Bovine Brain Mitochondrial Hexokinase

Redkar, Vilas D.; Kenkare, Umakant W.

doi:10.1016/s0021-9258(19)44564-x

Cited by 69 publications

(12 citation statements)

References 31 publications

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“…Effect of Various Metabolites on the Reactivity of Sulfhydryl Residues of the Enzyme. Sulfhydryl residues in bovine brain hexokinase have differing degrees of reactivity toward DTNB (Redkar and Kenkare, 1972). These reactivities are modified differently by various metabolites related to hexokinase.…”

Section: Resultsmentioning

confidence: 99%

“…The protective effects of these ligands against inactivation by DTNB also vary to a considerable extent. We described these effects in qualitative terms in our previous communication (Redkar and Kenkare, 1972).…”

Section: Resultsmentioning

confidence: 99%

“…Crystalline bovine albumin used as a standard for measurement of protein concentration was obtained from Nutritional Biochemicals Corporation. Bovine brain mitochondrial hexokinase was prepared by a previously published procedure (Redkar and Kenkare, 1972). Enzyme Assays.…”

Section: Methodsmentioning

confidence: 99%

“…Enzyme Assays. Hexokinase was assayed either by coupling the formation of Glc-6-P to the reduction of triphosphopyridine nucleotide (Sharma et al, 1963) as described previously (Redkar and Kenkare, 1972), or by coupling the formation of ADP to the oxidation of NADH as described by Grossbard and Schimke (1966). Unit of activity has been defined earlier (Redkar and Kenkare, 1972).…”

Section: Methodsmentioning

confidence: 99%

“…These were estimated by the method of Ellman (1959) using DTNB as described in detail by Redkar and Kenkare (1972). For these experiments, the stock solution of the enzyme (Redkar and Kenkare, 1972) was dialyzed against 0.05 M Tris-chloride buffer (pH 8.0) containing 0.5 mM EDTA. Experiments involving DTNB were carried out at 25°.…”

Section: Methodsmentioning

confidence: 99%

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

Redkar¹,

Kenkare²

1975

Biochemistry

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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 gamma phosphate of ATP and that nucleotides ATP and ADP bind to the enzyme in the absence of Mg2+.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%