X-ray structure indicated that cimetidine fit well into the substrate binding site. The substitution on the thiazole ring of nizatidine, however, prevented docking into the binding site. Cimetidine inhibition of ethanol oxidation by sigma sigma and beta 2 beta 2 was competitive with varied ethanol, exhibiting Ki values of 2.8 +/- 0.4 mM and 0.77 +/- 0.07 mM, respectively. Cimetidine inhibition of ethanol oxidation by pi pi was noncompetitive with varied ethanol (Ki = 0.50 +/- 0.03 mM). Inhibition of ethanol oxidation by sigma sigma and beta 2 beta 2 with varied NAD+ was competitive. These results, together with the cimetidine inhibition kinetics of acetaldehyde reduction by sigma sigma and beta 2 beta 2, with either varied NADH or varied acetaldehyde, are consistent with cimetidine binding to two enzyme species. These species are free enzyme and the productive enzyme.NAD+ complex.
Biochemistry 04/1995; 34(12):4008-14. · 3.42 Impact Factor
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ABSTRACT: A full-length 1966-base pair clone of the human class IV alcohol dehydrogenase (sigma-ADH) was isolated from a human stomach cDNA library. The 373-amino acid sigma-ADH encoded by this cDNA was expressed in Escherichia coli. The specific activity of the recombinant enzyme for ethanol oxidation at pH 7.5 and 25 degrees C, calculated from active-site titration of NADH binding, was 92 +/- 9 units/mg. Kinetic analysis of the catalytic efficiency (kcat/KM) of recombinant sigma-ADH for oxidation of primary alcohols indicated broad substrate specificity. Recombinant human sigma-ADH exhibited high catalytic efficiency for oxidation of all-trans-retinol to all-trans-retinal. This pathway is important in the synthesis of the transcriptional regulator all-trans-retinoic acid. Secondary alcohols and 3 beta-hydroxysteroids were inactive with sigma-ADH or were oxidized with very low efficiency. The KM of sigma-ADH for ethanol was 25 mM, and the KM for primary straight chain alcohols decreased substantially as chain length increased. There are important amino acid differences in the alcohol-binding site between the human class IV (sigma) and human class I (beta) alcohol dehydrogenases that appear to explain the high catalytic efficiency for all-trans-retinol, the high kcat for ethanol, and the low catalytic efficiency for secondary alcohols of sigma-ADH relative to beta 1-ADH. For example, modeling the binding of all-trans-retinol in the human beta 1-ADH structure suggested that coordination of retinol to the active-site zinc is hindered by a loop from residues 114 to 120 that is at the entrance to the alcohol-binding site. The deletion of Gly-117 in human sigma-ADH and a substitution of Leu for the bulky Tyr-110 appear to facilitate retinol access to the active-site zinc.
Journal of Biological Chemistry 03/1995; 270(8):3625-30. · 4.77 Impact Factor
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ABSTRACT: Molecular cloning has provided evidence for a new family of protein kinases in eukaryotic cells. These kinases show no sequence similarity with other eukaryotic protein kinases, but are related by sequence to the histidine protein kinases found in prokaryotes. These protein kinases, responsible for phosphorylation and inactivation of the branched-chain alpha-ketoacid dehydrogenase and pyruvate dehydrogenase complexes, are located exclusively in mitochondrial matrix space and have most likely evolved from genes originally present in respiration-dependent bacteria endocytosed by primitive eukaryotic cells. Long-term regulatory mechanisms involved in the control of the activities of these two kinases are of considerable interest. Dietary protein deficiency increases the activity of branched-chain alpha-ketoacid dehydrogenase kinase associated with the branched-chain alpha-ketoacid dehydrogenase complex. The amount of branched-chain alpha-ketoacid dehydrogenase kinase protein associated with the branched-chain alpha-ketoacid dehydrogenase complex and the message level for branched-chain alpha-ketoacid dehydrogenase kinase are both greatly increased in the liver of rats starved for protein, suggesting increased expression of the gene encoding branched-chain alpha-ketoacid dehydrogenase kinase. The increase in branched-chain alpha-ketoacid dehydrogenase kinase activity results in greater phosphorylation and lower activity of the branched-chain alpha-ketoacid dehydrogenase complex. The metabolic consequence is conservation of branched chain amino acids for protein synthesis during periods of dietary protein deficiency. Two isoforms of pyruvate dehydrogenase kinase have been identified and cloned. Pyruvate dehydrogenase kinase 1, the first isoform cloned, corresponds to the 48 kDa subunit of the pyruvate dehydrogenase kinase isolated from rat heart tissue. Pyruvate dehydrogenase kinase 2, the second isoform cloned, corresponds to the 45 kDa subunit of this enzyme. In addition, it also appears to correspond to a possibly free or soluble form of pyruvate dehydrogenase kinase that was originally named kinase activator protein. Assuming that differences in kinetic and/or regulatory properties of these isoforms exist, tissue specific expression of these enzymes and/or control of their association with the complex will probably prove to be important for the long term regulation of the activity of the pyruvate dehydrogenase complex. Starvation and the diabetic state are known to greatly increase activity of the pyruvate dehydrogenase kinase in the liver, heart and muscle of the rat. This contributes in these states to the phosphorylation and inactivation of the pyruvate dehydrogenase complex and conservation of pyruvate and lactate for gluconeogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)
Advances in Enzyme Regulation 02/1995; 35:147-62.
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ABSTRACT: The branched-chain alpha-ketoacid dehydrogenase complex, catalyst for the rate-limiting step of branched-chain amino acid catabolism, is controlled by a highly specific protein kinase (branched-chain alpha-ketoacid dehydrogenase kinase) that associates tightly with the complex. The activity state (proportion of the enzyme in its active, dephosphorylated state) of the complex varies dramatically in different rat tissues. The activity state of the complex in the liver is greater than that in any other tissue, and liver contains the lowest amount of kinase protein and kinase mRNA. However, protein malnutrition, a condition under which the complex is largely phosphorylated and inactive, resulted in a three- to fourfold increase in hepatic kinase activity with an accompanying increase in amounts of kinase protein and mRNA. Refeeding a 50% protein diet restored the normal activity state and the original levels of kinase protein and mRNA. The amount of kinase protein associated with the complex rather than changes in specific activity of the kinase appears responsible for observed differences in activity states of the complex in several rat tissues tested. Accordingly, the levels of kinase protein and mRNA measured are highest in tissues with greatest kinase activity (heart > kidney > liver), correlating reasonably well inversely with activity state of the branched-chain alpha-ketoacid dehydrogenase complex in the respective tissues. These observations suggest that the amount of kinase protein expressed in various tissues and in response to dietary protein deficiency is an important factor determining the activity state of the complex.
Archives of Biochemistry and Biophysics 02/1995; 316(1):148-54. · 2.93 Impact Factor
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Advances in experimental medicine and biology 02/1995; 372:341-7. · 1.09 Impact Factor
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ABSTRACT: 3T3-L1 fibroblasts have limited enzymatic capacity to oxidize valine. Enzymes expressed in these cells allow efficient oxidation of only the first carbon of this branched chain amino acid. The pathway is effectively truncated at the level of 3-hydroxyisobutyrate because of very low expression of two enzymes required for the complete pathway, 3-hydroxyisobutyrate dehydrogenase and methylmalonate semialdehyde dehydrogenase. These two enzymes, as well as the branched chain alpha-ketoacid dehydrogenase, are markedly induced upon differentiation of 3T3-L1 fibroblasts into adipocytes. Flux through the first two decarboxylation steps of valine catabolism is increased dramatically after differentiation, particularly through the step catalyzed by methylmalonate semialdehyde dehydrogenase. Activation of the distal portion of the valine catabolic pathway correlates with significant increases in enzyme protein and mRNA levels for 3-hydroxyisobutyrate dehydrogenase and methylmalonate semialdehyde dehydrogenase, and this establishes the pathway in 3T3-L1 adipocytes for utilization of valine carbon for lipogenesis. The induction profiles of 3-hydroxyisobutyrate dehydrogenase and methylmalonate semialdehyde dehydrogenase are very similar, suggesting coordinate regulation of the expression of these two valine pathway-specific enzymes. Induction of valine catabolism in 3T3-L1 cells is solely differentiation dependent, suggesting regulation by the same factors that govern differentiation of 3T3-L1 fibroblasts into adipocytes.
Archives of Biochemistry and Biophysics 01/1995; 315(2):317-22. · 2.93 Impact Factor
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ABSTRACT: Purified preparations of rat heart pyruvate dehydrogenase kinase have two polypeptides with molecular weights of 48,000 (p48) and 45,000 (p45). Recently, we reported the primary structure of p48 (Popov, K. M., Kedishvili, N. Y., Zhao, Y., Shimomura, Y., Crabb, D. W., and Harris, R. A. (1993) J. Biol. Chem. 268, 26602-26606) and presented evidence that (i) it exhibits kinase activity for pyruvate dehydrogenase and (ii) it belongs to a family of mitochondrial protein kinases unique from other eukaryotic protein kinases. Here, we report the molecular cloning and deduced amino acid sequence of p45. The protein sequence of p45 has 70% identity to the protein sequence of p48. Minor differences exist throughout the protein sequences with the greatest difference occurring at the amino termini. Recombinant p45 protein, expressed in Escherichia coli and purified to homogeneity, catalyzed the phosphorylation and inactivation of kinase-depleted pyruvate dehydrogenase complex, indicating that p45 and p48 correspond to different isoforms of pyruvate dehydrogenase kinase. Northern blot analysis revealed a single hybridizing species of 2.5 kilobases. The highest level of p45 message expression was found in heart and skeletal muscle and the lowest in spleen and lung. Liver, kidney, brain, and testis express intermediate amounts of p45 mRNA. In contrast, p48 mRNA is predominantly expressed in heart, with other tissues expressing only a modest amount of this message. Tissue-specific expression of isoforms of pyruvate dehydrogenase kinase may indicate the existence of tissue-specific mechanisms for the regulation of pyruvate dehydrogenase activity.
Journal of Biological Chemistry 12/1994; 269(47):29720-4. · 4.77 Impact Factor
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ABSTRACT: Levels of expression of two subunits of the liver branched-chain alpha-ketoacid dehydrogenase complex in response to extremes of dietary protein intake (50% versus 0% protein diet) were determined by quantitative immunoblotting. Dietary protein deficiency decreased the amount of E1 alpha protein to a greater extent than E2 protein. The ratio of E1 alpha to E2 was below 1 in the liver of animals starved for protein and above 1 in the liver of animals fed the high-protein diet. Supplementation of the 0% protein diet with 5% leucine (but not 5% valine) had the same effect as the 50% protein diet. The extremes of dietary protein also resulted in a divergent pattern of expression of the mRNAs for the subunits of the complex. The E1 beta message showed the expected corollary of being greater in the liver of the high-protein-fed rats than the no-protein-fed rats. In contrast, the E2 message was not affected by the two extremes of dietary protein and the E1 alpha message was greater in the liver of the no-protein-fed rats than the high-protein-fed rats. Thus, coordinate regulation of gene expression of the subunits of the complex does not occur in response to dietary protein. Post-transcriptional regulatory mechanisms most likely determine the amount of the complex and the ratio of its subunits. The decrease in E1 alpha/E2 protein ratio that occurs in dietary protein deficiency may increase sensitivity of the complex to phosphorylation-mediated inhibition by branched-chain alpha-ketoacid dehydrogenase kinase.
Archives of Biochemistry and Biophysics 03/1994; 308(2):446-53. · 2.93 Impact Factor
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ABSTRACT: We recently reported molecular cloning of the branched chain alpha-ketoacid dehydrogenase kinase, the first mitochondrial protein kinase to be cloned (Popov, K. M., Zhao, Y., Shimomura, Y., Kuntz, M. J., and Harris, R. A. (1992) J. Biol. Chem. 267, 13127-13130). From a search for proteins related to the branched chain alpha-ketoacid dehydrogenase kinase, a cDNA encoding the 434 amino acid residues corresponding to pyruvate dehydrogenase kinase has been cloned from a rat heart cDNA library. Evidence that the clone codes for pyruvate dehydrogenase kinase includes: (a) the deduced amino acid sequence is identical to the partial sequence of the kinase determined by direct sequencing; (b) expression of the cDNA in Escherichia coli resulted in synthesis of a protein that phosphorylated and inactivated the pyruvate dehydrogenase complex; (c) kinase activity of the recombinant protein is sensitive to inhibition by a specific inhibitor of pyruvate dehydrogenase kinase; and (d) antiserum raised against the recombinant protein recognized the protein subunit known to correspond to pyruvate dehydrogenase kinase in a highly purified preparation of the pyruvate dehydrogenase complex. Like the branched chain alpha-ketoacid dehydrogenase kinase, pyruvate dehydrogenase kinase lacks motifs usually associated with eukaryotic Ser/Thr-protein kinases. Considerable sequence similarity exists between these mitochondrial protein kinases and members of the prokaryotic histidine kinase family, a diverse set of sensing and response systems important in the regulation of bacterial processes. Thus, molecular cloning of these proteins establishes a new eukaryotic family of protein kinases that is related to a prokaryotic family of protein kinases.
Journal of Biological Chemistry 01/1994; 268(35):26602-6. · 4.77 Impact Factor
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ABSTRACT: The complete amino acid sequence of rat liver CoA-dependent methylmalonate semialdehyde dehydrogenase, the enzyme responsible for the oxidative decarboxylation of malonate- and methylmalonate semialdehydes to acetyl- and propionyl-CoA in the distal portions of the valine and pyrimidine catabolic pathways, has been deduced from overlapping cDNAs obtained by screening a lambda gt11 library with nondegenerate oligonucleotide probes synthesized according to PCR-amplified portions coding for the N-terminal amino acid sequence of the enzyme. Although unique because of its requirement for coenzyme A, the methylmalonate semialdehyde dehydrogenase clearly belongs to the aldehyde dehydrogenase superfamily of enzymes. Quantitation of mRNA and protein levels indicates tissue-specific expression of methylmalonate semialdehyde dehydrogenase. A large increase in expression of methylmalonate semialdehyde dehydrogenase occurs during 3T3-L1 preadipocyte differentiation into adipocytes. The complete amino acid sequence of rat liver branched-chain alpha-ketoacid dehydrogenase kinase, the enzyme responsible for phosphorylation and inactivation of the branched-chain alpha-ketoacid dehydrogenase complex, was deduced from a cDNA cloned by a procedure similar to that described above for the methylmalonate semialdehyde dehydrogenase. Expression of the cDNA in E. coli yielded a protein that phosphorylated and inactivated the branched-chain alpha-ketoacid dehydrogenase complex. Very little sequence similarity between branched-chain alpha-ketoacid dehydrogenase kinase and other eukaryotic protein kinases could be identified. However, a high degree of similarity within subdomains characteristic of prokaryotic histidine protein kinases was apparent. Thus, this first mitochondrial protein kinase to be cloned appears closer, evolutionarily, to the prokaryotic histidine protein kinases than eukaryotic ser/thr protein kinases.
Advances in Enzyme Regulation 02/1993; 33:255-65.
