Assay of Mitochondrial ATP Synthesis in Animal Cells and Tissues
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Adenosine triphosphate
Hexokinase
Abstract Soluble hexokinase from frog skeletal muscle has been purified 180-fold. Interconvertible molecular forms of hexokinase were found in the 105,000 x g supernatant. They may be separated by Sephadex G-200 gel filtration into a heavier molecule, hexokinase-H, and a lighter molecule, hexokinase-L (apparent mol wt, 100,000). On sucrose gradient, hexokinase-H may be separated into polyaggregates of hexokinase-L with apparent molecular weights ranging from 300,000 to 3 million. The molecular species are interconvertible with changes in ionic strength and appear to be in equilibrium with each other. Hexokinase-H is activated by orthophosphate, particularly at high ATP concentration, whereas hexokinase-L is not. Thus 8 mm orthophosphate raised the Vmax 2-fold for hexokinase-H. 1,5-Anhydroglucitol-6-P (analogue of glucose-6-P) served as an inhibitor competitive with respect to ATP and noncompetitive with respect to glucose. Hexokinase-H was more resistant to the noncompetitive inhibition of glucose. The Ki values for noncompetitive inhibition of glucose by 1,5-anhydroglucitol-6-P for hexokinase-H and hexokinase-L were 0.62 and 0.32 mm, respectively. Similar kinetic constants for both the purified hexokinase-H and hexokinase-L were: Km for ATP, 1.48 mm; Ki of 1,5-anhydroglucitol-6-P for ATP, 0.058 mm; Km for glucose, 0.100 mm. Varying the ATP concentration had no effect on the Km for glucose. Physiological concentrations of orthophosphate could overcome the competitive or noncompetitive inhibition of both hexokinase-H and hexokinase-L by 1,5-anhydroglucitol-6-P.
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Hexokinase
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Adenosine triphosphate
Carbohydrate Metabolism
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SYNOPSIS. Glucose‐6‐phosphate and adenosine triphospbate levels in glucose‐abundant and glucose‐depleted preparations of the culture forms of Schizotrypanum cruzi were measured. Glucose‐6‐phosphatc formation was dependent upon an exogenous source of glucose. Adenosine triphosphate levels were maintained whether exogenous glucose was present or absent. Net synthesis of adenosine triphosphate in the presence of exogenous glucose was observed. The results favor the presence of a hexokinase in S. cruzi.
Hexokinase
Adenosine triphosphate
Carbohydrate Metabolism
Adenosine diphosphate
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Hexokinase
Carbohydrate Metabolism
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Hexokinase PII, but not isoenzyme PI, has a unique role in glucose repression in yeasts [Entian, K.-D. (1980) Mol. Gen. Genet. 178, 633–637; Entian, K.-D. and Mecke, D. (1982) J. Biol. Chem. 257, 870–874; Entian, K.-D. and Fröhlich, K.-U. (1984) J. Bacteriol. 158, 29–35]. The number of hexokinase isoenzymes in crude extracts was re-examined by chromatofocusing. In addition to the known isoenzymes PI and PII, a third isoenzyme, PIIM, was detected. The activity of this enzyme was only about 5–10% of that of hexokinase PII and was independent of growth conditions. Experiments with hexokinase transformants and purified hexokinase isoenzymes clearly indicated that the PIIM form is also present in vivo. Fingerprint mapping of purified hexokinases showed that hexokinase PIIM is closely related to PII. Hybridization experiments between totally restricted yeast DNA and the previously isolated PII gene clearly indicated that PIIM is also coded by one of the two known hexokinase genes. No mRNA specific for hexokinase PIIM was detected after hybridization experiments with the previously cloned hexokinase PII gene [Fröhlich et al. (1984) Mol. Gen. Genet. 194, 144–148]. Hexokinase PIIM appears to be derived from hexokinase PII by a posttranslational event. The Km values of each of the purified isoenzymes, PII and PIIM, were identical for glucose, fructose and ATP. Both isoenzymes were strongly inhibited by high physiological concentrations for ATP; such inhibition has not been described previously. The possible role of hexokinase PIIM in glucose repression is discussed.
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In the hexokinase reaction with equilibrated d ‐glucose (36.2%, α‐ d ‐glucopyranose and 63.8%β‐ d ‐glucopyranose) at saturating concentrations of ATP‐Mg, the ratio of phosphorylation of α‐ d ‐glucose and β‐ d ‐glucose was investigated. It is shown that, knowing the Michaelis constants K m,(α) , K m,(β) , and the maximum velocities V (α) , V (β) , of hexokinase with α‐ d ‐glucose and β‐ d ‐glucose respectively, one can calculate the ratio of phosphorylation of α‐ d ‐glucose and β‐ d ‐glucose starting from the equilibrium mixture of d ‐glucose. In addition, the ratio of generation of α‐and β‐ d ‐glucopyranose‐6‐phosphate in the hexokinase reaction was determined from steady‐state analysis of the hexokinase/glucose‐6‐phosphate dehydrogenase system with equilibrated d ‐glucose. The experimentally obtained and the calculated values were in agreement.
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Tumor cells show a higher glycolytic rate than normal cells. Of glycolytic enzymes, the activity of hexokinase, known as a rate limiting enzyme in glycolysis, is amazingly high in malignant tumor cells. In mammals, four isozymes of hexokinase are expressed but the question which isozyme is responsible for the high hexokinase activity observed in tumor cells was not yet clearly answered. By Northern blot analysis, we found that the type II isozyme, which is only slightly expressed in normal heart, muscle and adipose tissue, was remarkably expressed in malignant tumor cells. We next tried to understand how the expression of type II hexokinase gene is regulated in tumor cells. For this purpose, we first isolated the type II hexokinase gene and characterized its structural features. We further investigated the regulatory machanisms of the expression of type II hexokinase in tumor cells. Results indicate the potential involvement of a serum responsive factor in the regulation of the expression of type II hexokinase in tumor cells. In addition to the remarkable expression, binding of the type II hexokinase to mitochondria is another characteristic of tumor cells, however, the physiological meaning of hexokinase binding to mitochondria was not yet fully understood. Our results clearly showed that the mitochondria-bound hexokinase utilize mitochondrially generated ATP more preferentially under normal conditions. However, when the rate of extramitochondrial ATP generating system (glycolysis) exceed that of mitochondrial ATP generating system (oxidative phosphorylation), the mitochondria-bound hexokinase utilize extramitochondrial ATP. This result indicates that the hexokinase binding enables a cross talk between oxidative phosphorylation and glycolysis.
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Adenosine triphosphate
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