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Enzyme activities of human myocardium: creatine kinase and hexose phosphate isomerase.

Creatine kinase (CK) and hexose phosphate isomerase (PHI) activities were determined in 36 samples of human heart muscle obtained from patients during cardiovascular surgery. With one exception, CK activities were higher than any reported so far in the literature (papillary muscle, left ventricle: 2,521 +/- 776 IU/g wet weight at 37 degrees C). PHI Activities, the first time measured, are comparable to those in skeletal muscle (200-400 U/g, higher in papillary muscle than in the right auricle). The activities of both enzymes determined correlate significantly (all samples considered together: r = 0.773; p < 0.001). Results are discussed with respect to their possible clinical interest as well as their significance for myocardial metabolism.

Adult↗

Studies on the regulation of the three enzymes of the Leloir pathway in cultured mammalian cells. I. Effect of substitution of galactose for glucose as the sole hexose in the medium in human diploid cell strains and in a rat hepatoma line.

In human diploid cell strains, the substitution of galactose for glucose as the sole hexose in the medium had no measurable effect on the specific activity of the cell protein for any of the three enzymes of the Leloir pathway. These enzymes are galactokinase, alpha-D-galactose-1-phosphate: UDP glucose uridyl transferase and UDP galactose 4-epimerase. A cell strain from a patient with galactosemia had no detectable activity for the transferase. The substitution of galactose for glucose in the medium of these cells (which has been shown to cause the cells to accumulate galactose-1-phosphate) also failed to affect cellular activity for the three enzymes. Similarly, the three activities failed to respond to the substitution of galactose for glucose in cultures of a rat hepatoma line. Cells of this line have been shown by others to perform a number of the tissue-specific functions of liver. The failure of galactose to stimulate increasd cellular activity for the three enzymes represents a striking difference between the behavior of these enzymes in human diploid cell strains and their behavior in E. coli.

Bucladesine↗

Membrane transport by guinea pig peritoneal exudate leukocytes: effect of phagocytosis on hexose and amino acid transport.

Short term, carrier mediated transport of D-glucose, L-leucine and L-lysine by guinea pig peritoneal macrophages was characterized. Analysis of the amino acid transport demonstrated two-limbed double reciprocal plots suggesting two transport systems for each amino acid. The low concentration limb of the curves established a Km of 0.1 mM for L-leucine and 0.05 mM for L-lysine; Vmax values were 2.0 and 2.85 nmole/mg protein/90 seconds, respectively. Leucine and lysine were shown to be competitive inhibitors of each other. Further competition studies revealed that other amino acids also had affinity for these carriers. Amino acid transport was found to be sensitive to sulfhydryl active compounds. Colchicine treatment of peritoneal macrophages did not inhibit the transport of the amino acids tested. Preloading macrophages with latex beads or heat-killed staphylococci by phagocytosis stimulated 2-deoxy-D-glucose (2-dOG) uptake markedly, but had no measurable effect on amino acid transport. Although total transport of 2-dOG increased in post-phagocytic macrophages, the kinetics of the system were not altered significantly. The Km for both pre- and post-phagocytic transport of 2-dOG was shown to be 1.2 mM and the Vmax was shown to increase from a pre-phagocytic value of 20 nmoles/mg protein/90 seconds to a post-phagocytic 27 nmoles/mg protein/90 seconds. Phagocytosis of heat-killed staphylococci by guinea pig polymorphonuclear leukocytes (PMNs), however, did not cause an augmentation in hexose transport in the cells. The presence of colchicine during phagocytosis did not alter subsequent uptake of amino acids by the macrophages.

Animals↗

[In vitro incorporation of 14C-hexose-6-phosphate in mannan, beta-glucan and glycogen in Candida spec. H and its mutants].

Mannose-6-p is an activator of the 14C-mannose incorporation from GDP-14C-mannose in the mono- and oligosaccharides and in the mannopolymers of the cell wall proteophosphomannan produced by the food protein yeast Candida spec. H. Moreover, mannose-6-p is a precursor of proteophosphomannan: 14C-Mannose-6-p has been incorporated in absence of GTP. Corresponding behaviour shows glucose-6-p by synthesis of beta-glucan and glycogen. Mutants of Candida spec. H with different efficiency in the biosynthesis of mannan, beta-glucan and glycogen incorporate hexose-6-p in a different extent.

Candida↗

Effects of exogenous hexoses on bovine in vitro fertilized and cloned embryo development: Improved blastocyst formation after glucose replacement with fructose in a serum-free culture medium.

To evaluate the embryotrophic role of three hexoses (glucose, fructose, and galactose), bovine embryos derived from somatic cell nuclear transfer (SCNT) or in vitro-fertilization (IVF) were cultured in a modified synthetic oviductal fluid (mSOF), which contained either glucose (1.5 or 5.6 mM), fructose (1.5 or 5.6 mM), or galactose (1.5 or 5.6 mM). Compared to 1.5 mM glucose, use of 1.5 mM fructose significantly enhanced blastocyst formation in both SCNT (23 vs. 33%) and IVF embryos (26 vs. 34%), while 5.6 mM fructose did not improve blastocyst formation. Using 1.5 mM galactose did not improve blastocyst formation in SCNT embryos (22 vs. 23%), whereas it significantly inhibited blastocyst formation in IVF embryos (26 vs. 0%). In both SCNT and IVF embryos, 5.6 mM glucose or galactose significantly inhibited embryo development. In a second experiment, in glucose-free mSOF, fructose at concentrations of 0.75, 1.5, 3.0, or 5.6 mM was able to support to morula (32-42 vs. 12%) and blastocyst formation (30-38 vs. 12%) compared to 0 mM fructose. In Experiment 3, addition of fructose (1.5, 3.0, or 5.6 mM) to mSOF containing 1.5 mM glucose did not further promote blastocyst formation in SCNT embryos compared with replacement with 1.5 mM fructose only. Replacement of glucose with 1.5 mM fructose significantly increased total blastomeres (143 vs. 123 cells) and trophectodermal (TE) cells (116 vs. 94 cells) and decreased inner cell mass (ICM) to TE cell ratio (0.24 vs. 0.31) in blastocysts, compared to 1.5 mM glucose. The combined addition of 1.5 mM fructose and glucose significantly increased ICM cell number (36.7 cells) and ICM/TE ratio (0.46). In conclusion, fructose might be a more efficient energy substrate than glucose for producing large number of transferable blastocysts derived from SCNT.

Animals↗

Sequencing analysis of a 24.7 kb fragment of yeast chromosome XIV identifies six known genes, a new member of the hexose transporter family and ten new open reading frames.

The DNA sequence of a 24.7 kb region covering the left arm of chromosome XIV from Saccharomyces cerevisiae was determined. This region contains 17 open reading frames (ORFs) which code for proteins of more than 100 amino acids. Five ORFs correspond to the KRE1, ATP11, DAL82, RFA2 and MCK1 loci, described previously. Two ORFs present high similarity to known proteins: NO345 with the hexose transporter family, and NO351 with the yeast chorismate mutase/prephenate dehydratase enzyme encoded by PHA2. Six ORFs show limited similarity with known proteins or some specific features: NO339 presents 11 potential transmembrane domains. NO343, which is internal to NO345, presents a putative signal sequence and a potential transmembrane domain. NO348 shows similarity with YCW2, TUP1 and SEC13. NO364 reveals a signature for a pyridoxal-phosphate attachment site. Finally, NO384 and NO388 present a biased amino acid composition, being rich in Asn or Glu/Lys/Arg, respectively. Four other ORFs (NO342, NO376, NO381 and NO397) show no similarity to proteins within the databases screened.

Amino Acid Sequence↗

Human hexose-6-phosphate dehydrogenase (glucose 1-dehydrogenase) encoded at 1p36: coding sequence and expression.

Using the published protein sequence from a rabbit microsomal glucose-6-phosphate dehydrogenase G6PD we have isolated and sequenced a cDNA clone coding for its human equivalent, which is also known as hexose-6-phosphate dehydrogenase (H6PD) and glucose dehydrogenase. The corresponding genomic sequence is in the databases enabling its localization to chromosome 1p36. The gene spans 37 kb and consists of 5 exons, the fifth of which codes for more than half of the 89 kDa protein. The first intron is a 10 kb insertion in the 5' untranslated sequence. The predicted mRNA has an exceptionally long (6.5 kb) 3' untranslated sequence. The predicted protein shows extensive homology with X-linked G6PD, suggesting the two genes share a common ancestor but no intron positions are conserved between the two genes suggesting the gene duplication was an ancient event. The C-terminal portion of the protein is not homologous with G6PD but shows limited homology with proteins of unknown function found throughout evolution and encoded next to G6PD in various micro-organisms. Intriguingly this C-terminal portion has some homology with the N-terminal sequence of Plasmodium falciparum G6PD.

Amino Acid Sequence↗

Stimulation of the hexose monophosphate shunt activity in human polymorphonuclear leukocytes.

The glucose metabolism in human polymorphonuclear (PMN) leukocytes was stimulated by latex particles, zymosan particles, gamma-G globulin and formylmet-leu-phe (FMLP) peptide. Maximum stimulation occurred when zymosan particles in amounts above 0.25 mg/ml per 4 X 10(6) leukocytes were used. Latex particles, gamma-G globulin and FMLP stimulated the glucose oxydation less than zymosan particles. The glucose oxydation increase in proportion to the amount of latex particles and gamma-G globulin in the measured range, while the dose response curve using FMLP shows maximum activity at a consentration of the peptide of 5 X 10(-5)M per 4 X 10(6) leukocytes. It is likely that FMLP and gamma-G globulin act on the glucose metabolism via cell surface receptors. The synthetic polystyrene particles (latex) do not occupy these receptors, but stimulate the hexose monophosphate (HMP) shunt activity of the PMN via intracellular mechanisms. Opsonized zymosan particles seem to act on the leukocyte glucose metabolism via cell surface receptors as well as via intracellular mechanisms.

Glucose↗

Hexose metabolism in pancreatic islets. Activation of the Krebs cycle by nutrient secretagogues.

In rat pancreatic islets, a rise in extracellular D-glucose concentration is known to cause a greater increase in the oxidation of D-[6-14C]glucose than utilization of D-[5-3H]glucose. In the present study, such a preferential stimulation of acetyl residue oxidation relative to glycolytic flux was mimicked by nutrient secretagogues such as 2-aminobicyclo[2,2,1]heptane-2-carboxylate, 3-phenylpyruvate, L-leucine, 2-ketoisocaproate, D-fructose and ketone bodies. The preferential stimulation of D-[6-14C]glucose oxidation by these nutrients was observed at all hexose concentrations (0.5, 6.0 and 16.7 mM), coincided with an unaltered rate of D-[3,4-14C]glucose oxidation, was impaired in the absence of extracellular Ca2+, and failed to be affected by NH4+. Although the ratio between D-[6-14C]glucose oxidation and D-[5-3H]glucose utilization in islets exposed to other nutrient secretagogues could be affected by factors such as isotopic dilution and mitochondrial redox state, the present data afford strong support to the view that the preferential stimulation of oxidative events in the Krebs cycle of nutrient-stimulated islets is linked to the activation of key mitochondrial dehydrogenases, e.g. 2-ketoglutarate dehydrogenase. The latter activation might result from the mitochondrial accumulation of Ca2+, as attributable not solely to stimulation of Ca2+ inflow into the islet cells but also to an increase in ATP availability.

Amino Acids↗

Phosphoglucoisomerase-catalyzed interconversion of hexose phosphates. Study by 13C NMR of proton and deuteron exchange.

The exchange of protons and deuterons by phosphoglucoisomerase during the single passage conversion of D-[2-13C,1-2H]fructose 6-phosphate in H2O or D-[2-13C]fructose 6-phosphate in D2O to D-[2-13C]glucose 6-phosphate, as coupled with the further generation of 6-phospho-D-[2-13C]gluconate in the presence of excess glucose-6-phosphate dehydrogenase was investigated by 13C NMR spectroscopy of the latter metabolite. In H2O, the intramolecular deuteron transfer from the C1 of D-fructose 6-phosphate to the C2 of D-glucose 6-phosphate amounted to 65%, a value only slightly lower than the 72% intramolecular proton transfer in D2O. Both percentages, especially the latter one, were lower than those previously recorded during the single passage conversion of D-[1-13C,2-2H]glucose 6-phosphate in H2O or D-[1-13C]glucose 6-phosphate in D2O to D-fructose 6-phosphate and then to D-fructose 1,6-bisphosphate. These differences indicate that the sequence of interactions between the hexose esters and the binding sites of phosphoglucoisomerase is not strictly in mirror image during, respectively, the conversion of the aldose phosphate to ketose phosphate and the opposite process.

Binding Sites↗

Roles of cAMP and free fatty acids on the activity of the hexose monophosphate shunt.

Basal glucose utilization by isolated rat adipocytes have been found to be increased ten times in the presence of certain preparations of albumin. In these conditions the effects of several adrenergic agonists and related compounds on glucose oxidation, lipolysis and triacylglycerol synthesis in isolated fat cells have been studied. Oxidation of D(1(-14)C) glucose in rat adipocytes was almost completely inhibited by norepinephrine and isoproterenol when added to incubated fat cells. Agents able to modify intracellular AMP cyclic levels by different mechanisms display a similar ability to imitate the effect of lipolytic agents. The inhibition of glucose oxidation due to norepinephrine and isoproterenol is partially reverted by propanolol. Under the same conditions in which norepinephrine and isoproterenol markedly reduced glucose conversion to 14CO2, they stimulated lipolysis and triacylglycerol synthesis and in this case propanolol also reverted those actions. However, in these experimental conditions, norepinephrine and isoproterenol did not raise cAMP levels 10 min after hormone addition. It is concluded from these data that glucose oxidation through hexose monophosphate shunt, activation of lipolysis and triacylglycerol synthesis in isolated rat fat cells by lipolytic agents occurs by a mechanism(s) that depend(s) on intracellular free fatty acids levels.

3',5'-Cyclic-AMP Phosphodiesterases↗

Hexose metabolism in pancreatic islets. Galactose transport, phosphorylation and oxidation.

In rat pancreatic islets, the apparent space of distribution of galactose is not different from that of other hexoses. In homogenates of islets or tumoral insulin-producing cells, galactose is phosphorylated at a very low rate relative to either glucose phosphorylation in the same tissues or galactose phosphorylation by liver homogenates. In intact islets, galactose increases modestly the glucose 6-phosphate content and is oxidized at a much lower rate than glucose. Galactose slightly increases insulin output in the presence of a stimulatory concentration of glucose but fails to provoke insulin release in the absence of glucose, whether in islets removed from rats fed a normal or galactose-rich diet. The low rate of galactose oxidation and its poor insulinotropic capacity appear attributable to the weak activity of galactokinase in pancreatic islets.

Animals↗

Changes in serum creatine kinase and hexose phosphate isomerase activity with exercise duration.

In 18 groups of trained healthy male subjects (n = 166; age 17--42 years) the influence of type and duration of long-lasting exercise on the changes of creatine kinase (CPK) and hexose phosphate isomerase (PHI) activity in serum was studied. Above 65--70% of maximal aerobic capacity, a serum enzyme activity increase proportional to the exercise time was found with a different slope in each of the three exercise types examined [a) bicycle ergometer; b) ski race; c) impact-type exertion]. Work duration of more than 300 min induced in the third type of exertion an accelerated increase in serum CPK. A third degree equation best describes the changes in serum CPK for nine of our groups and five others from the literature. To ascertain the possible influence of intravascular hemolysis, sera were mixed with autologous erythrocyte hemolysates and enzyme activity measured. The findings are discussed with reference to critical aspects of the analytical methods, possible causes of serum enzyme changes, and possible use of enzyme measurements in evaluating adaptation of the organism to prolonged exercise.

Adolescent↗

Genetics of male-specific glucose oxidase and the identification of other unusual hexose enzymes in Drosophila melanogaster.

A glucose oxidase (GO) has been identified in the ejaculatory duct of male Drosophila melanogaster. Evidence is given that this enzyme was previously misidentified as HEX-1. Genetic analysis indicates that the Go structural gene is located on the third chromosome at 48 + or - 0.5 cm. Go is polymorphic in males in populations of D. melanogaster and D. simulans located in Athens, Georgia. Two other hexose enzymes have also been tentatively identified for the first time in Drosophila. These are NAD(P)-glucose dehydrogenase (GODH) and NAD-gluconate dehydrogenase (GNDH). GODH and GNDH are found in both males and females and may circumvent the initial steps in the pentose shunt.

Animals↗

Activities of enzymes of the hexose monophosphate pathway in nervous system tumors induced by ethylnitrosourea.

The activities of three enzymes of the hexose monophosphate pathway (HMP) were measured in frozen samples of rat cerebral cortex and ethylnitrosourea-induced tumors of the rat nervous system. Results show that in most tumors, adequate amounts of glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), and transketolase (TK) were present so as not to be rate-limiting for utilization of the HMP. When enzyme activites were expressed on the basis of fresh weight, TK was increased in most tumors as compared with cerebral cortex; G6PDH and 6PGDH were more variable, showing either higher or lower activities in tumors as compared to control. Tumors in general had a lower protein content than cerebral cortex. When activities were expressed in terms of this protein content, mean values for the dehydrogenases in all groups of tumors were higher than the average value in cortex, indicating that G6PDH and 6PGDH were spared in tumors relative to other proteins. TK activities in tumors, expressed in terms of cytosol protein were lower than or in the same range as cortex. Increased activities of HMP enzymes in some tumors indicated that the potential activity of the HMP in some (but not all) tumors of the nervous system is greater than that of cerebral cortex.

Animals↗

Hexose metabolism in pancreatic islets: effect of D-glucose on the mitochondrial redox state.

The mitochondrial NADH/NAD+ ratio for free nucleotides in rat pancreatic islets was judged from the cell content in L-glutamate and L-alanine, 2-ketoglutarate and pyruvate, and NH4+. At a physiological concentration of D-glucose, such a ratio averaged 9.6 +/- 1.1%. A rise in hexose concentrations, above a threshold value in excess of 5.6 mM, caused a rapid, sustained and rapidly reversible decrease in the mitochondrial NADH/NAD+ ratio. It is speculated that in the process of glucose-stimulated insulin release, the latter change participates in the coupling between metabolic and secretory events by favouring both the activity of key mitochondrial dehydrogenases and the translocation of Ca2+ from the mitochondria into the cytosol.

Animals↗

Kinetic properties of hexose-monophosphate dehydrogenases. II. Isolation and partial purification of 6-phosphogluconate dehydrogenase from rat liver and kidney cortex.

6-Phosphogluconate dehydrogenase (6PGDH) from rat-liver and kidney-cortex cytosol has been partially purified and almost completely isolated (more than 95%) from glucose-6-phosphate dehydrogenase activity. The purification and isolation procedures included high-speed centrifugation, 60-75% ammonium-sulphate fractionation, by which both hexose-monophosphate dehydrogenases activities were separated, and finally the protein fraction was applied to a chromatographic column of Sephadex G-25 equilibrated with 10 mM Tris-EDTA-NADP buffer, pH 7.6, to eliminate any contaminating metabolites. The kinetic properties of the isolated partially purified liver and renal 6PGDH were examined. The saturation curves of this enzyme in both rat tissues showed a typical Michaelis-Menten kinetic, with no evidence of co-operativity. The optimum pH for both liver and kidney-cortex 6PGDH was 8.0. The Km values of liver 6PGDH for 6-phosphogluconate (6PG) and for NADP were 157 microM and 258 microM respectively, while the specific activity measured at optimum conditions (pH 8.0 and 37 degrees C) was 424.2 mU/mg of protein. NADPH caused a competitive inhibition against NADP with an inhibition constant (Ki) of 21 microM. The Km values for 6PG and NADP from kidney-cortex 6PGDH were 49 microM and 56 microM respectively. The specific activity at pH 8.0 and 37 degrees C was 120.7 mU/mg of protein. NADPH also competitively inhibited 6PGDH activity, with a Ki of 41 microM. This paper describes a quick, easy and reliable method for the separation of the two dehydrogenases present in the oxidative segment of the pentose-phosphate pathway in animal tissues, eliminating interference in the measurements of their activities.

Animals↗

Hexose transport by brain slices: further studies on energy dependence.

We studied the uptake of [3H]2-deoxyglucose [( 3H]2DG) by slices of rat cerebral cortex in vitro as a model of glucose transport by brain. Slices were incubated with [3H]2DG, or with L-[3H]glucose as a marker for diffusion; the difference between [3H]2DG uptake and L-[3H]glucose uptake was defined as net [3H]2DG transport. Net [3H]2DG transport was a function of incubation temperature, with an estimated temperature coefficient of 1.87 from 15 degrees C to 25 degrees C. The net uptake of [3H]2DG was not inhibited by phlorizin or phloretin in concentrations well above the reported Ki of these inhibitors for hexose uptake in other systems. To examine the hypothesis that [3H]2DG transport by brain slices is dependent on mitochondrial energy, we studied net [3H]2DG uptake by slices which had been preincubated in media designed to alter intracellular ATP stores. The transport process was very sensitive to inhibition by DNP, but the correlation between [3H]2DG transport and ATP levels was unclear. In contrast to our published hypothesis that the transport process required mitochondrial energy, these data indicate that dependence on energy is not absolute.

Adenosine Triphosphate↗