Mitochondrial ATP:AMP phosphotransferase from beef heart: purification and properties

Abstract

ATP:AMP phosphotransferase has been purified 186-fold from beef heart mitochondria in an overall yield of 30%. The purified enzyme, Mr 31 500, has a specific activity of 800 U/mg and was found to be a homogeneous protein by constancy of specific activity across the elution peak following passage through an Affi-Gel Blue agarose column and by dodecylsulfate gel electrophoresis. Isoelectrofocusing shows a minor peak of activity that arises again when the major peak is re-focused. The pI of the enzyme is 9.30 and the optimum pH of activity is 5.8 in the forward reaction (formation of ADP) and 8.0 in the reverse reaction. The enzyme requires the presence of a divalent metal ion, namely Mg2+, Co2+, Ca2+ Mn2+, Ni2+ and Zn2+ in decreasing order of activity and is influenced by NaCl. It shows rigorous specificity for AMP (dAMP) was phosphate acceptor but is rather unspecific for the triphosphate donor since, UTP, CTP, ITP and GTP may substitute for ATP (dATP). ATP and MgATP (dATP was not tested) bind to the enzyme with dissociation constants of 0.122 and 0.132 mM respectively, while AMP (dAMP was not tested), UTP, CTP, ITP and GTP binding could not be detected by the dialysis equilibrium technique used. Amino acid analysis gave the following composition as residues: 26 aspartic acid or asparagine, 14 threonine, 17 serine, 27 glutamic acid or glutamine, 24 proline, 19 glycine, 25 alanine, 17 valine, 8 methionine, 14 isoleucine, 31 leucine, 5 tyrosine, 9 phenylalanine, 6 histidine, 25 lysine, 15 arginine, 4 half cystine (no free sulfhydryls) and no tryptophan, giving a total of 286 residues. The purified mitochondrial adenylate kinase is not a contaminating heart cytosolic enzyme. ATP:AMP phosphotransferase was extracted from beef heart cytosol depleted of mitochondria and brought to homogeneity. Determinations of pI, Mr and amino acid composition showed that the heart mitochondrial and cytosolic adenylate kinases are different isozymes.