title: Subunit structure of bovine milk xanthine oxidase. Effect of limited cleavage by proteolytic enzymes on activity and structure.
abstract: Bovine milk xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) has been purified by a modified method without the use of proteases, and its structure has been analyzed by polyacrylamide gel electrophoresis. Native xanthine oxidase is found to consist of only two polypeptide chains A with molecular weights of 150 000 each. These chains have NH2-terminal methionine. Limited proteolysis with trypsin, chymotrypsin, or subtilisin at pH 8 did not affect molecular weight and activities of the enzyme while each of the A chains was cleaved under these conditions to three fragments C, E, and F with molecular weights of 92 00, 42 000 and 20 000, respectively. These fragments remained bound to each other and were relatively resistant to subsequent proteolysis. The isolation of xanthine oxidase in the presence of pancreatin as described by Hart et al. (1970, Biochem. J. 116, 851) gives partially digested enzyme composed mainly of chains C, E (Mr 35 000) and a small component (Mr approx. 15 0-0). The action of subtilisin on xanthine oxidase at pH 11 resulted in complete digestion of E chains, FAD separation, and total loss of xanthine:oxygen oxidoreductase activity while xanthine:indophenol oxidoreductase activity was relatively little affected. The residual enzyme has a molecular weight of about 200 000, is composed mainly of two C chains (and may probably contain F and/or proteolytic fragments of low molecular weight), contains molybdenum, and does not contain FAD.
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005182:Flavin-Adenine Dinucleotide; D007700:Kinetics; D046911:Macromolecular Substances; D008892:Milk / Q000201:enzymology*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D013381:Subtilisins; D014357:Trypsin; D014969:Xanthine Oxidase / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000596:Amino Acids; D046911:Macromolecular Substances; D010446:Peptide Fragments; D005182:Flavin-Adenine Dinucleotide; D014969:Xanthine Oxidase; D013381:Subtilisins; D014357:Trypsin