Brain microvessel hexokinase: Kinetic properties

Summary Kinetic differences between brain capillary and parenchymal hexokinase in the presence of glucose, ATP, fructose, potassium, sodium and different pH were established. Parenchymal hexokinase is more susceptible to glucose inhibition, can tolerate greater variations in the ATP concentration, is inhibited by increasing concentrations of fructose and potassium, and showed greater activity on the lower pH values. The data suggest that in brain parenchyma and endothelial cells of brain microvessels, there are 2 different enzymes with regard to the kinetic properties.     

Brain microvessel hexokinase: kinetic properties.

Kinetic differences between brain capillary and parenchymal hexokinase in the presence of glucose, ATP. fructose, potassium, sodium and different pH were established. Parenchymal hexokinase is more susceptible to glucose inhibition, can tolerate greater variations in the ATP concentration, is inhibited by increasing concentrations of fructose and potassium, and showed greater activity on the lower pH values. The data suggest that in brain parenchyma and endothelial cells of brain microvessels, there are 2 different enzymes with regard to the kinetics properties.     

The involvement of fructose 2,6-bisphosphate in substrate cycle control in the nonoxidative stage of the pentose phosphate pathway. A phosphorus magnetic resonance spectroscopy study

The role of fructose 2,6-bisphosphate in the interconversion of sedoheptulose 7-phosphate and sedoheptulose 1,7-bisphosphate in rat liver cytosol fractions was studied by means of phosphorus magnetic resonance spectroscopy. When the activit of 6-phosphofructo-1-kinase was inhibited by a high concentration of ATP, the addition of fructose 2,6-bisphosphate led to a marked decrease in sedopheptulsoe 7-phosphate levels, accompanied by an increased concentration of ADP. Frructose 2,6-bisphosphate essentially inhibited both the decrease in sedoheptulose 1,7-disphosphate concentration and the accumulation of P_i in the incubation mixture. The data provided evidence that fructose 2,6-bisphosphate can regulate the substrate cycle; sedoheptulose 7-phosphate sedoheptulose 1,7-bisphosphate in the liver, and thus control the flux through the nonoxidative stage of the pentose phosphate pathway.     

Stimulation by D-glucose of mitochondrial respiration

D-glucose increases O2 uptake by cerebellum mitochondria. This effect is abolished by D-glucose-6-phosphate and D-mannoheptulose. It is proposed that the phosphorylation of D-glucose as catalyzed by bound hexokinase directly affects mitochondrial respiration.     

Stimulation by D-glucose of mitochondrial respiration

Summary D-glucose increases O₂ uptake by cerebellum mitochondria. This effect is abolished by D-glucose-6-phosphate and D-mannoheptulose. It is proposed that the phosphorylation of D-glucose as catalyzed by bound hexokinase directly affects mitochondrial respiration.     

Interaction of mitochondrial porin with cytosolic proteins.

Intracellular phosphorylation is an important step in active uptake and utilization of carbohydrates. For example glucose and glycerol enter the liver cell along the extra intracellular gradient by facilitated diffusion through specific carriers and are concentrated inside the cell by phosphorylation via hexokinase or glycerol kinase. Depending on the function of the respective tissue the uptake of carbohydrates serves different metabolic purposes. In brain and kidney medulla cells which depend on carbohydrates, glucose and glycerol are taken up according to the energy demand. However, in tissues such as muscle which synthesize glycogen or like liver which additionally produce fat from glucose, the uptake of carbohydrates has to be regulated according to the availability of glucose and glycerol. How the reversible coupling of the kinases to the outer membrane pore and the mitochondrial ATP serves to fulfil these specific requirements will be explained as well as how this regulates the carbohydrate uptake in brain according to the activity of the oxidative phosphorylation and how this allows glucose uptake in liver and muscle to persist in the presence of high glucose 6-phosphate without activating the rate of glycolysis.     

Interaction of mitochondrial porin with cytosolic proteins

Summary Intracellular phosphorylation is an important step in active uptake and utilization of carbohydrates. For example glucose and glycerol enter the liver, cell along the extra intracellular gradient by facilitated diffusion through specific carriers and are concentrated inside the cell by phosphorylation via hexokinase or glycerol kinase. Depending on the function of the respective tissue the uptake of carbohydrates serves different metabolic purposes. In brain and kidney medulla cells which depend on carbohydrates, glucose and glycerol are taken up according to the energy demand. However, in tissues such as muscle which synthesize glycogen or like liver which additionally produce fat from glucose, the uptake of carbohydrates has to be regulated according to the availability of glucose and glycerol. How the reversible coupling of the kinases to the outer membrane pore and the mitochondrial ATP serves to fulfil these specific requirements will be explained as well as how this regulates the carbohydrate uptake in brain according to the activity of the oxidative phosphorylation and how this allows glucose uptake in liver, and muscle to persist in the presence of high glucose 6-phosphate without activating the rate of glycolysis.     

Biochemical polymorphism of Japanese quail (Coturnix coturnix japonica): comparison of functionally different proteins (author's transl)]

The polymorphism observed among the enzymes involved in the respiratory metabolism (lactate dehydrogenase, malate dehydrogenase, phosphoglucomutase, phosphohexoseisomerase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase fructose 1-6 diphosphate dehydrogenase) is less important than that of the enzymes physiologically less essential, such as the various esterases, the alkaline phosphatase, the alcohol dehydrogenase, and of the non-enzymatic proteins (ovalbumin, ovoglobulins, ovomucoid, conalbumin, transferrin, etc.).     

Effects of glycolytic metabolites on preservation of high energy phosphate level and synaptic transmission in the granule cells of guinea pig hippocampal slices

The present study was undertaken to investigate whether neural activity of hippocampal slices can be preserved after replacingd-glucose with glycolytic intermediate metabolites such as lactate, pyruvate and citrate or with other sugars such as fructose, mannose, maltose, glucosamine, sucrose and galactose. As an index of neural activity, population spikes (PS) were recorded in the granule cell layers after electrical stimulation to the perforant path of guinea pig hippocampal slices. In addition, we determined the levels of ATP and creatine phosphate (CrP) in each slice after the replacement ofd-glucose with these substrates, and correlated it with the neural activity. Substrates other thand-glucose could not maintain the PS for even 20 min although the slices perfused with medium containing lactate, pyruvate, galactose, fructose and maltose maintained similar levels of ATP and CrP as in slices incubated in thed-glucose-containing medium. These results indicate thatd-glucose is essential for the preservation of synaptic activity in addition to its main role as the substrate for energy production to maintain the levels of high energy phosphates.     

Control of adenine nucleotide metabolism and glycolysis in vertebrate skeletal muscle during exercise

The turnover of adenosine triphosphate (ATP) in vertebrate skeletal muscle can increase more than a hundredfold during high-intensity exercise while the content of ATP in muscle may remain virtually unchanged. This requires that the rates of ATP hydrolysis and ATP synthesis are exactly balanced despite large fluctuations in reaction rates. ATP is regenerated initially at the expense of phosphocreatine (PCr) and then mainly through glycolysis from muscle glycogen. The increased ATP turnover in contracting muscle will cause an increase in the contents of adenosine diphosphate (ADP), adenosine monophosphate (AMP) and inorganic phosphate (P_i), metabolites that are substrates and activators of regulatory enzymes such as glycogen phosphorylase and phosphofructokinase. An intracellular metabolic feedback mechanism is thus activated by muscle contraction. How muscle metabolism is integrated in the intact body under physiological conditions is not fully understood. Common frogs are suitable experimental animals for the study of this problem because they can readily be induced to change from rest to high-intensity exercise, in the form of swimming. The changes in metabolites and effectors in gastrocnemius muscle were followed during exercise, post-exercise recovery and repeated exercise. The results suggest that glycolytic flux in muscle is modulated by signals from outside the muscle and that fructose 2,6-bisphosphate is a key signal in this process.     

Restoration of the poor oxygen transport function of ACD-stored blood by pyridoxal 5′-phosphate

Summary Pyridoxal 5-phosphate is easily incorporated into ACD-stored erythrocytes without decrease of ATP, and restores the poor oxygen transport function with a similar effect to 2,3-diphosphoglycerate.     

Glucose-6-phosphate dehydrogenase of rat liver peroxisomes

Summary A study was made of the effect of chronic administration of clofibrate on the activity and intracellular localization of rat liver glucose-6-phosphate dehydrogenase. Clofibrate-activated glucose-6-phosphate dehydrogenase was found to be located in peroxisomes.     

The purine nucleoside cycle in cell-free extracts of rat brain: evidence for the occurrence of an inosine and a guanosine cycle with distinct metabolic roles

The purine nucleoside cycle is a cyclic pathway composed of three cytosolic enzymes, hypoxanthine-guanine phosphoribosyltransferase, IMP-GMP specific 5'-nucleotidase, and purine-nucleoside phosphorylase. It may be considered a 'futile cycle', whose net reaction is the hydrolysis of 5-phosphoribosyl-1-pyrophosphate to inorganic pyrophosphate and ribose 1-phosphate. The availability of a highly purified preparation of cytosolic 5'-nucleotidase prompted us to reconstitute the purine nucleoside cycle. Its kinetics were strikingly similar to those observed when dialyzed extracts of rat brain were used. Thus, when the cycle is started by addition of inorganic phospate (Pi) and hypoxanthine or inosine (the 'inosine cycle'), steady-state levels of the intermediates are observed and the cycle 'turns over' as far as 5-phosphoribosyl-1-pyrophosphate is being consumed. In the presence of ATP, which acts both as an activator of IMP-GMP-specific 5'-nucleotidase and as substrate of nucleoside mono- and di-phosphokinases, no IDP and ITP are formed. The inosine cycle is further favored by the extremely low xanthine oxidase activity. Evidence is presented that ribose 1-phosphate needed to salvage pyrimidine bases in rat brain may arise, at least in part, from the 5-phosphoribosyl-1-pyrophosphate hydrolysis as catalyzed by the inosine cycle, showing that it may function as a link between purine and pyrimidine salvage. When the cycle is started by addition of Pi and guanine (the 'guanosine cycle'), xanthine and xanthosine are formed, in addition to GMP and guanosine, showing that the guanosine cycle 'turns over' in conjunction with the recycling of ribose 1-phosphate for nucleoside interconversion. In the presence of ATP, GDP and GTP are also formed, and the velocity of the cycle is drastically reduced, suggesting that it might metabolically modulate the salvage synthesis of guanyl nucleotides.     

The susceptibility to exercise-induced muscle damage increases as rats grow larger

Glucose-6-phosphate dehydrogenase and N-acetyl-beta-glucosaminidase activities were both elevated after eccentric exercise indicating that this type of exercise causes muscle damage. Muscle damage as measured by glucose-6-phosphate dehydrogenase activity in the vastus intermedius was greater and occurred later in larger rats indicating that the susceptibility to muscle damage is increased and the repair process delayed in older and larger animals.     

An improved assay for UDPglucose pyrophosphorylase and other enzymes that have nucleotide products

UDPglucose pyrophosphorylase catalyses the interconversion UDPglucose plus pyrophosphate and glucose 1-phosphate plus UTP. Several assay methods for this enzyme have been described but the only one that can be used to investigate the specificity with respect to various UDPsugars is based on coupling to UTP formation. This assay employs phosphoglycerate kinase to catalyse the formation 1,3-bisphosphoglycerate which is then used to oxidise NADH in the presence of glyceraldehyde 3-phosphate dehydrogenase. We have found that the activity of phosphoglycerate kinase towards UTP is low which limits the usefulness of the assay to very low rates, in agreement with the published recommendation of Hansen et al.⁵. Here it is shown that the dynamic range of the assay is increased by more than five fold on addition of nucleoside diphosphate kinase and ADP, which convert UTP to the preferred phosphoglycerate kinase substrate, ATP. It is also shown that the improved assay is suitable for enzymes with other nucleotide triphosphate products.     

The susceptibility to exercise-induced muscle damage increases as rats grow larger

Summary Glucose-6-phosphate dehydrogenase and N-acetyl--glucosaminidase activities were both elevated after eccentric exercise indicating that this type of exercise causes muscle damage. Muscle damage as measured by glucose-6-phosphate dehydrogenase activity in the vastus intermedius was greater and occurred later in larger rats indicating that the susceptibility to muscle damage is increased and the repair process delayed in older and larger animals.     

Effect of 5-thio-D-glucose on testicular lipids of mice

Summary Spermatogenesis is reported to be completely inhibited by 5-thio-D-glucose in mice. In an investigation of this inhibition, testicular lipid constituents, namely, total lipids, phospholipids, triacylglycerol, free and total cholesterol, 3-hydroxy-3-methylglutaryl-coenzyme A reductase and NADPH generators like glucose-6-phosphate dehydrogenase, intubation for 21 days. Significant increase in cholesteryl ester, glucose-6-phosphate dehydrogenase activity and malic enzyme and a decrease in free cholesterol and phospholipids were observed.Acknowledgments. We are thankful to the Director, Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry 6, India for permission to do the work and Mr T. Natarajan, Department of Biochemistry of ministerial assistance.     

Eptoformazione non ossidativa nel muscolo scheletrico

Summary The so-called non-oxydative heptoformation has been shown in enzymatic preparations of rat skeletal muscle, using as substrates glucose-6-phosphate and fructose-6-phosphate.     

Synergistic effect of acute hypoxia on flow-induced release of ATP from cultured endothelial cells

Human umbilical vein endothelial cells (HUVECs) in primary cultures were perfused under normoxic or hypoxic conditions. These cells were stimulated twice for 3 min by increased flow (from 0.5 to 3.0 ml/min). Under hypoxic conditions the basal release of ATP was the same as under normoxic conditions, but during increased flow the release was greater (0.58±0.07>0.32±0.04 pmoles/ml/10⁶ cells (+78%), for the first period of stimulation; 0.39±0.05>0.22±0.03 pmoles/ml/10⁶ cells (+79%) for the second period). Further experiments with sequential increments in flow rate showed that under both normoxic and hypoxic conditions, a positive correlation existed between ATP release and the rate of flow but there was always more ATP released under hypoxic conditions regardless of the flow rate.HUVECs in secondary culture (second passage) were similarly stimulated. No differences were observed between normoxic and hypoxic conditions. In both cases, the quantity of ATP released during high flow (0.050±0.004 pmoles/ml/10⁶ cells) was significantly smaller than the quantity of ATP released during low flow (0.09±0.01 pmoles/ml/10⁶ cells).To conclude, since hypoxia alone did not affect ATP release, there appears to be a synergistic relationship between increased shear stress and hypoxia in the stimulation of ATP release from HUVECs. Moreover, the release of ATP under these conditions seems to be a property of highly differentiated endothelial cells.     

Sul meccanismo della eptoformazione non ossidativa nel muscolo scheletrico

Summary F-1,6-P has been shown to increase the rate of non-oxydative heptoformation from glucose-6-phosphate and fructose-6-phosphate in enzymatic preparations of rat skeletal muscle. The mechanism of this increase — probably due to triose-phosphate, formed by aldolase action on F-1,6-P — is discussed.