S-Adenosylhomocysteinase from rat liver. Evidence for structurally identical and catalytically equivalent subunits.
Lines of evidence are presented which indicate that rat liver S-adenosylhomocysteinase consists of four identical or nearly identical subunits. Cross-linking of the enzyme with dimethyl suberimidate followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis yields four distinct protein bands with molecular weights of 47,000, 93,000, 145,000, and 190,000. The molecular weight of the largest protein is in excellent agreement with that of the native enzyme. Carboxypeptidase A liberates 4 mol of COOH-terminal tyrosine/mol of enzyme, and the number of arginine-containing peptides in a tryptic digest of the enzyme is one-fourth of that arginine residues present in the enzyme. The enzyme reversibly binds 4 mol of the substrate adenosine in a noninteracting manner, and the binding is associated with the reduction of 3.2 mol of enzyme-bound NAD+. However, in the presence of dithiothreitol, the same compound causes a time-dependent irreversible loss of enzyme activity concomitant with the formation of 3.6 mol of enzyme-bound NADH/mol of enzyme. Studies with adenine-labeled adenosine shows that radioactivity corresponding to 3.8 mol of substrate is tightly bound to the inactivated enzyme. Since the inactivation is apparently the consequence of reaction of dithiothreitol with an enzyme-bound intermediate as revealed by the kinetics of inactivation, these results support the conclusion that the four subunits of rat liver S-adenosylhomocysteinase are functionally equivalent.
D000241:Adenosine / Q000378:metabolism; D043383:Adenosylhomocysteinase; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000032:analysis; D004120:Dimethyl Suberimidate / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006867:Hydrolases / Q000032:analysis*; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D051381:Rats; D014357:Trypsin / Q000378:metabolism
D046911:Macromolecular Substances; D009243:NAD; D004120:Dimethyl Suberimidate; D001120:Arginine; D006867:Hydrolases; D043383:Adenosylhomocysteinase; D014357:Trypsin; D000241:Adenosine; D004229:Dithiothreitol