Soluble and Particulate Hexokinase of Frog Skeletal Muscle

Abstract

Abstract Both particulate and soluble hexokinases from frog skeletal muscle were prepared by differential centrifugation and their distribution, properties, and kinetics were investigated. Approximately 50% of the hexokinase activity was in the soluble and 50% in the particulate form. Further fractionation of the particulate form yielded a sarcoplasmic vesicle fraction free of mitochondria. This comprised at least one-half of the complete particulate fraction and had a specific activity 4.5 times that of the soluble enzyme. Significant differences were noted between the interaction of the particulate and the soluble enzyme with their substrates and regulators: adenosine triphosphate, 1,5-anhydroglucitol 6-phosphate (analogue of glucose 6-phosphate), and orthophosphate. The Km of the particulate enzyme for MgATP, 0.28 mm, is approximately one-fifth of the soluble enzyme. MgATP at concentrations above 2.5 mm was inhibitory for the particulate enzyme. Inhibition by 1,5-anhydroglucitol-6-P appeared to be competitive with MgATP for the soluble enzyme, and for the particulate enzyme at concentrations below 2.5 mm. At MgATP concentration above 2.5 mm the particulate enzyme is more sensitive to inhibition by 1,5-anhydroglucitol-6-P. The particulate enzyme is more resistant to inhibition by 1,5-anhydroglucitol-6-P at MgATP concentration below 2.5 mm, its Ki, 0.08 mm, being 3 times that of the soluble enzyme. Orthophosphate relieved the inhibition by 1,5-anhydroglucitol-6-P by raising its Ki value. Orthophosphate alone had no effect on the soluble enzyme but activated the particulate enzyme, particularly at high MgATP concentrations. Anaerobiosis and muscle contraction, conditions which result in the apparent activation of hexokinase, had no effect on the distribution and activity of the particulate and soluble enzyme following extraction and isolation. Concentrations of substrates and regulators found in intact frog sartorius during anaerobiosis, and during anaerobiosis plus muscle contraction, were duplicated in the assay system in order to simulate activation states. The velocity of the reactions catalyzed by both soluble and particulate hexokinase decreased with increasing simulated activation state when compared to maximal control values. During these simulated conditions, the decrease in velocity of the reaction catalyzed by the soluble enzyme was twice as great as the corresponding decrease for the particulate enzyme.