Studies on hexokinase: 1. The hexokinase activity of rat-brain extracts

Weil‐Malherbe, H.; Bone, A. D.

doi:10.1042/bj0490339

Cited by 154 publications

(37 citation statements)

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“…This parallel measurement of both activities allows the relative rates of each to be compared, based on the assumption that the glucose transported out of the cell and the glucose undergoing phosphorylation are derived from the same intracellular pool. As was expected from previous studies comparing trans port and phosphorylation rates in other neuronal cells (Weil-Malherbe and Bone, 1951;Fromm and Zewe, 1962 ;Katzen and Schimke, 1965 ;Minna et a!., 1975 ;Lange et a!., 1982;Pardridge, 1983), both NCB20 and DRG cells had rates of glucose efflux severalfold greater than rates of phosphorylation, even at extracellular glucose concentrations of 1 mM or less (Fig. 6).…”

Section: Discussionsupporting

confidence: 85%

“…4) is lower than would be expected for a transport limited process, which should have had an apparent Km 10-20-fold greater, given a Km of GLUTl and GLUn of 4-10 mM (Keller et a!., 1989 ;Gould et a!., 1991;Colville et a!., 1993). Nonetheless, the apparent Km is also almost an order of magnitude greater than would be expected if hexokinase I were totally rate-limiting (Weil-Malherbe and Bone, 1951;Fromm and Zewe, 1962 ;Katzen and Schimke, 1965). This suggests phosphorylation of glucose was not totally rate-limiting, but that either glucose transport or diffusion barriers to glucose within cells did impede access of glucose to the hexokinase.…”

Section: Discussionmentioning

confidence: 96%

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Coupled Glucose Transport and Metabolism in Cultured Neuronal Cells: Determination of the Rate-Limiting Step

Whitesell

Ward

McCall

et al. 1995

J Cereb Blood Flow Metab

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Summary: In brain and nerves the phosphorylation of glucose, rather than its transport, is generally considered the major rate-limiting step in metabolism. Since little is known regarding the kinetic coupling between these pro cesses in neuronal tissues, we investigated the transport and phosphorylation of [2-3H)glucose in two neuronal cell models: a stable neuroblastoma cell line (NCB20), and a primary culture of isolated rat dorsal root ganglia cells. When transport and phosphorylation were measured in series, phosphorylation was the limiting step. because in tracellular glucose concentrations were the same as those outside of cells, and because the apparent Km for glucose utilization was lower than expected for the transport step. However, the apparent Km was still severalfold higher than the Km of hexokinase I. When [2-3H)glucose efflux and phosphorylation were measured from the same intra- 814 cellular glucose pool in a parallel assay, rates of glucose efflux were three-to-fivefold greater than rates of phos phorylation. With the parallel assay, we observed that activation of glucose utilization by the sodium channel blocker veratridine caused a selective increase in glucose phosphorylation and was without effect on glucose trans port. In contrast to results with glucose, both cell types accumulated 2-deoxY-D-[1 4 C)glucose to concentrations severalfold greater than extracellular concentrations. We conclude from these studies' that glucose utilization in neuronal cells is phosphorylation-limited, and that the coupling between transport and phosphorylation depends on the type of hexose used.

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Section: Discussionsupporting

confidence: 85%

Section: Discussionmentioning

confidence: 96%

Coupled Glucose Transport and Metabolism in Cultured Neuronal Cells: Determination of the Rate-Limiting Step

Whitesell

Ward

McCall

et al. 1995

J Cereb Blood Flow Metab

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“…ligand complex has been reviewed extensively [8,9,. The effect of a paramagnetic ion on the spin-lattice relaxation rate (T;:) of a ligand nucleus in a macromolecular complex is given by 3 where TTL is the relaxation rate of the observed nucleus in the paramagnetic complex; T M is the life-time of the complex and p is the mole fraction of the ligand in the macromolecular paramagnetic complex. Clearly, if T~ % TIM (i.e.…”

Section: Theoretical Basis For Distance Determinationmentioning

confidence: 99%

“…An understanding of the molecular mechanism of brain hexokinase regulation requires knowledge of the nature of Glc6P binding to the enzyme. On the basis of the non-competitive nature of Glc6P inhibition with respect to glucose [3] and observation of differences in molecular specificities of sugar substrate and inhibitor [4], an allosteric inhibitory site, spatially distinct from the catalytic site for Glc6P, was proposed. However, several kinetic studies by other workers [5,61 and the observation of a reverse hexokinase reaction [7], strongly support the binding of Glc6P at the product site.…”

Section: Mn(i1)mentioning

confidence: 99%

Mapping of glucose and glucose‐6‐phosphate binding sites on bovine brain hexokinase

Jarori

Iyer

Kasturi

et al. 1990

European Journal of Biochemistry

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Inhibition of bovine brain hexokinase by its product, glucose 6‐phosphate, is considered to be a major regulatory step in controlling the glycolytic flux in the brain. Investigations on the molecular basis of this regulation, i.e. allosteric or product inhibition, have led to various proposals. Here, we attempt to resolve this issue by ascertaining the location of the binding sites for glucose and glucose 6‐phosphate on the enzyme with respect to a divalent‐cation‐binding site characterized previously[Jarori, G. K., Kasturi, S. R. & Kenkare, U. W.(1981) Arch. Biochem. Biophys. 211, 258–268]. The paramagnetic effect of enzyme‐bound Mn(II) on the spinlattice relaxation rates (T−11) of ligand nuclei (1H and31P)in E. Mn(II). Gle and E. Mn(II). Glc66 complexes have been measured. The paramagnetic effect of Mn(II) on the proton relaxation rates of C1‐Hα, C1‐Hβ and C2‐Hβ of glucose in the E · Mn(II) · Glc complex was measured at 270 MHz and 500 MHz. The temperature dependence of these rates was also studied in the range of 5–30°C at 500 MHz. The ligand nuclear relaxation rates in E · Mn(II) · Glc are field‐dependent and the Arrhenius plot yields an activation energy (ΔE) of 16.7–20.9 kJ/mol. Similar measurements have also been carried out on C1‐Hα, C1‐Hβ and C6‐31P at 270 MHz (1H and 202.5 MHz (31P) for the E · Mn(II) · Glc6P complex. The temperature dependence of 31P relaxation rates in this complex was measured in the range 5–30°C, which yielded Δ= 9.2 kJ/mol. The electron‐nuclear dipolar correlation time (τc), determined from the field‐dependent measurements of proton relaxation rates in the E · Mn(II)· Gle complex, is 0.22–1.27 ns. The distances determined between Mn(II) and C1‐H of glucose and glucose 6‐phosphate are ∼ 1.1 nm and ∼ 0.8 nm, respectively. These data, considered together with our recent results [Mehta, A., Jarori, G. K. & Kenkare. U. M.(1988) J. Biol. Chem. 263, 15492–15498], suggest that glucose and glucose 6‐phosphate may bind to very nearly the same region of the enzyme. The structure of the binary Glc6P· Mn(II) complex has also been determined. The phosphoryl group of the sugar phosphate forms a first co‐ordination complex with the cation. However, on the enzyme, the phosphoryl group is located at a distance of ∼ 0.5–0.6 nm from the cation.

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“…Animal hexokinases generally are markedly inhibited by low concentrations of glucose 6-phosphate (Crane & Sols, 1954;Fromm & Zewe, 1962a) whereas yeast hexokinase (Colowick & Kalckar, 1943;Weil-Malherbe & Bone, 1951) and hepatic glucokinase (Salas, Salas, Viniuela & Sols, 1965) are not. Likewise, different hexokinases are inhibited to different degrees by ADP.…”

mentioning

confidence: 99%

Kinetic studies with skeletal-muscle hexokinase

Toews

1966

Biochemical Journal

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Rat skeletal-muscle hexokinase was partially purified by ammonium sulphate fractionation and gel filtration. The mechanism of the skeletal-muscle hexokinase was studied kinetically by initial-velocity analysis and product inhibition. Glucose 6-phosphate was a non-competitive inhibitor of glucose and ATP. ADP was a non-competitive inhibitor of glucose and a competitive inhibitor of ATP. The data on product inhibition and initial-velocity analysis of skeletal-muscle hexokinase support an ordered sequential mechanism (ordered Bi Bi) where the addition of substrates and release of products is in the order: ATP, glucose, glucose 6-phosphate and ADP.

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Studies on hexokinase: 1. The hexokinase activity of rat-brain extracts

Cited by 154 publications

References 4 publications

Coupled Glucose Transport and Metabolism in Cultured Neuronal Cells: Determination of the Rate-Limiting Step

Coupled Glucose Transport and Metabolism in Cultured Neuronal Cells: Determination of the Rate-Limiting Step

Mapping of glucose and glucose‐6‐phosphate binding sites on bovine brain hexokinase

Kinetic studies with skeletal-muscle hexokinase

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