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Factors affecting the glucose 6-phosphate inhibition of hexokinase from cerebral cortex tissue of the guinea pig.

ABSTRACT: 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.

SUBMITTER: Newsholme EA

PROVIDER: S-EPMC1198486 | biostudies-other | 1968 Jan

REPOSITORIES: biostudies-other

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Project description:1. The metabolism of glucose 6-phosphate in rat cerebral-cortex slices in vitro was compared with that of glucose. It was found that a glucose 6-phosphate concentration of 25mm was required to achieve maximal oxygen uptake rates and ATP concentrations, whereas only 2mm-glucose was required. 2. When 25mm-[U-(14)C]glucose 6-phosphate was used as substrate, the pattern of labelling of metabolites was found to be quantitatively and qualitatively similar to the pattern found with 10mm-[U-(14)C]glucose, except that incorporation into [(14)C]lactate was decreased, and significant amounts of [(14)C]glucose and [(14)C]mannose phosphate and [(14)C]fructose phosphate were formed. 3. Unlabelled glucose (10mm) caused a tenfold decrease in the incorporation of 25mm-[U-(14)C]glucose 6-phosphate into all metabolites except [(14)C]glucose and [(14)C]mannose phosphate and [(14)C]fructose phosphate. In contrast, unlabelled glucose 6-phosphate (25mm) had no effect on the metabolism of 10mm-[U-(14)C]glucose other than to increase markedly the incorporation into, and amount of, [(14)C]lactate, the specific radioactivity of this compound remaining approximately the same. 4. The effect of glucose 6-phosphate in increasing lactate formation from glucose was found to occur also with a number of other phosphate esters and with inorganic phosphate. Further investigation indicated that the effect was probably due to binding of medium calcium by the phosphate moiety, thereby de-inhibiting glucose uptake. 5. Incubations carried out in a high-phosphate high-potassium medium gave a pattern of metabolism similar to that found when slices were subjected to depolarizing conditions. Tris-buffered medium gave similar results to bicarbonate-buffered saline, except that it allowed much less lactate formation from glucose. 6. Part of the glucose formed from glucose 6-phosphate was extracellular and was produced at a rate of 12mumol/h per g of tissue in Krebs tris medium when glycolysis was blocked. The amount formed was much less when 25mm-P(i) or 26mm-HCO(3) (-) was present, the latter being in the absence of tris. 7. Glucose 6-phosphate also gave rise to an intracellular glucose pool, whereas no intracellular glucose was detectable when glucose was the substrate.

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Factors affecting the glucose 6-phosphate inhibition of hexokinase from cerebral cortex tissue of the guinea pig.

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