Ultrastructural demonstration of glucose 6-phosphatase in cerebral cortex

Summary A two-stage fixation technique has been developed to obtain morphological preservation and retention of glucose 6-phosphatase (G6-Pase) activity for its demonstration in rat cerebral cortex. The technique was then employed to localize the enzyme in the cortex where it produced a dense reaction over the well developed granular endoplasmic reticulum cisternae in nerve cells and oligodendrocytes which contrasted with a thin reaction in astrocytes. Other membranous organelles showed no reaction.     

Ultrastructural demonstration of dehydrogenases in rat cerebral cortex

Summary Techniques for the ultrastructural demonstration of dehydrogenases in cerebral cortex are described. The best fixation for good fine structural preservation and retension of LDH and NADH-diaphorase was obtained by perfusion with a mixture of formaldehyde and glutaraldehyde and for SDH by perfusion with formaldehyde. Comparison of incubation conditions showed that consistent results were obtained using enzyme markers NBT and DS-NBT for LDH and NADH-diaphorase; DS-NBT was more satisfactory than NBT and BSPT for SDH. Penetration of incubation media was improved by Triton X-100; DMSO and ultrasonic treatment were less effective. The techniques enabled the first electron cytochemical demonstration of dehydrogenases in different elements of prefixed cerebral cortex. Ultrastructural sites of enzyme activities were localized within cristae and intermembrane spaces of mitochondria in nerve cell cytoplasm and its processes, oligodendrocytes and astrocytes. Authenticity of the ultrastructural sites was confirmed by four different control experiments.     

Ultrastructural demonstration of dehydrogenases in rat cerebral cortex.

Techniques for the ultrastructural demonstration of dehydrogenases in cerebral cortex are described. The best fixation for good fine structural preservation and retention of LDH and NADH-diphorase was obtained by perfusion with a misture of formaldehyde and glutaraldehyde and for SDH by perfusion with formaldehyde. Comparison of incubation conditions showed that consistent results were obtained using enzyme markers NBT and DS-NBT for LDH and NADH-diaphorase: DS-NBT was more satisfactory than NBT and BSPT for SDH. Penetration of incubation media was improved by Triton X-100: DMSO and ultrasonic treatment were less effective. The techniques enabled the first electron cytochemical demonstration of dehydrogenases in different elements of prefixed cerebral cortex. Ultrastructural sites of enzyme activities were localized within cristae and inter-membrane spaces of mitochondria in nerve cell cytoplasm and its processes, oligodendrocytes and astrocytes. Authenticity of the ultrastructural sites was confirmed by four different control experiments.     

Ultrastructural demonstration of cytochrome oxidase via cytochrome C in cerebral cortex.

A procedure for the ultrastructural cytochemical localization of cytochrome oxidase via cytochrome c in the cerebral cortex is described. Vascular perfusion fixation by formaldehyde and glutaraldehyde of different concentrations and mixtures of the two gave varying results. A mixture of 4% formaldehyde and 0.5% glutaraldehyde gave the best combination of ultrastructural preservation and retention of enzyme activity. Histochemical methods were examined for optimum incubation conditions, based on the oxidative polymerization of 3,3'-diaminobenzidine (DAB) to an osmiophilic product. The reaction product was discretely localized within intercristate and the intermembrane space of mitochondria. The staining pattern was the same in nerve cells and in neuroglia and their processed. The DAB reaction product was also found in mitochondria of the endothelial cells.     

Cytochemical demonstration of glucose-6-phosphatase in human liver

To determine the cytochemical localization of glucose-6-phosphatase in the human hepatocyte, lead - based and cerium - based media were used. By studying the effects of systematic variation of the incubation medium components, the optimal experimental conditions were determined. The exclusive localization of the cytochemical reaction in the endoplasmic reticulum and nuclear envelope, together with the results of control experiments ensured that these findings could be correlated with the phosphohydrolase activity of the multicomponent glucose-6-phosphatase system.     

The metabolism of glucose 6-phosphate by mammalian cerebral cortex in vitro

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.     

Sensitivity of glucose 6-phosphatase activity to glutaraldehyde.

The effect of glutaraldehyde fixation on glucose 6-phosphatase activity in mouse liver was investigated. After transparenchymal perfusion with 2% glutaraldehyde for 1.5 minutes, the activity of the recovered enzyme was higher than those reported for acid phosphatase and aryl sulfatase activities after fixation under similar condition, and an abundant deposition of reaction product was observed in hepatocytes. Subsequent immersion in the same fixative solution for 30 minutes after 4 degrees C resulted in only a slight decrease in the activity. However, the activity was almost completely destroyed after 3 hours of immersion fixation at 4 degrees C following the perfusion. Therefore, the enzyme can be said to be aldehyde-sensitive when a long fixation time is used, but not aldehyde-sensitive during a short fixation time.     

An assay for glucose 6-phosphatase based on the formation of glucose

A rapid, convenient method for the assay of glucose 6-phosphatase dependent on the removal of radioactive substrate from radioactive product by Dowex 2 fluoride is described. The enzymatic reaction is stopped by the addition of an ethanolic slurry of the resin. After the tubes are shaken, the radioactivity of glucose in the clarified supernatant layer is measured. The release of glucose is directly proportional to time and enzyme concentration. Detergents do not interfere with the method.     

Improved method for the histochemical demonstration of glucose-6-phosphatase activity

Summary Methodological studies on the histochemical technique for the demonstration of G6Pase activity showed that the occurrence of common artifacts: morphological destruction, extracellular precipitation of reaction product and nuclear staining are dependent on the concentration of lead nitrate, buffer and substrate. By studying the effects of systematic variation of the incubation media on the histochemical reaction optimal concentrations of either of these components were determined. An improved medium containing 3.6 mM lead nitrate, 40 mM tris-maleate buffer, pH 6.5, 10 mM G6P and 300 mM sucrose was used for the study of G6Pase distribution patterns in liver acini of juvenile and adult rats of both sexes and in those of starved adult female rats. The results obtained indicate sex dependent differences in the functional organization of the liver acinus and furthermore demonstrate the rapid functional adaptability of liver parenchyma to changes of the nutritional situation.Supported by a grant of the Deutsche Forschungsgemeinschaft     

Induction of glucose 6-phosphatase in cultured hepatoma cells by dexamethasone

The synthetic glucocorticoid, dexamethasone, induced high levels of glucose 6-phosphatase in a line of cultured hepatoma cells (2S FAZA). This conflicts with current theory that glucocorticoids induce a microsomal translocase, specific for glucose 6-phosphate, but not the hydrolase itself. The earlier conclusion was based on experiments with rat livers in vivo in which cortisone was the inducing agent. In the present study, 5 X 10(-6) M dexamethasone induced a tenfold increase in glucose 6-phosphatase activity in "intact" as well as disrupted microsomes using either glucose 6-phosphate or mannose 6-phosphate as substrate. This induction was blocked by cycloheximide (50 micrograms/ml). The broad pH maxima between 5.5 and 7.0 were similar for both "intact" and disrupted microsomes.