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

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, S20,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

Characterization of P0, a ribosomal phosphoprotein of Plasmodium falciparum: antibody against amino-terminal domain inhibits parasite growth

A cDNA expression clone of the human malarial parasite Plasmodium falciparum, λPf4, which was reactive only to the immune sera and not to the patient sera, has recently been found to be the P. falciparum homologue of the P0 ribosomal phosphoprotein gene. A Northern analysis of the P0 gene revealed the presence of two transcripts, both present in all the different intraerythrocytic stages of the parasite life cycle. A 138-base pair amino-terminal domain of this gene was expressed as a fusion protein with glutathione S-transferase in Escherichia coli. Polyclonal antibodies raised against this domain immunoprecipitated the expected 38-kDa P0 protein from the 35S-labeled as well as 32P-labeled P. falciparum cultures. Monospecific human immune sera affinity-purified using the expression clone λPf4 also immunoprecipitated the same size protein from [35S]methionine-labeled P. falciparum protein extract. Purified IgG from polyclonal antibodies raised against the amino-terminal domain of P0 protein completely inhibited the growth of P. falciparum in vitro. This inhibition appears to be mainly at the step of erythrocyte invasion by the parasites

Effect of ligands on the reactivity of essential sulfhydryls in brain hexokinase. Possible interaction between substrate binding sites

Inactivation of bovine brain mitochondrial hexokinase by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), a sulfhydryl specific reagent, has been investigated. The study shows that the inactivation of the enzyme by DTNB proceeds by way of prior binding of the reagent to the enzyme and involves the reaction of 1 mol of DTNB with a mol of enzyme. At stoichiometric levels of DTNB, the inactivation of the enzyme is accompanied by the formation of a disulfide bond. But it is not clear whether the disulfide bond or the mixed disulfide intermediate formed prior to it causes inactivation. On the basis of considerable protection afforded by glucose against this inactivation it is tentatively concluded that the sulfhydryl residues involved in this inactivation are at the glucose binding site of the enzyme, although other possibilities are not ruled out. An analysis of effects of various substrates and inhibitors on the kinetics of inactivation and sulfhydryl modification by DTNB has led to the proposal that the binding of substrates to the enzyme is interdependent and that glucose and glucose 6-phosphate produce slow conformational changes in the enzyme. Protective effects by ligands have been employed to calculate their dissociation constant with respect to the enzyme. The data also indicate that glucose 6-phosphate and inorganic phosphate share the same locus on the enzyme as the γ phosphate of ATP and that nucleotides ATP and ADP bind to the enzyme in the absence of Mg2+