S-Adenosylhomocysteine hydrolase from human placenta. Affinity purification and characterization.
S-Adenosylhomocysteine hydrolase (EC 3.3.1.1) was purified to homogeneity from human placenta by using S-adenosylhomocysteine-agarose affinity chromatography. The enzyme is a tetramer with a native Mr of 189 000 and subunit Mr of 47 000-48 000; there were nine cysteine residues per subunit and no disulphide bonds. The pI was 5.7. H.p.l.c. analysis revealed that the enzyme contained four molecules of tightly bound cofactor (NAD) per tetramer, of which 10-50% was in the reduced form. The enzyme had four binding sites per tetramer for adenosine, of which 10-35% were found to be occupied. Two types of adenosine-binding sites could be distinguished on the basis of differences in rates of dissociation of the enzyme-adenosine complex, and by examining binding of adenosine at 0 degree C and 37 degrees C. The enzyme catalysed the interconversion of adenosine and 4',5'-dehydroadenosine; the equilibrium constant for this reaction was 2.1 and favoured 4',5'-dehydroadenosine formation. Variability in the specific activity of preparations of S-adenosylhomocysteine hydrolase was related to the NAD+/NADH ratio of the preparation. The capacity to bind radioactively labelled adenosine depended on the adenosine content of the purified enzyme. The rate of adenosine binding and the sensitivity of S-adenosylhomocysteine hydrolase to inactivation by adenosine were both diminished in the absence of dithiothreitol.
D000241:Adenosine / Q000378:metabolism; D043383:Adenosylhomocysteinase; D000596:Amino Acids / Q000032:analysis; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004229:Dithiothreitol / Q000494:pharmacology; D005260:Female; D006801:Humans; D006867:Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D009243:NAD / Q000378:metabolism; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011485:Protein Binding / Q000187:drug effects
D000596:Amino Acids; D008024:Ligands; D009243:NAD; D006867:Hydrolases; D043383:Adenosylhomocysteinase; D000241:Adenosine; D004229:Dithiothreitol