Effects of hyperthyroidism on the sensitivity of glycolysis and glycogen synthesis to insulin in the soleus muscle of the rat.

1. The effects of hyperthyroidism on the sensitivity and responsiveness of glycolysis and glycogen synthesis to insulin were investigated in the isolated incubated soleus muscle of the rat. 2. Hyperthyroidism, which was induced by administration of tri-iodothyronine (T3) to rats for 2, 5 or 10 days, increased fasting plasma concentrations of glucose, insulin and free fatty acids. 3. Administration of T3 for 2 or 5 days increased the rates of glycolysis at all insulin concentrations studied: this was due to increased rates of both glucose phosphorylation and glycogen breakdown, but there was no effect of T3 on the sensitivity of glycolysis to insulin. However, administration of T3 for 10 days increased the sensitivity of the rate of glycolysis to insulin. 4. The concentration of adenosine in the gastrocnemius muscles of the rats was not different from controls after 5 days, but it was markedly decreased after 10 days of T3 administration. If these changes are indicative of changes in the soleus muscle, the increased sensitivity of glycolysis to insulin found after 10 days' T3 administration could be due to the decrease in the concentration of adenosine. 5. Administration of T3 decreased the sensitivity of glycogen synthesis to insulin and the glycogen content of the soleus muscles. This may explain the decreased rates of non-oxidative glucose disposal found in spontaneous and experimental hyperthyroidism in man. 6. The rates of glucose oxidation did not change after 2 days, but they were increased after 5 and 10 days of T3 administration.     

Effect of exercise on insulin action in human skeletal muscle

The effect of 1 h of dynamic one-legged exercise on insulin action in human muscle was studied in 6 healthy young men. Four hours after one-legged knee extensions, a three-step sequential euglycemic hyperinsulinemic clamp combined with arterial and bilateral femoral vein catheterization was performed. Increased insulin action on glucose uptake was found in the exercised compared with the rested thigh at mean plasma insulin concentrations of 23, 40, and 410 microU/ml. Furthermore, prior contractions directed glucose uptake toward glycogen synthesis and increased insulin effects on thigh O2 consumption and at some insulin concentrations on potassium exchange. In contrast, no change in insulin effects on limb exchange of free fatty acids, glycerol, alanine or tyrosine were found after exercise. Glycogen concentration in rested vastus lateralis muscle did not increase measurably during the clamp even though indirect estimates indicated net glycogen synthesis. In contrast, in exercised muscle estimated and biopsy-verified increases in muscle glycogen concentration agreed. Local contraction-induced increases in insulin sensitivity and responsiveness play an important role in postexercise recovery of human skeletal muscle.     

Effects of monocarboxylates and external pH on some parameters of insect muscle fibres

The resting membrane potential and the conductance of flight muscle fibres of the butterfly Pieris brassicae were measured by means of two intracellular electrodes. The intracellular potassium, chloride, and hydrogen ion concentrations were estimated by measuring the concentrations in diluted muscle homogenates. Chloride in the bath was replaced in part by monocarboxylates and the pH was subsequently lowered. Replacement of chloride by propionate caused decrease in the internal chloride concentration, a marked hyperpolarization and a small increase in conductance. Lowering the pH in the propionate saline caused an additional decrease in the internal chloride concentration, an increase in the internal hydrogen ion concentration, a marked depolarization and a concurrent decrease in conductance. This was followed by an irreversible increase in conductance. With glycolate the effects on the membrane parameters were small and with pyruvate no effect was observed.     

Inhibition of glucose uptake and glycogenolysis by availability of oleate in well-oxygenated perfused skeletal muscle

The effects of exogenous oleate on glucose uptake, lactate production and glycogen concentration in resting and contracting skeletal muscle were studied in the perfused rat hindquarter. In preliminary studies with aged erythrocytes at a haemoglobin concentration of 8g/100ml in the perfusion medium, 1.8mm-oleate had no effect on glucose uptake or lactate production. During these studies it became evident that O(2) delivery was inadequate with aged erythrocytes. Perfusion with rejuvenated human erythrocytes at a haemoglobin concentration of 12g/100ml resulted in a 2-fold higher O(2) uptake at rest and a 4-fold higher O(2) uptake during muscle contraction than was obtained with aged erythrocytes. Rejuvenated erythrocytes were therefore used in subsequent experiments. Glucose uptake and lactate production by the well-oxygenated hindquarter were inhibited by one-third, both at rest and during muscle contraction, when 1.8mm-oleate was added to the perfusion medium. Addition of oleate also significantly protected against glycogen depletion in the fast-twitch red and slow-twitch red types of muscle, but not in white muscle, during sciatic-nerve stimulation. In the absence of added oleate, glucose was confined to the extracellular space in resting muscle. Addition of oleate resulted in intracellular glucose accumulation in red muscle. Contractile activity resulted in accumulation of intracellular glucose in all three muscle types, and this effect was significantly augmented in the red types of muscle by perfusion with oleate. The concentrations of citrate and glucose 6-phosphate were also increased in red muscle perfused with oleate. We conclude that, as in the heart, availability of fatty acids has an inhibitory effect on glucose uptake and glycogen utilization in well-oxygenated red skeletal muscle.     

Zonation of glycogen and glucose syntheses, but not glycolysis, in rat liver.

We have investigated the cause of defective glycogen synthesis in hepatocyte preparations enriched with cells from the periportal or perivenous zones obtained by the methods of Lindros & Penttila [Biochem. J. (1985) 228, 757-760] and of Quistorff [Biochem. J. (1985) 229, 221-226]. A modified procedure which yields hepatocytes capable of consistent rates of glycogen synthesis is described, and the rates of glucose and glycogen syntheses and of glycolysis in hepatocytes from the two zones are compared. Glycogen synthesis in cells was greatly impaired by very low concentrations (0.01-0.05 mg/ml) of digitonin, which had little effect on glucose and protein syntheses and Trypan Blue exclusion. Cells exposed to such low concentrations of digitonin lose all their synthetic capacity and ability to exclude Trypan Blue when incubated with EGTA, which does not affect cells not exposed to digitonin. With a modified procedure based on this phenomenon, our study reveals that hepatocyte preparations enriched with cells from the periportal zone synthesized glucose from lactate and alanine at rates twice those by cells from the perivenous zone, whereas the rate of glycogen synthesis from C3 precursors in periportal cells was 4 times that in the perivenous preparations. With substrates entering the pathway at the triose phosphate level, gluconeogenesis in periportal-cell preparations was 20% higher, and glycogen synthesis was twice that in perivenous preparations. Glycolysis was studied by the formation of 3HOH from [2-3H]glucose, the yield of lactate, and the conversion of [14C]glucose into [14C]lactate. In cell preparations from both zones glycolysis by all criteria was negligible at 10 mM-glucose, but was substantial at higher concentrations. However, there was no difference between the zones. We confirm that the capacities for glucose and glycogen syntheses in periportal cells are higher than in perivenous cells, but that at physiological glucose concentrations there is negligible glycolysis in liver parenchyma in both zones. The metabolic pattern in the perivenous cells is not glycolytic.     

Substrate specific activation by glucose 6-phosphate of the dephosphorylation of muscle glycogen synthase.

The activation of glycogen synthase by insulin is in many instances stimulated by the presence of extracellular glucose. Previous observations in cell extracts, glycogen pellets and other crude systems suggest that this stimulation may be due to an increase in glucose 6-phosphate, which activates the dephosphorylation of glycogen synthase by protein phosphatases. Using purified rabbit muscle glycogen synthase D and protein phosphatases 1 and 2A, the types responsible for the activation of muscle synthase, it was found that glucose 6-phosphate, at low, physiological concentrations, stimulated the dephosphorylation of glycogen synthase. Both types of phosphatase were stimulated to the same extent when acting on glycogen synthase. The dephosphorylation of other protein substrates of the phosphatases was either not affected or inhibited by glucose 6-phosphate. It appears that the stimulatory effect of glucose 6-phosphate at physiological concentrations is apparently specific for glycogen synthase, and most likely due to an allosteric configuration change of this enzyme which facilitates its dephosphorylation. In addition, the effects of other reported modulators of glycogen synthase dephosphorylation, AMP, ATP and Mg2+, were studied in this 'in vitro' system.     

Effects of okadaic acid, an inhibitor of protein phosphatases-1 and -2A, on glucose transport and metabolism in skeletal muscle.

The effect of okadaic acid, an inhibitor of protein phosphatases-1 and -2A, was studied on glucose transport and metabolism in soleus muscles isolated from lean and insulin-resistant obese mice. In muscles from lean mice, the uptake of 2-deoxyglucose, an index of glucose transport and phosphorylation, was increased by okadaic acid in a concentration-dependent manner. At 5 microM, okadaic acid was as efficient as a maximally effective insulin concentration. Glucose metabolism (glycolysis and glycogen synthesis) was also measured. Whereas glycolysis was stimulated by okadaic acid, glycogen synthesis was unchanged. When okadaic acid and insulin were added together in the incubation medium, the rates of glucose transport, glycolysis, and glycogen synthesis were similar to those obtained with insulin alone, whether maximal or submaximal insulin concentrations were used. Furthermore, okadaic acid did not activate the kinase activity of the insulin receptor studied in an acellular system or in intact muscles. These results indicate that a step in the insulin-induced stimulation of muscle glucose transport involves a serine/threonine phosphorylation event that is regulated by protein phosphatases-1 and/or -2A. In muscles of insulin-resistant obese mice, the absolute values of deoxyglucose uptake stimulated by okadaic acid were lower than in muscles from lean mice. However, the okadaic acid effect, expressed as a fold stimulation, was normal. These observations suggest that in the insulin-resistant state linked to obesity, the serine/threonine phosphorylation event is likely occurring normally, but a defect at the level of the glucose transporter itself would prevent a normal response to insulin or okadaic acid.     

Regulation of glycogen synthase activity in the isolated mouse soleus muscle. Effect of insulin, epinephrine, glucose and anti-insulin receptor antibodies

The effects of insulin, epinephrine, glucose and anti-insulin receptor antibodies on enzymes involved in the regulation of glycogen synthesis were investigated in the isolated mouse soleus muscle. Insulin maximally increased the percentage of glycogen synthase active form after 15 min in the absence of glucose in the extracellular medium; half-maximal and maximal effects were obtained with 1.5 and 33 mM insulin, respectively. The basal percentage of glycogen phosphorylase active form was not altered by insulin. Antibodies to the insulin receptor had similar effects to those of insulin on both enzymes. The percentage of glycogen synthase active form was maximally decreased and that of phosphorylase maximally increased after a 2 min exposure to epinephrine in the absence of extracellular glucose. Glucose alone had no effect on muscle glycogen synthase. When muscles were incubated with insulin (33 nM) plus glucose (20 mM) for 5-10 min, the increase in the percentage of glycogen synthase active form was greater than with insulin alone. This enhancing effect of glucose on insulin activation of glycogen synthase disappeared after 20 min. The results suggest the existence of two mechanisms whereby insulin activates muscle glycogen synthase. The main effect is operative in the absence of extracellular glucose and occurs at insulin concentrations close to the physiological range. The other effect requires glucose and may result from the stimulation by insulin of glucose transport and/or metabolism.     

The stimulation of glycolysis by previous aerobiosis in rat-liver slices

1. An investigation has been made on the stimulation of the anaerobic glycolysis by rat-liver slices caused by previous incubation in oxygen. 2. The stimulation is sustained partly by endogenous carbohydrates and partly by added glucose. The effect of glucose reaches a maximum at a concentration of 20mm; it is more pronounced when glucose is present in the actual glycolytic phase and not during the aerobic preincubation. The conversion of fructose and pyruvate into lactic acid is not affected by the preincubation in oxygen. 3. The stimulation occurs also when preincubation is carried out in a medium that blocks the action of phosphorylase. 4. Preincubation for 2-3min. at 37 degrees is enough to ensure maximum stimulation. The main effect of the aerobic incubation is on the initial velocity of the anaerobic glycolysis. 5. The stimulation depends on the nutritional state of the animal: it is decreased practically to nil in rats starved overnight. In starved animals glycogen content and basal and stimulated glycolysis decline progressively with the same trend. If starved animals are injected with glucose, liver glycogen concentration increases but basal glycolysis remains at a low level; however, the rate of stimulated glycolysis becomes progressively higher and correlates with the amount of liver glycogen. 6. It is suggested that the aerobic preincubation modifies the factors that regulate glycolysis in liver slices at steps above the level of triose phosphates.     

The pH-dependence of sugar-transport and glycolysis in cultured Ehrlich ascites-tumour cells.

1. pH-dependence of glycolysis has generally been ascribed to the effects of pH on the activities of glycolytic enzymes. The present study shows that sugar transport is pH-dependent in cultured Ehrlich ascites-tumour cells. 2. The rates of glucose consumption, of 3-O-methylglucose transport, and of 2-deoxyglucose transport and phosphorylation increased as linear functions of pH, as the pH of the cell culture medium was increased from 6.1 to 8.5. Transport of glucose, as measured in ATP-depleted cells, was pH-dependent to the same extent as transport of the non-metabolizable sugars. 3. Glucose consumption rates were about 8-fold higher at pH 8.5 than at pH 6.4. About 65-85% of glucose was converted into lactate. Sugar transport rates were 2.5-fold higher at pH 8.5 than at pH 6.3. 4. pH affected both simple diffusion and facilitated diffusion. pH effect was mainly on the Vmax. of 2-deoxyglucose uptake, and on the rapid-uptake phase of 3-O-methylglucose transport. 5. It was estimated that about 70% of the pH effect on the rates of glucose consumption may be due to the effect on sugar transport and the remainder to the effect on the activities of glycolytic enzymes.     

Post-mortem glycolysis in ox skeletal muscle. Effect of pre-rigor freezing and thawing on the intermediary metabolism

1. Ox sternomandibularis muscle was ;slow-frozen' by placing it in air at -22 degrees or ;fast-frozen' by immersion in liquid air or acetone-solid carbon dioxide. In all cases muscles were frozen pre-rigor. Changes in length, pH and the concentrations of P(i), creatine phosphate, hexose monophosphate (glucose 1-phosphate+glucose 6-phosphate+fructose 6-phosphate), fructose diphosphate (fructose 1,6-diphosphate+(1/2) triose phosphate), lactate, ATP, ADP, AMP and NAD(+) during freezing and during subsequent thawing were determined. In addition some measurements were made of the changes in alpha-glycerophosphate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate and pyruvate concentrations during slow freezing. 2. Appreciable shortening and marked changes in chemical composition took place during slow freezing but not during fast freezing. 3. During slow freezing the hexose monophosphate concentration fell and fructose 1,6-diphosphate and triose phosphate increased substantially. Increases also took place in 3-phosphoglycerate, 2-phosphoglycerate and phosphoenolpyruvate, but not in pyruvate. 4. On thawing, most of the chemical changes were similar to those in unfrozen muscle post mortem, but took place much more rapidly; loss of NAD(+) was particularly rapid. Fast-frozen muscle metabolized at a faster rate on thawing than did slow-frozen muscle. 5. The overall changes in length during freezing and thawing were about the same in slow-frozen as in fast-frozen muscle.     

Mechanism of muscle glycogen autoregulation in humans

To examine the mechanism by which muscle glycogen limits its own synthesis, muscle glycogen and glucose 6-phosphate (G-6-P) concentrations were measured in seven healthy volunteers during a euglycemic ( approximately 5.5 mM)-hyperinsulinemic ( approximately 450 pM) clamp using (13)C/(31)P nuclear magnetic resonance spectroscopy before and after a muscle glycogen loading protocol. Rates of glycogen synthase (V(syn)) and phosphorylase (V(phos)) flux were estimated during a [1-(13)C]glucose (pulse)-unlabeled glucose (chase) infusion. The muscle glycogen loading protocol resulted in a 65% increase in muscle glycogen content that was associated with a twofold increase in fasting plasma lactate concentrations (P < 0.05 vs. basal) and an approximately 30% decrease in plasma free fatty acid concentrations (P < 0.001 vs. basal). Muscle glycogen loading resulted in an approximately 30% decrease in the insulin-stimulated rate of net muscle glycogen synthesis (P < 0.05 vs. basal), which was associated with a twofold increase in intramuscular G-6-P concentration (P < 0.05 vs. basal). Muscle glycogen loading also resulted in an approximately 30% increase in whole body glucose oxidation rates (P < 0.05 vs. basal), whereas there was no effect on insulin-stimulated rates of whole body glucose uptake ( approximately 10.5 mg. kg body wt(-1). min(-1) for both clamps) or glycogen turnover (V(syn)/V(phos) was approximately 23% for both clamps). In conclusion, these data are consistent with the hypothesis that glycogen limits its own synthesis through feedback inhibition of glycogen synthase activity, as reflected by an accumulation of intramuscular G-6-P, which is then shunted into aerobic and anaerobic glycolysis.     

Regulation of glycogen synthase activity in the isolated mouse soleus muscle effect of insulin,epinephrine, glucose and anti-insulin receptor antibodies

The effects of insulin, epinephrine, glucose and anti-insulin receptor antibodies on enzymes involved in the regulation of glycogen synthesis were investigared in the isolated mouse soleus muscle. Insulin maximally increased the percentage of glycogen synthase active form after 15 min in the absence of glucose in the extracellular medium; half-maximal and maximal effects were obtained with 1.5 and 33 nM insulin, respectively. The basal percentage of glycogen phosphorylase active form was not altered by insulin. Antibodies to the insulin receptor had similar effects to those of insulin on both enzymes. The percentage of glycogen synthase active form was maximally decreased and that of phosphorylase maximally increased after a 2 min exposure to epinephrine in the absence of extracellular glucose. Glucose alone had no effect on muscle glycogen synthase. When muscles were incubated with insulin (33 nM) plus glucose (20 mM) for 5–10 min, the increase in the percentage of glycogen synthase active form was greater than with insulin alone. This enhancing effect of glucose on insulin activation of glycogen synthase disappeared after 20 min. The results suggest the existence of two mechanisms whereby insulin activates muscle glycogen synthase. The main effect is operative in the absence of extracellular glucose and occurs at insulin concentrations close to the physiological range. The other effect requires glucose and may result from the stimulation by insulin of glucose transport and/or metabolism.     

Effect of acetate on glucose utilization by isolated rat thymocytes

The effects of acetate and ammonium salts on glucose metabolism, aminoisobutyric acid influx, and radioiodinated insulin binding in isolated thymocytes were studied. Acetate in the concentration range, 0.1–30 mm, was found to inhibit basal and insulin-stimulated CO₂ production whereas ammonium chloride at concentrations greater than 0.3 mm was slightly stimulatory. Ammonium salts inhibited glucose incorporation into glycogen and aminoisobutyric acid influx only at high concentration (30 mm). Neither acetate nor ammonium salts had significant effects on glucose incorporation into glycogen or aminoisobutyric acid influx at lower concentrations. No effect on insulin binding was observed. The observation that very low concentrations of acetate can perturb these biological assay systems suggests that other biological functions may be affected by trace amounts of buffer salts carried over from protein isolation steps.     

Regulation of Glycogen Content in Primary Astrocyte Culture: Effects of Glucose Analogues, Phenobarbital, and Methionine Sulfoximine

Compounds known to affect glycogen metabolism in vivo or in cell-free preparations were used to investigate the regulation of glycogen content in intact astrocytes cultured from newborn rat cortex. Compounds were added with fresh medium to culture dishes, and astrocyte glucose and glycogen content determined 24 h later. Increasing the medium glucose concentration from 7.5 mM to 30 mM increased cell glycogen content 80%. Addition of 2-deoxyglucose or 3-O-methyl glucose (2.5-10 mM) also increased cell glycogen content, 50-100%, suggesting a regulatory rather than mass action effect of glucose on astrocyte glycogen content. The phosphorylase b inhibitors 2,2',4,4',5,5'-hexabromobiphenyl and riboflavin had no effect on astrocyte glycogen content, consistent with negligible phosphorylase b activity in normal astrocytes. Phenobarbital and L-methionine-DL-sulfoximine (MSO) are both known to induce astrocyte glycogen accumulation in vivo. The addition of phenobarbital (2 mM) had no effect on the glycogen content of cultured astrocytes, suggesting an indirect mechanism for the in vivo effect. MSO at 1 mM, however, induced a 300% increase in glycogen content. The time course of glucose and glycogen content after MSO administration suggests this increase to be the result of slowed glycogenolysis rather than accelerated glycogen synthesis.     

Kinetics of glycogen phosphorylase a from the muscle of the blue crab, Callinectes danae

The kinetics of purified glycogen phosphorylase a from the muscle of the blue crab (Callinectes danae) were studied in the direction of glycogen synthesis, and in the direction of glycogen degradation with P_i or arsenate as substrates. The effects of AMP, UDPG, G-6-P, glucose, and arsenate on the appropriate systems were studied. AMP is an activator of the enzyme. Inhibition by UDPG with respect to P_i changes from noncompetitive to competitive when AMP is added; it changes from noncompetitive to mixed with respect to glycogen when AMP is added. G-6-P is a competitive inhibitor of G-1-P and arsenate. Inhibition by glucose with respect to glycogen changes from noncompetitive to competitive when AMP is added in the direction of glycogen breakdown; it is noncompetitive with respect to P_i. Arsenate is a competitive inhibitor with respect to P_i. The K_m for AMP increases in the presence of UDPG, and decreases with increasing concentrations of P_i or glycogen. We propose a model in which the enzyme bears three interacting sites: an active site, an activator (AMP) site, and an inhibitor (glucose) site. The active site has three subsites: one for P_i, one for glycogen, and one for a glucose moiety which may be part of the substrates or inhibitors.     

Characteristics of film based on protein isolate from red tilapia muscle with negligible yellow discoloration

The properties of film from fish protein isolate (FPI) prepared by prior washing followed by alkaline solubilization process (ASP) from red tilapia muscle were monitored during the storage of 20 days at 50% RH and 25°C, in comparison with those of films from washed mince. Lipid, heme iron and non-heme iron contents in FPI were decreased by 98.8, 36.8 and 91.9%, respectively in comparison with those of washed mince (p<0.05). Films from FPI had higher tensile strength (TS) and elongation at break (EAB) than those from washed mince for both pH (3 and 11) used for film preparation (p<0.05). Film from FPI at pH 3 showed the highest TS, while that from washed mince at pH 11 had the lowest TS (p<0.05). Nevertheless, films from FPI had higher WVP than those from washed mince for both pH used (p<0.05). At the same pH used for film preparation (3 or 11), films from FPI showed the lower TBARS values than those from washed mince (p<0.05). Nevertheless, films from both FPI and washed mince had the higher TBARS values when pH 3 was used for film preparation, compared with pH 11 (p<0.05). Among all films, those from FPI prepared at pH 3 had the highest transparency and no yellow discoloration was observed during the storage of 20 days, in comparison with other films (p<0.05). Conversely, film from washed mince prepared at pH 3 had the higher increase in b*-value and ΔE*-value than other films. Therefore, FPI could serve as a potential material for film preparation with lower contents of lipid and prooxidants, thereby preventing the yellow discoloration of the fish myofibrillar protein-based film during extended storage.     

Modeling cancer glycolysis

Most cancer cells exhibit an accelerated glycolysis rate compared to normal cells. This metabolic change is associated with the over-expression of all the pathway enzymes and transporters (as induced by HIF-1α and other oncogenes), and with the expression of hexokinase (HK) and phosphofructokinase type 1 (PFK-1) isoenzymes with different regulatory properties. Hence, a control distribution of tumor glycolysis, modified from that observed in normal cells, can be expected. To define the control distribution and to understand the underlying control mechanisms, kinetic models of glycolysis of rodent AS-30D hepatoma and human cervix HeLa cells were constructed with experimental data obtained here for each pathway step (enzyme kinetics; steady-state pathway metabolite concentrations and fluxes). The models predicted with high accuracy the fluxes and metabolite concentrations found in living cancer cells under physiological O(2) and glucose concentrations as well as under hypoxic and hypoglycemic conditions prevailing during tumor progression. The results indicated that HK≥HPI>GLUT in AS-30D whereas glycogen degradation≥GLUT>HK in HeLa were the main flux- and ATP concentration-control steps. Modeling also revealed that, in order to diminish the glycolytic flux or the ATP concentration by 50%, it was required to decrease GLUT or HK or HPI by 76% (AS-30D), and GLUT or glycogen degradation by 87-99% (HeLa), or decreasing simultaneously the mentioned steps by 47%. Thus, these proteins are proposed to be the foremost therapeutic targets because their simultaneous inhibition will have greater antagonistic effects on tumor energy metabolism than inhibition of all other glycolytic, non-controlling, enzymes.     

The allosteric regulation of hexokinase C from amphibian liver.

A type C hexokinase (ATP:D-hexose-6-phosphotransferase EC 2.7.1.1) was partially purified from the liver of the frog Calyptocephalella caudiverbera. The enzyme is inhibited by glucose levels in the range of normal blood sugar concentrations. The extent of the inhibition by glucose depends on the concentration of ATP, being most marked between 1 and 5 mM ATP. Fructose, although a substrate, was not inhibitory of its own phosphorylation. The inhibitory effect of high glucose levels exhibited a strong, reversible pH dependence being most marked at pH 6.5. At pH 7.5 the inhibition by high glucose levels was a function of the enzyme concentration, the effect being stronger at high enzyme concentrations, whereas no inhibition was observed when assaying very diluted preparations. At all enzyme concentrations studied, high levels of glucose caused no inhibition at pH 8.5, whereas at pH 6.5 strong inhibition was always observed. Short times of photooxidation of hexokinase C as well as incubation with low concentrations of p-chloromercuribenzoate resulted in the loss of the inhibition by excess of glucose. Glucose-6-phosphate was found to be a strong inhibitor of hexokinase C but only at high glucose levels. The inhibitory effect of glucose-6-P follows sigmoidal kinetics at low (about 0.02 mM) glucose concentrations, the Hill coefficient being 2.3. The kinetics of the inhibition became hyperbolic at high (greater than 0.2 mM) glucose levels. These results suggest that the inhibition of hexokinase C by excess glucose is due to the interaction of glucose with a second, aldose-specific, regulatory site on the enzyme. The modification of the inhibitory effect by ATP, glucose-6-P, enzyme concentration, and pH, all of them at physiological levels, indicates a major role for hexokinase C in the regulation of glucose utilization by the liver.     

Calcium transport in intact human erthrocytes

Intact human erythrocytes can be readily loaded with calcium by incubation in hypersomotic media at alkaline pH. Erythrocyte calcium content increases from 15-20 to 120-150 nmol/g hemoglobin after incubation for 2 h at 20 degree C in a 400 mosmol/kg, pH 7.8 solution containing 100 mM sodium chloride, 90 mM tetramethylammonium chloride, 1 mM potassium chloride, and 10 mM calcium chloride. Calcium uptake is a time-dependent process that is associated with an augmented efflux of potassium. The ATP content in these cells remains at more than 60% of normal and is not affected by calcium. Calcium uptake is influenced by the cationic composition of the external media. The response to potassium is diphasic. With increasing potassium concentrations, the net accumulation of calcium initially increases, becoming maximal at 1 mM potassium, then diminishes, falling below basal levels at concentrations above 3 mM potassium. Ouabain inhibits the stimulatory effect of low concentrations of potassium. The inhibitory effects of higher concentrations of potassium are ouabain insensitive and independent of the external calcium concentration. Sodium also inhibits calcium uptake but this inhibition can be modified by altering the external concentration of calcium. The effux of calcium from loaded erythrocytes is not significantly altered by changes in osmolality, medium ion composition, or ouabain. It is concluded that hypertonicity increases the net uptake of calcium by increasing the influx of calcium and that some part of the sodium potassium transport system is involved in this influx process.