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Hexokinase and glucokinase binding in permeabilized guinea-pig hepatocytes.

ABSTRACT: The release of glucokinase (hexokinase IV) from digitonin-permeabilized hepatocytes from rat, guinea pig or mouse liver is inhibited by physiological concentrations of Mg2+ (> 0.25 mM). Preincubation of hepatocytes with fructose increases glucokinase release during permeabilization in the presence of Mg2+ but decreases glucokinase release in the absence of Mg2+, suggesting that fructose causes translocation of glucokinase from the Mg(2+)-dependent site. Glucose (25 mM) and sorbitol (1 mM) also induce translocation of glucokinase from the Mg(2+)-dependent site in guinea-pig, as in rat hepatocytes, but glucose is less effective than fructose or sorbitol, and the concentrations of fructose and sorbitol that cause half-maximal activation (A50) are 3-fold and 20-fold higher, respectively, in guinea-pig than in rat hepatocytes (170 microM and 257 microM, compared with 61 microM and 13 microM). Dihydroxyacetone and glycerol have no effect on fructose-induced or sorbitol-induced translocation in guinea-pig hepatocytes, in contrast with the potentiation and inhibition, respectively, by these substrates in rat hepatocytes. Some, but not all, of the differences between rat and guinea-pig hepatocytes could be due to the more reduced cytoplasmic NADH/NAD+ redox state in guinea-pig cells. The activity of low-Km hexokinases accounts for 30% of total hexokinase activity (low-Km hexokinases + glucokinase) in guinea-pig hepatocytes. Of the low-Km hexokinase activity, approx. 30% is released in the presence of Mg2+, 9% shows Mg(2+)-dependent binding and 60% shows Mg(2+)-independent binding. There was no substrate-induced translocation of low-Km hexokinase activity, indicating that translocation is specific for hexokinase IV.

SUBMITTER: Agius L

PROVIDER: S-EPMC1137623 | biostudies-other | 1994 Nov

REPOSITORIES: biostudies-other

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Project description:Metabolism of inositol 1,4,5-trisphosphate was investigated in permeabilized guinea-pig hepatocytes. The conversion of [3H]inositol 1,4,5-trisphosphate to a more polar 3H-labelled compound occurred rapidly and was detected as early as 5 s. This material co-eluted from h.p.l.c. with inositol 1,3,4,5 tetrakis[32P]phosphate and is presumably an inositol tetrakisphosphate. A significant increase in the 3H-labelled material co-eluting from h.p.l.c. with inositol 1,3,4-trisphosphate occurred only after a definite lag period. Incubation of permeabilized hepatocytes with inositol 1,3,4,5-tetrakis[32P]phosphate resulted in the formation of 32P-labelled material that co-eluted with inositol 1,3,4-trisphosphate; no inositol 1,4,5-tris[32P]phosphate was produced, suggesting the action of a 5-phosphomonoesterase. The half-time of hydrolysis of inositol 1,3,4,5-tetrakis[32P]phosphate of approx. 1 min was increased to 3 min by 2,3-bisphosphoglyceric acid. Similarly, the rate of production of material tentatively designed as inositol 1,3,4-tris[32P]phosphate from the tetrakisphosphate was reduced by 10 mM-2,3-bisphosphoglyceric acid. In the absence of ATP there was no conversion of [3H]inositol 1,4,5-trisphosphate to [3H]inositol tetrakisphosphate or to [3H]inositol 1,3,4-trisphosphate, which suggests that the 1,3,4 isomer does not result from isomerization of inositol 1,4,5-trisphosphate. The results of this study suggest that the origin of the 1,3,4 isomer of inositol trisphosphate in isolated hepatocytes is inositol 1,3,4,5-tetrakisphosphate and that inositol 1,4,5-trisphosphate is rapidly converted to this tetrakisphosphate. The ability of 2,3-bisphosphoglyceric acid, an inhibitor of 5-phosphomonoesterase of red blood cell membrane, to inhibit the breakdown of the tetrakisphosphate suggests that the enzyme which removes the 5-phosphate from inositol 1,4,5-trisphosphate may also act to convert the tetrakisphosphate to inositol 1,3,4-trisphosphate. It is not known if the role of inositol 1,4,5-trisphosphate kinase is to inactivate inositol 1,4,5-trisphosphate or whether the tetrakisphosphate product may have a messenger function in the cell.

Project description:1. The inhibition of hexokinase by glucose 6-phosphate has been investigated in crude homogenates of guinea-pig cerebral cortex by using a sensitive radio-chemical technique for the assay of hexokinase activity. 2. It was observed that 44% of cerebral-cortex hexokinase activity did not sediment with the microsomal or mitochondrial fractions (particulate fraction), and this is termed soluble hexokinase. The sensitivities of soluble and particulate hexokinase, and hexokinase in crude homogenates, to the inhibitory actions of glucose 6-phosphate were measured; 50% inhibition was produced by 0.023, 0.046 and 0.068mm-glucose 6-phosphate for soluble, particulate and crude homogenates respectively. 3. The optimum Mg(2+) concentration for the enzyme was about 10mm, and this appeared to be independent of the ATP concentration. In the presence of added glucose 6-phosphate, raising the Mg(2+) concentration to 5mm increased the activity of hexokinase, but above this concentration Mg(2+) potentiated the glucose 6-phosphate inhibition. When present at a concentration above 1mm, Ca(2+) ions inhibited the enzyme in the presence or absence of glucose 6-phosphate. 4. When the ATP/Mg(2+) ratio was 1.0 or below, variations in the ATP concentration had no effect on the glucose 6-phosphate inhibition; above this value ATP inhibited hexokinase in the presence of glucose 6-phosphate. ATP had an inhibitory effect on soluble hexokinase similar to that on a whole-homogenate hexokinase, so that the ATP inhibition could not be explained by a conversion of particulate into soluble hexokinase (which is more sensitive to inhibition by glucose 6-phosphate). It is concluded that ATP potentiates glucose 6-phosphate inhibition of cerebral-cortex hexokinase, whereas the ATP-Mg(2+) complex has no effect. Inorganic phosphate and l-alpha-glycerophosphate relieved glucose 6-phosphate inhibition of hexokinase; these effects could not be explained by changes in the concentration of glucose 6-phosphate during the assay. 5. The inhibition of hexokinase by ADP appeared to be independent of the glucose 6-phosphate effect and was not relieved by inorganic phosphate. 6. The physiological significance of the ATP, inorganic phosphate and alpha-glycerophosphate effects is discussed in relation to the control of glycolysis in cerebral-cortex tissue.

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Hexokinase and glucokinase binding in permeabilized guinea-pig hepatocytes.

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