Identification of essential lysyl and cysteinyl residues, and the amino acid sequence at the substrate-binding site of retinal oxidase.
Retinal oxidase, which synthesizes all-trans and 9-cis retinoic acid from all-trans and 9-cis-retinal, has been purified from rabbit liver cytosol. The substrate-binding site of the retinal oxidase was investigated with some chemical modification reagents. Lysyl-specific pyridoxal 5'-phosphate (PLP) and cysteinyl-specific p-chloromercuribenzoate (PCMB) competitively inhibited the activity of the retinal oxidase, and the inhibition could be prevented by the presence of all-trans-retinal or its derivatives. Treatment with sodium borohydride (NaBH4) resulted in covalent attachment of PLP to the lysyl residue of the retinal oxidase and the PLP modified-retinal oxidase was cut with cyanogen bromide, and the polypeptides modified with PLP were further digested with trypsin. Two of the peptides modified with PLP were purified from the digested polypeptide mixture and their amino acid sequences were determined.
Huang
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Department of Biochemistry, Kagawa Medical School, Japan.
Ichikawa
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D000445:Aldehyde Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001894:Borohydrides / Q000494:pharmacology; D002729:Chloromercuribenzoates / Q000494:pharmacology; D003488:Cyanogen Bromide; D003545:Cysteine / Q000032:analysis* / Q000737:chemistry; D003600:Cytosol / Q000201:enzymology; D008099:Liver / Q000201:enzymology*; D008239:Lysine / Q000032:analysis* / Q000737:chemistry; D008297:Male; D008969:Molecular Sequence Data; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D011817:Rabbits; D050697:Retinal Dehydrogenase; D017421:Sequence Analysis; D014357:Trypsin / Q000378:metabolism; D020245:p-Chloromercuribenzoic Acid
D001894:Borohydrides; D002729:Chloromercuribenzoates; D020245:p-Chloromercuribenzoic Acid; D011732:Pyridoxal Phosphate; C025364:sodium borohydride; D000445:Aldehyde Oxidoreductases; D050697:Retinal Dehydrogenase; D014357:Trypsin; D008239:Lysine; D003545:Cysteine; D003488:Cyanogen Bromide