Subcellular localization of branched-chain amino acid aminotransferase and lactate dehydrogenase C4 in rat and mouse spermatozoa.

Spermatozoa isolated from rat and mouse epididymes show a relatively high branched-chain amino acid aminotransferase (leucine aminotransferase, EC 2.6.1.6) activity. There is a significant reduction of leucine aminotransferase and of the isoenzyme C4 of lactate dehydrogenase (EC 1.1.1.27) in the gametes during their epididymal transit. Studies of patterns of liberation of the leucine aminotransferase and of the lactate dehydrogenase C4 from intact spermatozoa, treated with increasing concentrations of digitonin, indicate that both enzymes have the same dual subcellular location, i.e. in the cytosol and in the mitochondria.     

A study on lactate dehydrogenase isozymes in rat ova

The presence of subunits A and B has been demonstrated in mature rat ova by several means, including the immunohistochemical method of Coons, use of antisera against LDH-1 (B₄) and LDH-5 (A₄) isozymes, and polyacrylamide gel electrophoresis.     

Structural relatedness of mouse lactate dehydrogenase isozymes,A4 (muscle), B4 (heart), and C4 (testis)

Three homotetrameric lactate dehydrogenase isozymes, LDH-M(A₄), LDH-H(B₄), and LDH-X(C₄), from DBA/2J mice have been purified by affinity chromatography. The amino acid compositions of the subunits A, B, and C, based on a molecular weight of 36,000, have been determined. The compositional relatedness of these isozymes indicates that subunits A (muscle) and B (heart) are more closely related to each other than to subunit C (testis). Tryptic peptide maps and amino acid compositions of some active site peptides appear to confirm the compositional relatedness among these isozymes. The sequence of the loop region of mouse C subunit seems to be markedly different from all known A and B sequences, and the structural and functional implications are discussed.     

Comparative biochemical studies of isozymes of isocitrate dehydrogenase from the mouse

Summary Biochemical properties of cytoplasmic and mitochondrial isozymes of isocitrate dehydrogenase from DBA/2J mice were compared under various experimental conditions. These included K_m determinations, coenzyme specificity, pH dependence, urea, iodoacetate and thermal inactivation and fluorescence titration studies. From these comparative studies each isozyme was found to have distinct coenzyme specificity, thermal stability and sensitivity to alkylation. In the case of the cytoplasmic isozyme, both NADP⁺ and isocitrate protect the enzyme against thermal denaturation but not iodoacetate inactivation. On the contrary, neither NADP⁺ nor isocitrate protects the mitochondrial enzyme against thermal or iodoacetate inactivation. Both isozymes exhibit similar fluorescence properties. NADP⁺ and NADPH, but not isocitrate, cause quenching of protein fluorescence. Enhancement of coenzyme fluorescence and protein energy transfer was observed when either isozyme was added to NADPH solutions. Further addition of isocitrate or isocitrate-Mg⁺⁺ to a NADPH-enzyme solution caused a decrease of the enhancement of coenzyme fluorescence and protein energy transfer, but not quenching of protein fluorescence, indicating the formation of a ternary complex. This observation precludes the mechanism of mutual exclusion between NADPH and isocitrate in the active site of the enzyme.Abbreviations used IDH isocitrate dehydrogenase - NHDP⁺ nicotinamide-hypoxanthine dinucleotide phosphate - TNADP⁺ thionicotinamide-adenine dinucoleotide phosphate - AcPyADP⁺ 3-acetylpyridine-adenine dinucleotide phosphate NIH Visiting Fellow.     

The amino acid sequence of the tryptic peptides isolated from dogfish M4 lactate dehydrogenase.

The peptides which result from treatment of the S-[14C]carboxymethyl derivative of dogfish M4 lactate dehydrogenase (EC 1.1.1.27) with trypsin have been isolated and their sequences have been elucidated. Each identical subunit has a molecular weight of 36,000 and on the basis of the amino acid composition 40 unique tryptic peptides are anticipated. Thirty-seven of these peptides have been isolated and have been completely characterized. The amino acid sequences of these peptides are presented.     

Cloning and characterization of lactate dehydrogenase C4 from Ochotona curzoniae

Lactate dehydrogenase C4 (LDH-C4) is considered to be a good target protein for the development of contraceptive drugs. To develop contraceptive rodenticide against pika (Ochotona curzoniae) LDH-C4, the pika LDH-C gene was cloned and expressed in Escherichia coli. The recombinant protein was purified and characterized. The cDNA of pika LDH-C gene was cloned by the RACE method. The cDNA was 1498 bp in length and contained an ORF of 996 bp, which encoded a polypeptide of 332 amino acids. The ORF of pika LDH-C was introduced in E. coli and expressed with no fusion tags added. The recombinant LDH-C4 protein was purified by heating, affinity chromatography and ion-exchange chromatography. The recombinant pika LDH-C4 was a tetramer with a molecular weight of approximately 140 kDa, and it had temperature-dependent catalytic activity, as it was thermally stable up to 60°C. The optimal pH values in the forward and backward reactions were around 7.48 and 10.28, respectively. The apparent Michaelis constants for pyruvate and lactate were 51.2 ± 3.8 and 8568.8 ± 409 μM respectively. The inhibition constant for oxalic acid was 11.8 ± 3.5 mM. This study laid a solid foundation for contraceptive rodenticide development against pika LDH-C4.     

Amino acid sequence of ovine 6-phosphogluconate dehydrogenase

The amino acid sequence of the NADP+-dependent enzyme ovine 6-phosphogluconate dehydrogenase has been determined by conventional direct protein sequence analysis of peptides resulting from digestion of the protein with trypsin and chemical cleavages with cyanogen bromide, hydroxylamine, and iodosobenzoic acid. The polypeptide contains 466 amino acids and its NH2 terminus is acetylated. The Candida utilis enzyme is inactivated by reaction of pyridoxal phosphate with two lysine residues (Minchiotti, L., Ronchi, S., and Rippa, M. (1981) Biochim. Biophys. Acta 657, 232-242). These residues are conserved in the ovine enzyme. In contrast to NAD+ dehydrogenases which have weakly related sequences and spatially related folds in their nucleotide-binding sites, no significant sequence homologies were detected between 6-phosphogluconate dehydrogenase and any of three other NADP+-requiring enzymes, glutamate dehydrogenase, p-hydroxybenzoate hydroxylase, and dihydrofolate reductase. This is in accord with structural data that show no spatial relationship between NADP+-binding sites in these enzymes.     

Biosynthesis and localization of lactate dehydrogenase X in pachytene spermatocytes and spermatids of mouse testes

The presence and biosynthesis of the testis-specific isozyme of lactate dehydrogenase (LDH-X) in cells at various stages of spermatogenesis have been examined. Enrichment of testicular cells in various stages of spermatogenesis has been achieved by two methods: (1) cell separation by velocity sedimentation in the Elutriator rotor and (2) γ irradiation of testes to eliminate specific classes of testicular cells. Separation of cells from immature mice indicated that cells prior to the midpachytene stage contain no LDH-X. Measurement of LDH-X levels in cells separated from adult mice and in testicular homogenates prepared at various times after irradiation indicated that the highest level of LDH-X per cell (normalized for DNA content) was in spermatids. Synthesis of LDH-X was determined, after in vivo injection of [³H]valine, by measurement of the radioactivity in LDH-X precipitated with specific antiserum. After irradiation, the rate of LDH-X synthesis remained constant, despite the loss of early primary spermatocytes. In separated cells, the rate of LDH-X synthesis was highest in late pachytene spermatocytes, lower in round spermatids, and even lower, but still significant, in elongated spermatids. Therefore, the synthesis of LDH-X begins at a specific point during spermatogenesis, the midpachytene stage of spermatocyte development, and continues throughout spermatid differentiation.     

Amino acid sequence of alcohol dehydrogenase from the thermophilic bacterium Thermoanaerobium brockii

The complete amino acid sequence of alcohol dehydrogenase of Thermoanaerobium brockii (TBAD) is presented. The S-carboxymethylated protein was cleaved at methionine residues (with cyanogen bromide) to provide a set of 10 nonoverlapping fragments accounting for 90% of the sequence. These fragments were then overlapped and aligned, and the sequence was completed by using peptides generated by proteolytic cleavage at lysine residues (with Achromobacter protease I). The protein subunit contained 352 amino acid residues corresponding to a molecular weight of 37,652. The sequence showed about 35% identity with that of the prokaryotic Alcaligenes eutrophus alcohol dehydrogenase and about 25% identity with any one of the eukaryotic alcohol/polyol dehydrogenases known today. Of these, only 18 residues (5%) are strictly conserved: 11 Gly, 2 Asp, and 1 each of Cys, His, Glu, Pro, and Val.