Purification, characterization, and cDNA cloning of opine dehydrogenases from the polychaete rockworm Marphysa sanguinea.
Alanopine dehydrogenase (AlDH) and three isoforms of strombine/alanopine dehydrogenase (St/AlDH) were purified from muscle tissue of the polychaete rockworm Marphysa sanguinea. The four enzymes, which can be distinguished by the isoelectric point, are monomeric 42 kDa proteins, possess similar pH-activity profiles, and display specificity for pyruvate and NAD(H). The three isoforms of St/AlDH show equivalent Km and Vmax for glycine and L-alanine and for D-strombine and meso-alanopine. Free amino acid levels in the muscle and D-strombine accumulation in vivo during muscle activity suggest that St/AlDHs function physiologically as StDH. AlDH shows specificity for L-alanine and meso-alanopine, but not for glycine or D-strombine. The amino acid sequences of AlDH and one of the St/AlDH isoforms were determined by a combination of amino acid sequence analysis and cDNA cloning. St/AlDH cDNA consisted of 1586 bp nucleotides that encode a 399-residue protein (43,346.70 Da), and AlDH cDNA consisted of 1587 bp nucleotides that encode a 399-residue protein (43,886.68 Da). The two amino acid sequences deduced from the cDNA displayed 67% amino acid identity, with greatest similarity to that of tauropine dehydrogenase from the polychaete Arabella iricolor.
Endo
Noriyuki
N
Department of Marine Bioscience, School of Fisheries Sciences, Kitasato University, Iwate 022-0101, Japan.
Kan-no
Nobuhiro
N
Nagahisa
Eizoh
E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000235:genetics; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics*; D011077:Polychaeta / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D013045:Species Specificity
D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes; D000587:Oxidoreductases Acting on CH-NH Group Donors; C030014:alanopine dehydrogenase