Studies on adenosine triphosphate transphosphorylases. Isolation and several properties of the crystalline calf ATP-AMP transphosphorylases (adenylate kinases) from muscle and liver and some observations on the rabbit muscle adenylate kinase.

Abstract Detailed procedures are described for the large-scale isolation of crystalline calf muscle and calf liver adenylate kinase isoenzymes applicable to frozen tissue. Proposed modifications in the large-scale isolation of the crystalline rabbit muscle adenylate kinase are also described. Although a great deal of homology exists between the muscle types, calf and rabbit, as revealed by their amino acid compositions (differing in only 10 out of 193 residues), the calf muscle adenylate kinase acts as a potent antigen in the rabbit. A description for the purification (ca. 600-fold) from the rabbit antiserum of the anti-enzyme globulin is given. The anti-enzyme shows a remarkable specificity in that it is largely unreactive as an inhibitor toward the calf liver-type enzyme, but is a powerful inhibitor of the calf muscle type (or human muscle type) with a kinetically evaluated dissociation constant of ca. 3 × 10−14 m 2 (for the case of a stoichiometry of 2 Ag to 1 Ab), a second-order k ⋍ 5 × 10 6 M −1 · min −1 at 30 ° C , and an Arrhenius energy of activation of ca. 6.3 kcal/mol. Dodecyl sulfate polyacrylamide gel electrophoresis revealed only a single heavy chain (Mr, ~42,000) and a single light chain (Mr, ~25,000) of the antibody, which together with studies employing 3.5% stacking gels lead to a molecular weight of approximately 134,000 for the native antibody. Similarly, by dodecyl sulfate polyacrylamide gel electrophoreses, only single bands were obtained for the calf liver and rabbit muscle adenylate kinase (Mr, ca. 21,000–23,000) and the calf liver adenylate kinase (Mr, ca. 25,000). Sedimentation velocity of the calf muscle-type and calf liver-type proteins yielded single sedimenting boundaries, with s 0 20,w ⋍ 2.1 6 and 2.37 S, respectively. Estimates of the molecular weights of the native structure of the calf isoenzymes by sedimentation equilibrium gave M w = 21,200 ± 200 for the calf muscle enzyme and 25,600 ± 200 for the calf liver adenylate kinase. Electrophoresis by liquid boundary of the rabbit muscle enzyme at protein concentrations of 5–8 mg/ml resulted in a nonideal system, as a result largely of charge effects and interactions with buffer components. Similarly, isoelectric points determined by isoelectric focusing were very dependent on the protein load, but could be extrapolated to zero protein to approximately pI0 of 10.6. However, electrophoresis on cellulose acetate at microgram to nanogram levels of protein under conditions where electroendosmosis seemed absent did permit an accurate estimate of the pI0 by a plot of pIapp vs ( Γ 2 ) 1 2 to a pI0 = 10.60. This value is now in good agreement with an estimate made earlier from its amino acid composition (T. A. Mahowald et al., 1962, J. Biol. Chem.237, 1138–1145). By this technique, the pIapp values at 0.05 ( Γ 2 ) for the calf isoenzymes were estimated and found to differ only slightly (viz., 9.6 for the liver adenylate kinase vs 10.1 for the muscle myokinase). While inhibition of the rabbit and calf muscle adenylate kinases (which are apparently cytoplasmic enzymes) by Ap5A [p1,p5-di(adenosine-5′)pentaphosphate] was very significant at even 10−8 m and similar to that previously reported for the rabbit muscle adenylate kinase ( Lienhard and Secemski, 1973 , J. Biol. Chem.248, 1121–1123), inhibition of the liver-type adenylate kinase (which is apparently a mitochondrial enzyme) required almost 10−6 m (Ap5A) for similar percentage inhibitions. For the forward reactions (i.e., MgATP2− + AMP2− →), Ap5A acts as a competitive inhibitor with respect to either substrate with K i ⋍ (0.5−1) × 10 −8 M for both of the muscle enzymes, but yields a K i ⋍ (3−8) × 10 −7 M for the liver enzyme. However, in the reverse direction (MgADP− + ADP3− →), inhibition by Ap5A appears to be noncompetitive with respect to either substrate, with a K i ⋍ (0.6−2) × 10 −8 M for the muscle enzymes, but only (3–4) × 10−6 m for the liver enzyme. Thus, although Ap5A may act in part as a transition state analog, the explanation for its “multisubstrate inhibition” (Lienhard and Secemski, see above) may also lie in the structure of its metal chelates, e.g., of Mg2(Ap5A)−.