Adenosylhomocysteinase (bovine)

This chapter describes the assay and purification procedures of bovine liver adenosylhomocysteinase, of which much of the enzymology is described. Enzyme activity can be assayed both, in the synthetic direction and in the hydrolytic direction. The synthetic direction using ion-exchange column is the method of choice because it does not require laborious preparation of radioactive S-adenosylhomocysteine. However, inhibitors of adenosine deaminase must be included in the incubation mixture when assaying crude extracts. Because the adenosylhomocysteinase is a very active enzyme, linearity with respect to protein concentrations should always be established. Adenosylhomocysteine can be synthesized by the method of de la Haba and Cantoni, and then purified by repeated crystallization. A faster alternative is to purify the synthesized product by high-pressure liquid chromatography. Another enzymatic synthesis of radioactive S-adenosylhomocysteine labeled by L-[2(n)- 3 H]homocysteine is described. It is based on the conversion of S- adenosyl-L-[2(n)- 3 H]methionine to S-adenosyl-L-[2(n)- 3 H]homocysteine by glycine N-methyltransferase using glycine as the acceptor. A convenient chemical synthesis of S-adenosylhomocysteine may also be applied to prepare its radioactive counterpart.