Purification of rat liver adenylate kinase isozyme II and comparison with isozyme III

Abstract

1. 1.|Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) isozyme II from rat liver was purified 136-fold to a final specific activity of 60 μmoles of ADP produced per min per mg of protein at 25°. This represented a purification to at least 85% homogeneity. Further purification was hampered by the instability of the enzyme. 2. 2.|The final preparation behaved as a single peak in the analytical ultracentrifuge, and gave a single major band and three trace bands on disc gel electrophoresis using sodium dodecul sulfate-urea. 3. 3.|The molecular weight, which was estimated to be 46 000 by Sephadex G-100 chromatography and to be 49 000 by analytical ultracentrifugation was similar to the previously published molecular weight (46 000) of the dimer form of rat liver adenylate kinase isozyme III. 4. 4.|Both isozymes from rat liver can use ATP and dATP as phosphoryl donors. The K m values for ATP was 0.39 mM with isozyme II and 0.43 mM with isozyme III. The K m for dATP was 1.8 mM with isozyme II and 0.83 mM with isozyme III. Both isozymes exhibited about 30 to 35% greater maximal velocity with dATP as a substrate than with ATP. 5. 5.|GTP, dGTP, CTP, UTP, and ITP did not serve as substrates for either isozyme. 6. 6.|Of several nucleoside monophosphates tested with isozyme II, only 5′-AMP served as a phosphoryl acceptor, with a K m value of 0.073 mM. 7. 7.|In the reverse direction (production of ATP), the K m for ADP was 0.3 mM. 8. 8.|Kinetic comparison of isozymes II and III revealed similar substrate specificities, Michaelis constants, inhibition to AMP, stability in acid, and lability in alkali. However, differences were observed in stability in phosphate buffer at 4°, sensitivity to mercurial reagents, and sensitivity to free fatty acids and detergents. 9. 9.|Comparison of physical parameters showed no significant differences between the two isozymes in terms of diffusion coefficients, partial specific volumes, frictional or axial ratios. Whereas isozyme III had the ability to aggregate, isozyme II did not. Also, there were differences in isoelectric points and immunological cross reactivity with antiserum prepared against isozyme III.