Purification and Characterization of S-Adenosylhomocysteine Hydrolase from Mouse Mastocytoma P-815 Cells. Evidence for Cell Cycle-Specific Fluctuation of the Enzyme Activity

Abstract

S-Adenosylhomocysteine hydrolase [EC 3. 3. 1. 1] was purified to electrophoretic homogeneity from mastocytoma P-815 cells. The purified enzyme had a molecular weight of 190, 000, as estimated by Sephadex G-200 chromatography, and a monomer molecular weight of 45, 000, as determined by polyacrylamide gel electrophoresis in the presence of SDS. The K_m value for adenosine was 0.29 μM and the V_max value 4.5 μmol S-adenosylhomocysteine•min^-1•mg^-1 in the synthetic reaction, while the K_m value for S-adenosylhomocysteine was 0.77 μM and the V_max 0.48 μmol adenosine•min^-1•mg^-1 in the hydrolytic reaction. The purified enzyme also had one binding site for adenosine (K_D=2.61×10^-7M) and one for cAMP (K_D=1.6×10^-7M). Using rabbit antiserum raised against the purified enzyme, it was shown that the enzyme activity and enzyme synthesis fluctuated during the cell cycle of mastocytoma cells, reaching the maximum levels as the cells changed from the G_l/S phase to the G₂ phase.