Adenosine Triphosphate-Adenosine 5'-Monophosphate Phosphotransferase of Bovine Liver Mitochondria

Abstract

Abstract Adenylate kinase has been purified approximately 700-fold from bovine liver mitochondria. The purity of the final preparation was evaluated by studies with ion exchange and molecular sieve chromatography and by sedimentation studies. By these criteria the enzyme is estimated to be 85 to 95% pure. The purified enzyme catalyzes transphosphorylation reactions between adenosine triphosphate and adenosine monophosphate, ATP and deoxy-AMP, and inosine triphosphate and AMP. The nearly constant ratios of these three activities throughout the purification scheme suggests that all are catalyzed by the same enzyme. Studies of the physical properties of the purified enzyme indicate it to possess a sedimentation constant of 2.49 S, a diffusion constant of 10.3 x 10-7 cm2 sec-1, a partial specific volume of 0.73 ml per g, and a molecular weight of 21,500. These properties are identical with those previously reported for crystalline adenylate kinase from skeletal muscle. Amino acid analysis of the purified enzyme indicate its composition to be: Asp18, Thr9, Ser11, Glu20, Pro13, Gly15, Ala17, Val13, Met5, Leu20, Illeu9, Phe7, Tyr5, Lys15, His4, Arg10, Try2, and CyS4, totaling 197 residues. Spectral studies with p-chloromercuribenzoate indicate that the enzyme does not contain free sulfhydryl groups. This finding is consistent with the lack of inhibition of catalytic activity by this and other sulfhydryl reagents and indicates that whereas the skeletal enzyme is sensitive to sulfhydryl reagents and is known to contain free sulfhydryl groups, the liver enzyme contains 4 half-cystine residues in some other form, possibly as two disulfide groups.