Purification and properties of buffalo (Bubalus bubalis) erythrocyte hexokinase

Buffalo erythrocytes contain one isozyme of hexokinase that apparently lacks microheterogeneity as shown by chromatographic properties. A single protein band was detected by means of Western blotting using an antibody raised in rabbits against homogeneous rat brain hexokinase I. The native protein has a molecular weight of 200,000 +/- 2880 by gel filtration. Partial purification of erythrocyte hexokinase by a combination of several procedures, including affinity chromatography, which was previously applied successfully to the purifica tion of other mammalian type I hexokinases, produced a partially purified enzyme that showed several contami nants after SDS-polyacrylamide gel electrophoresis. The affinity of buffalo erythrocyte hexokinase for glucose (K-m = 0.012 +/- 0.001 mM) is lower than most other mammal hexokinases type I. It phosphorylates other sugars, with considerably higher K-m values. This isozyme is able to use MgATP but does not use MgGTP, MgCTP or MgUTP. We used inhibition patterns, obtained with products to elucidate enzyme sequential mechanisms. Our results are clearly in agreement with a random sequential mechanism and in disagreement with an ordered sequential mechanism with either glucose or ATP as the obligatory first substrates. The ADP inhibition was of mixed type with both ATP and glucose as substrates. (C) 1997 Elsevier B.V.

During the initiation of fermentation overexpression of hexokinase PII in yeast transiently causes a similar deregulation of glycolysis as deletion of Tps1

In the yeast Saccharomyces cerevisiae a novel control exerted by TPS1 (=GGS1=FDP1=BYP1=CIF1=GLC6=TSS1)-encoded trehalose-6-phosphate synthase, is essential for restriction of glucose influx into glycolysis apparently by inhibiting hexokinase activity in vivo. We show that up to 50-fold overexpression of hexokinase does not noticeably affect growth on glucose or fructose in wild-type cells. However, it causes higher levels of glucose-6-phosphate, fructose-6-phosphate and also faster accumulation of fructose-1,6-bisphosphate during the initiation of fermentation. The levels of ATP and Pi correlated inversely with the higher sugar phosphate levels. In the first minutes after glucose addition, the metabolite pattern observed was intermediate between those of the tps1Δ mutant and tile wild-type strain. Apparently, during the start-up of fermentation hexokinase is more rate-limiting in the first section of glycolysis than phosphofructokinase. We have developed a method to measure the free intracellular glucose level which is based on the simultaneous addition of D-glucose and an equal concentration of radiolabelled L-glucose. Since the latter is not transported, the free intracellular glucose level can be calculated as the difference between the total B-glucose measured (intracellular + periplasmic/extracellular) and the total L-glucose measured (periplasmic/extracellular). The intracellular glucose level rose in 5 min after addition of 100 mM-glucose to 0.5-2 mM in the wild-type strain, ± 10 mm in a hxk1Δ hxk2Δ glk1Δ and 2-3 mM in a tps1Δ strain. In the strains overexpressing hexokinase PII the level of free intracellular glucose was not reduced. Overexpression of hexokinase PII never produced a strong effect on the rate of ethanol production and glucose consumption. Our results show that overexpression of hexokinase does not cause the same phenotype as deletion of Tps1. However, it mimics it transiently during the initiation of fermentation. Afterwards, the Tps1-dependent control system is apparently able to restrict Properly up to 50-fold higher hexokinase activity.

Occurrence of sibling species of Lutzomyia longipalpis (Diptera : Psychodidae) in Venezuela: First evidence from reproductively isolated sympatric populations

The delimitation of cryptic species within the main vector of the American visceral leishmaniasis, Lutzomyia longipalpis, remains a topic of controversy. An analysis of generic variability based on 8 enzymatic loci revealed fixed differences in 2 diagnostic loci, adenylate kinase (Ak) and hexokinase (Hk), between sympatric and allopatric populations at 4 localities in Venezuela. The absence of heterozygotes for these 2 loci within 1 locality indicates, for the first time, the presence of 2 sympatric reproductively isolated populations or cryptic species within L. longipalpis. Significant differences were also detected between these cryptic species in the allele frequencies of glucose-6-phosphate isomerase (Gpi) and malate dehydrogenase, decarboxylating (Me). One species showed mean heterozygosities that ranged between 6.6% and 6.7%, with 1.6-1.9 alleles detected per locus, while the other had mean heterozygosities that ranged from 4.3% to 6.3%, with 1.3-1.6 alleles per locus. Comparisons of isozyme profiles with published data suggests that 1 species is similar to the L. longipalpis described in Colombian and Brazilian populations, whereas the other has not been previously reported.

Walker 256 tumour growth causes marked changes of glutamine metabolism in rat small intestine

The effect of Walker 256 tumour growth on the metabolism of glucose and glutamine in the small intestine of rats was examined. Walker 256 tumour has been extensively used as an experimental model to induce cancer cachexia in rats. Walker 256 tumour growth decreased body weight and small intestine weight and length. The activities of glucose-6-phosphate dehydrogenase and phosphate-dependent glutaminase were reduced in the proximal, median and distal portions of the intestine. Glutamine oxidation was reduced in the proximal portion only. The decrease in glutaminase activity was not due to a low synthesis of the protein as indicated by Western blotting analysis. Hexokinase and citrate synthase activities were not changed by the tumour. These findings led us to postulate that tumour growth impairs glutamine metabolism of small intestine but the mechanism involved remains to be elucidated. Copyright (C) 2001 John Wiley Sons, Ltd.

Glucose metabolism in the erythrocytes of the buffalo (Bubalus bubalis)

(CO2)-C-14 production from [1-C-14] glucose, the rate of glycolysis measured by the value of lactate production and the activities of various enzymes were determined in buffalo erythrocytes. Buffalo red cell glycolytic metabolites were estimated and used for the calculation of the mass action ratios of reactions catalyzed by the glycolytic enzymes of Bubalus bubalis. A comparison of the values of the mass action ratios with the equilibrium constants of the various glycolytic reactions indicate that hexokinase, phosphofructokinase, phosphoglycerate kinase and pyruvate kinase reactions are displaced from equilibrium, suggesting a regulatory role for each of these enzymes in buffalo erythrocyte glycolysis. (C) 1997 Elsevier B.V.

Toxicity of hypercaloric diet and monosodium glutamate: oxidative stress and metabolic shifting in hepatic tissue.

The present study examines the effects of a hypercaloric diet on hepatic glucose metabolism of young rats, with and without monosodium glutamate (MSG) administration, and the association of these treatments with evaluating markers of oxidative stress. Male weaned Wistar rats (21 days old) from mothers fed with a hypercaloric diet or a normal diet, were divided into four groups (n=6): control (C) fed with control diet; (MSG) treated with MSG (4 mg/g) and control diet; (HD) fed with hypercaloric diet and (MSG-HD) treated with MSG and HD. Rats were sacrificed after the oral glucose tolerance test (OGTT), at 45 days of treatments. Serum was used for insulin determination. Glycogen, hexokinase(HK), glucose-6-phosphatase(G6PH), lipid hydroperoxide, superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px) were determined in liver. HD rats showed hypoglycemia, hyperinsulinemia, and high hepatic glycogen, HK and decreased G6PH. MSG and MSG-HD had hyperinsulinemia, hyperglycemia, decreased HK and increased G6PH in hepatic tissue. These animals had impaired OGTT. HD, MSG and MSG-HD groups had increased lipid hydroperoxide and decreased SOD in hepatic tissue. Hypercaloric diet and monosodium glutamate administration induced alterations in metabolic rate of glucose utilization and decreased antioxidant defenses. Therefore, the hepatic glucose metabolic shifting induced by HD intake and MSG administration were associated with oxidative stress in hepatic tissue.

Ajuste metabólico e respostas imunes de pacus juvenis alimentados com diferentes níveis de carboidratos e submetidos a jejum prolongado

Pós-graduação em Zootecnia - FCAV