Rat Liver Adenosine Triphosphate:Adenosine Monophosphate Phosphotransferase Activity: I. PURIFICATION AND PHYSICAL AND KINETIC CHARACTERIZATION OF ADENYLATE KINASE III

Abstract

Abstract Rat liver adenylate kinase III (ATP:AMP-phosphotransferase, EC 2.7.4.3) has been purified and characterized. The enzyme was purified 1500-fold to a final specific activity of 1000 µmoles of ADP produced per min per mg of protein at 25°. The preparation was homogeneous by polyacrylamide gel disc electrophoresis and at 10 mg per ml was essentially a single component by analytical ultracentrifugation. Molecular weight studies with Sephadex G-150 column chromatography revealed a Vo:Ve ratio of 1.94 which corresponded to a molecular weight of 46,000 for the enzyme. A range of Svedberg values as a function of protein concentration was obtained by ultracentrifugation and established the presence of two molecular weight forms of the enzyme; extrapolation to zero protein concentration indicated two S020, w values of 1.23 and 3.52. With the extrapolated value of the diffusion coefficient of 4.8 x 10-7 cm2 sec-1 and the partial specific volume of 0.74 calculated from the amino acid analysis, molecular weights of 23,000 and 68,000, respectively, were obtained. Frictional and axial ratios were found to be 1.1 and 4.0, respectively. We propose that rat liver adenylate kinase III is a globular protein existing as monomer, dimer, or trimer in very rapid equilibrium. Amino acid analysis revealed a total of 216 amino acids with a calculated minimum molecular weight of 23,400. The protein was found to be high in glutamic acid, alanine, aspartic acid, and leucine residues; it was low in histidine, methionine, half-cystine, and phenylalanine residues. Initial velocity studies revealed a narrow specificity for adenine nucleotides. The respective Km values for ATP, dATP, and dGTP were 0.12, 0.77, and 1.77 mm with respective Vmax values of 16,000, 40,000, and 25,000 moles of triphosphate min-1 mole-1 of enzyme. The only monophosphate acceptor, with a Km of 0.12 mm was 5'-AMP. The Km for ADP was 0.18 mm with a Vmax of 10,700 moles of diphosphate min-1 mole-1 of enzyme. It would appear that rat liver adenylate kinase III slightly favors the forward reaction (conversion of ATP).