Abstract
Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) from the mantle muscle of the squid, Loligo pealeii, was purified over 170-fold to homogeneity as judged by polyacrylamide and starch gel electrophoresis. The tissue contains a single isozyme of adenylate kinase, the enzyme from cytoplasmic and mitochondrial compartments (90 and 10% of total activity, respectively) being identical in physical and kinetic properties. Molecular weight was found to be 27,000 +/- 400. The enzyme shows a pH optimum of 8.2 in the forward (APD utilizing) and 7.4 in the reverse direction. Michaelis constants for ADP, ATP, and AMP are 0.70, 0.13, and 0.15 mM, respectively, with optimal Mg2+:adenylate ratios being 1:2 for ADP and 1:1 for ATP. A comparison of mass action ratios with the equilibrium constant indicated that squid adenylate kinase is held out of equilibrium in resting, but not active, muscle. A search for metabolic modulators of adenylate kinase revealed that NADH (Ki of 0.1 mM) was the only modulator which exerted a significant effect within its in vivo concentration range. The data presented indicate that NADH inhibition is the factor maintaining adenylate kinase in a nonequilibrium state in resting muscle and that release of this inhibition can serve to integrate adenylate kinase into the known scheme of intermediary metabolism in this tissue. A sharp drop in NADH levels at the onset on muscular work co-ordinates that activation of aerobic metabolism in this tissue and allows adenylate kinase to return to equilibrium function. At equilibrium, the enzyme can function to ampligy the concentration of AMP, a potent activator and deinhibitor of key glycolytic and Krebs cycle enzymes. The effect of modulators of adenylate kinase in preventing denaturation by heat or proteolysis revealed that NADH and substrates induced conformational changes in the enzyme which rendered it less susceptible to denaturation. The conformation state induced by NADH differed from that induced by substrate.
Highlights
The data presented indicate that NADH inhibition is the factor maintaining adenylate kinase in a nonequilibrium state in resting muscle and that release of this inhibition can serve to integrate adenylate kinase into the known scheme of intermediary metabolism in this tissue
During metabolic activation, falling NADH concentrations deinhibit the enzyme and allow it to return to equilibrium where it can function in amplifying the AMP signal, thereby promoting AMP activation of metabolism
As the rate of proteolysis is dependent on the conformation of the protein substrate [39], it is clear, that under the conditions employed, NADH induces a conformational change in adenylate kinase
Summary
ROLE OF NADH IN CONTROL OF THE ENZYME (Received for publication, April 27, 1976). KENNETH B. A sharp drop in NADH levels at the onset of muscular work co-ordinates the activation of aerobic metabolism in this tissue and allows adenylate kinase to return to equilibrium function. It is assumed that in most tissues adenylate kinase operates at equilibrium [5,6,7] and as such functions (a) to maintain a constant energy charge [3] and (b) to amplify small changes in ATP and ADP concentrations into proportionally larger changes in AMP concentrations [8]. Adenylate kinase mass action ratios, determined from measurements of ATP, ADP, and AMP at rest and during exercise in squid muscle,’ indicate the. The data in this study of mantle muscle adenylate kinase from the squid, Loligo pealeii, show that the enzyme is held out of equilibrium under resting conditions by potent NADH inhibition. During metabolic activation, falling NADH concentrations deinhibit the enzyme and allow it to return to equilibrium where it can function in amplifying the AMP signal, thereby promoting AMP activation of metabolism
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