Affiliation:
1. Laboratoire de Cytogénétique Moléculaire et Oncologie (UMR 147), CNRS-Institut Curie, Section de Recherche, 26 rue d'Ulm, 75231 Paris Cedex 05, France
2. Laboratoire de Physico-Chimie Biomoléculaire et Cellulaire (URA 2056), CNRS-Institut Curie, Section de Recherche, 11 rue Pierre et Marie Curie, 75231 Paris Cedex 05, France
Abstract
Hexokinase plays a key role in regulating cell energy metabolism. Hexokinase is mainly particulate, bound to the mitochondrial outer membrane in brain and tumour cells. We hypothesized that the intracellular pH (pHi) controls the intracellular distribution of hexokinase. Using the SNB-19 glioma cell line, pHi variations were imposed by incubating cells in a high-K+ medium at different pH values containing specific ionophores (nigericin and valinomycin), without affecting cell viability. Subcellular fractions of cell homogenates were analysed for hexokinase activity. Imposed pHi changes were verified microspectrofluorimetrically by using the pHi-sensitive probe SNARF-1-AM (seminaphthorhodafluor-1-acetoxymethyl ester). Imposition of an acidic pHi for 30 min strongly decreased the particulate/total hexokinase ratio, from 63% in the control sample to 31%. Conversely, when a basic pHi was imposed, the particulate/total hexokinase ratio increased to 80%. The glycolytic parameters, namely lactate/pyruvate ratio, glucose 6-phosphate and ATP levels, were measured concomitantly. Lactate/pyruvate ratio and ATP level were both markedly decreased by acidic pHi and increased by basic pHi. Conversely, the glucose 6-phosphate level was increased by acidic pHi and decreased by basic pHi. To demonstrate that the change of hexokinase distribution was not due to altered metabolite levels of glycolysis, a pHi was imposed for a 5 min incubation time. Modification of the hexokinase distribution was similar to that noted after a 30 min incubation, whereas metabolite levels of glycolysis were not affected. These results provide evidence that the intracellular distribution of hexokinase is highly sensitive to variations of the pHi, and regulates hexokinase activity.
Subject
Cell Biology,Molecular Biology,Biochemistry
Cited by
54 articles.
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