Bovine brain mitochondrial hexokinase: solubilization, purification, and role of sulfhydryl residues

Abstract

Bovine brain mitochondrial hexokinase, type I, has been solubilized by extraction of the mitochondria in 0.2 m acetate buffer, pH 5.0, containing 0.9 m NaCl. The solubilized enzyme has been purified to apparent homogeneity as shown by ultracentrifugal and electrophoretic criteria. The purification procedure included fractionation of the solubilized enzyme with ammonium sulfate and two successive diethylaminoethyl cellulose chromatographic steps. The sedimentation coefficient, S_20,w, was found to be 5.9 S at a protein concentration of 1.7 mg per ml. The approximate molecular weight as determined by gel filtration on Sephadex G-200 is 107,000. The enzyme has 11 to 13 sulfhydryl residues per mole as determined by reaction of the denatured enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Almost all of these residues react with DTNB in the native enzyme though with differing degrees of reactivity. Reaction of the enzyme with excess DTNB caused its rapid inactivation. A comparison of the progress of this inactivation with the progress of the reaction of the sulfhydryl residues of the enzyme with DTNB showed that a maximum of only 2 residues could be involved in the inactivation process. If 2-mercaptoethanol is added to the enzyme immediately after complete inactivation, a rapid and total recovery of enzyme activity ensues. These results have been analyzed in terms of involvement of sulfhydryl residues, in the active conformation of the enzyme. Substrate glucose partially protects the enzyme against inactivation by DTNB and also modifies the reactivity of the sulfhydryl residues of the enzyme toward this reagent. MgATP, MgADP, and inorganic phosphate even at 10 mm concentration do not protect the enzyme against inactivation by DTNB. Product inhibitor glucose 6-phosphate affords a complete protection to the enzyme against inactivation by DTNB and drastically changes the reactivity of its sulfhydryl residues. Fructose 6-phosphate is without a comparable effect.