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PubMed1992

Spectrophotometric pyrophosphate assay of 2',5'-oligoadenylate synthetase.

A nonradioactive multiwell spectrophotometric assay for the interferon-induced enzyme 2',5'-oligoadenylate synthetase measuring the inorganic pyrophosphate produced during oligoadenylate synthesis has been developed. A coupled enzymatic reaction results in a mole to mole formation of NADPH compared to the inorganic pyrophosphate through the use of the three enzymes UDP-Glc pyrophosphorylase (EC2.7.7.9), phosphoglucomutase (EC5.4.2.2), and glucose-6-phosphate dehydrogenase (EC1.1.1.49). The assay is at least as sensitive for measurements of 2',5'-oligoadenylate synthetase activity as the conventional assays using radioactive nucleotides as substrates. Even higher sensitivity of the assay can be obtained by taking advantage of the strong fluorescence of NADPH.

2',5'-Oligoadenylate Synthetase↗

PubMed1983

Vanadate affects glucose metabolism of human erythrocytes.

Vanadate causes a rapid breakdown of 2,3-bisphosphoglycerate in intact erythrocytes. This metabolite is nearly stoichiometrically transformed into pyruvate, which changes the cell redox state and enhances the glycolytic flux. The results show that the vanadate effect on 2,3-bisphosphoglycerate, also evident in hemolysates, is attributable to the stimulation of a phosphatase activity of the phosphoglycerate mutase. In agreement with others (J. Carreras, F. Climent, R. Bartrons, and G. Pons (1982) Biochim. Biophys. Acta 705, 238-242), vanadate is thought to destabilize the phosphoryl form of this enzyme which shows competitive inhibition between the ion and 2,3-bisphosphoglycerate in the mutase reaction. A competitive inhibition between vanadate and glucose 1,6-bisphosphate is also found for phosphoglucomutase, without evidence for phosphatase activity toward the bisphosphate cofactor.

2,3-Diphosphoglycerate↗

PubMed1983

Relative phosphate contents of phosphofructokinase and other soluble phosphoproteins in skeletal muscle.

In vivo phosphorylation of muscle proteins has been studied by incorporation of [32P]phosphate with emphasis placed upon the phosphorylation of glycolytic enzymes. Of the approximately 25 soluble proteins resolved by two-dimensional electrophoresis that contain significant 32P, phosphofructokinase was the sole glycolytic enzyme identified as a phosphoprotein. The extent of phosphorylation found for this enzyme was the same as determined previously for purified phosphofructokinase and was about the same as the extent of phosphorylation of phosphorylase in resting muscle. Subsequent partial purification of several glycolytic enzymes confirmed the absence of significant amount of phosphate. However, phosphoglycerate mutase contained small amounts of covalently bound 32P that was exchangeable with 3-phosphoglycerate and therefore, most likely was incorporated during the catalytic reaction cycle. Analogous results were obtained for phosphoglucomutase. Both mutases were also phosphorylated at the same sites by the catalytic subunit of cyclic AMP-dependent protein kinase.

Animals↗

PubMed1985

Glucose metabolism in insulin-producing tumoral cells.

Homogenates of insulin-producing tumoral cells catalyzed the phosphorylation of glucose, mannose, and fructose. The kinetics of phosphorylation at increasing glucose concentrations, the inhibitory effect of glucose 6-phosphate, and the comparison of results obtained with distinct hexoses indicated the presence of both low-Km hexokinase-like and high-Km enzymatic activities, the results being grossly comparable to those collected in normal pancreatic islets. Relative to protein content, the glucose-phosphorylating enzymatic activity was higher in tumoral than normal islet cells. The activity of other enzymes was either lower (glutamate dehydrogenase), moderately higher (phosphoglucomutase, lactate dehydrogenase) or considerably greater (ornithine decarboxylase) in tumoral than in normal islet cells. In intact tumoral cells, incubated under increasing glucose concentrations, the oxidation of D-[U-14C]glucose and the output of lactic and pyruvic acids reached a close-to-maximal value at 2.8 mM glucose. The ratios for glucose oxidation/utilization and lactate/pyruvate output were much lower in tumoral than in normal islet cells. Although glucose caused a modest increase in insulin output from the tumoral cells, this effect was saturated at a low glucose concentration (2.8 mM) and less marked than that of other secretagogues (e.g., L-leucine, L-ornithine, or forskolin). Thus, despite a close-to-normal enzymatic equipment for glucose phosphorylation, the tumoral cells displayed severe abnormalities in the metabolism and secretory response to this hexose. These findings point to regulatory mechanisms distal to glucose phosphorylation in the control of glucose metabolism in insulin-producing cells.

Animals↗

PubMed1987

Effect of orally administered secondary autoxidation products of linoleic acid on carbohydrate metabolism in rat liver.

The effects of orally administered secondary autoxidation products of linoleic acid in rat liver were investigated. Their administration led to two toxic effects on hepatic carbohydrate metabolism, as compared to the administration of saline or linoleic acid used as controls. One effect was depletion of glucose 6-phosphate and fructose 6-phosphate caused by the reduction of glycolysis and glycogenolysis, accompanied by decreases in glycogen synthesis and pentose phosphate cyclic activity. The reduction in these metabolic systems seems unlikely to occur because phosphofructokinase was regulated by ATP or citrate enzymatically, because their accumulation in the liver was not detected in the secondary products. Another toxic effect was the depletion of oxaloacetate and isocitrate caused by the reduction in enzyme activity of the mitochondrial citrate cycle. On the basis of these results, the hepatotoxic effects of secondary products are discussed as follows: the incorporated secondary products impaired the activities of hexokinase and phosphoglucomutase in the liver. The reduction in these enzyme activities resulted in the depletion of glucose 6-phosphate and fructose 6-phosphate, which led ultimately to decreases in the activities of phosphofructokinase, the pentose phosphate cycle, and glycogen synthesis. Moreover, the secondary products disturbed the mitochondrial membrane, resulting in a decrease in the activity of the citrate cycle, which was accompanied by depletion of its metabolites.

Adenine Nucleotides↗

PubMed1991

Stimulation of mutases and isomerases by vanadium.

The effects of vanadate and vanadate complexes on the rates of exchange of phosphoryl groups in the reactions catalyzed by the enzymes phosphoglucomutase and the coupled system formed by phosphoglycerate mutase and enolase, and the effects of vanadyl complexes on the interconversion of aldehyde and keto groups catalyzed by the enzymes phosphomannose isomerase, phosphoribose isomerase, and phosphoglucose isomerase, were measured using one-dimensional 31P nuclear magnetic resonance spectroscopy. Chemical exchange was investigated by observing the transfer of magnetization achieved by selective irradiation of resonances using the DANTE pulse sequence. The presence of vanadium stimulated the catalytic activity of the enzymes in vitro, with the exception of enolase whose activity was not affected. Addition of vanadate also increased the rate constants of the interconversion of glucose 6-phosphate and fructose 6-phosphate in hemolysates. 51V nuclear magnetic resonance spectroscopy and electron paramagnetic resonance spectroscopy were employed to investigate the interactions between ammonium vanadate and sugar phosphates and the formation of vanadium--sugar phosphate complexes that may be involved in the stimulation of the catalytic activity of the isomerases.

Animals↗

PubMed1987

Cerebral glycogenolysis and glycolysis in malathion-treated hyperglycaemic animals.

Treatment with malathion resulted in an increase in the level of blood glucose and lactate and reduced cerebral glycogen, 2 hr after its administration. The blood pyruvate level was not changed. The activities of glycogenolytic enzymes (glycogen phosphorylase and phosphoglucomutase) were increased significantly in the brain, whereas that of glucose-6-phosphatase remained unchanged. The activity of the glycolytic enzyme-hexokinase was increased significantly in malathion-treated animals, whereas those of the glucose-6-phosphate and lactate dehydrogenases were not significantly changed. The changes in enzyme activities may be a compensatory mechanism to provide energy in the form of glucose to cerebral tissue on account of stimulatory effects in malathion-treated animals.

Animals↗

PubMed1990

Modification of diazinon-induced changes in carbohydrate metabolism by adrenalectomy in rats.

Treatment with diazinon (40 mg/kg, i.p.) resulted in hyperglycemia and depletion of glycogen from cerebral and peripheral tissues 2 hr after its administration in rats. The activities of the glycogenolytic enzymes glycogen phosphorylase and phosphoglucomutase were increased significantly in brain and liver, whereas that of glucose-6-phosphatase was not altered. The activities of the glycolytic enzymes hexokinase and lactate dehydrogenase were increased only in the brain. The cholinesterase activity of the brain was reduced by treatment with diazinon. The activities of the hepatic gluconeogenic enzymes fructose 1,6-diphosphatase and phosphoenolpyruvate carboxykinase were also increased significantly in diazinon-treated animals. The level of lactate was increased in brain and blood, whereas that of pyruvate was not changed. The activity of glucose-6-phosphate dehydrogenase was not changed significantly. The cholesterol and ascorbic acid contents of adrenals were depleted in diazinon-treated animals. The hyperglycemia and changes in carbohydrate metabolism were abolished by adrenalectomy, suggesting the possible involvement of the adrenals in the induced changes in diazinon-treated animals.

Adrenal Glands↗

PubMed1978

An improved quantitative assay of galactose-1-phosphate uridyltransferase activity in erythrocytes based on the determination of glucose 1-phosphate generation.

An improved quantitative method for measuring galactose-1-phosphate uridyltransferase (EC 2.7.7.12, Gal-PUT) activity in erythrocytes was developed based on the detection of glucose 1-phosphate generated under the catalytic influence of the enzyme. This is achieved by incubating the enzyme with galactose 1-phosphate and uridyldiphosphoglucose during 15 min, followed by deproteinisation. The glucose 1-phosphate generated is quantitated subsequently by measuring NADPH formation from added NADP+ in a second incubation step with added phosphoglucomutase (EC 2.7.5.1) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49). Gal-PUT activity is calculated from the increment in absorption at 340 nm. Because it is technically a simple assay that is sensitive, specific and not affected by UDPgalactose-4-epimerase (EC 5.1.3.2) activity in the erythrocytal lysates it is suggested to be the method of choice for measuring Gal-PUT activity. Activities in erythrocytes of controls varied from 264 to 556 U/kg hemoglobin; in obligate heterozygotes from 53 to 190 U/kg Hb and in homozygous deficient patients less than 5 U/kg Hb was measured at 37 degrees C.

Erythrocytes↗

PubMed1982

A new microfluorometric method for the measurement of galactose-1-phosphate in erythrocytes.

A new and sensitive assay for measuring galactose-1-phosphate in erythrocytes is described. Galactose-1-phosphate is determined by mixing an aliquot of deproteinized hemolysate with a reagent containing uridine diphosphoglucose, NADP+, hexose-1-phosphate uridylyltransferase, phosphoglucomutase, glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase and measuring the NADPH formed fluorometrically. Under the conditions of this assay 2 mol of NADPH are formed per mol of galactose-1-phosphate. The assay is linear from 0 to 1160 micrograms of galactose-1-phosphate per gram of hemoglobin. Recovery of galactose-1-phosphate added to four hemolysates averaged 99%. Galactose-1-phosphate concentrations were measured in erythrocytes from five heterozygous subjects not under dietary control and seven transferase-deficient galactosemic individuals who were receiving galactose restricted diets. In all samples from the heterozygous individuals, the galactose-1-phosphate concentrations were normal. Of the samples from galactosemic subjects, two showed extreme elevations of galactose-1-phosphate, four showed moderate elevations, and one was normal. Galactose-1-phosphate levels are used to monitor the degree of dietary control in the transferase-deficient galactosemic individual.

Adolescent↗

PubMed1987

Determination of erythrocyte glucose 1,6-bisphosphate--a comparison of two methods using a centrifugal analyzer.

Glucose 1,6-bisphosphate is a key effector of human erythrocyte glycolysis, yet to date its assay has been problematical. Two methods for measuring glucose 1,6-bisphosphate were modified and adapted to a centrifugal analyser and the inaccuracy and imprecision of each method were compared. One assay, based on stimulation of phosphoglucomutase, was shown to underestimate the erythrocyte levels by approximately 5% due to inhibition of the mutase by endogenous 2,3-bisphosphoglycerate. An alternative chemical/enzymic method, consisting of acid hydrolysis of glucose 1,6-bisphosphate to glucose 6-phosphate and subsequent determination of the monophosphate was modified by omitting an initial alkaline hydrolysis step and by increasing the duration of acid hydrolysis. The modified method also enabled the determination of erythrocyte glucose 6-phosphate. The normal concentration of glucose 1,6-bisphosphate in whole blood and in washed human erythrocytes, determined using the more accurate chemical/enzymic method was 83 +/- 5 mumol/l cells and 86 +/- 4 mumol/l cells, respectively; the corresponding concentrations of glucose 6-phosphate were 26 +/- 2 mumol/l cells and 15 +/- 3 mumol/l cells.

Centrifugation↗

PubMed1978

[Isolation by affinity chromatography of specialized membrane fractions from cat liver microsomes].

Microsomal glucokinase is solubilized by incubation in the presence of several metabolites. After solubilization of the enzymes, the membranes present free sites for specific binding of glucokinase, therefore, they can be purified by affinity chromatography on Sepharose--ATP-glucokinase. This method yields membranous vesicles which contain, in addition to glucokinase, uridylyl-transferase, phosphoglucomutase, sialyl-transferase and adenylate cyclase. Galactosyl-transferase, glucose-6-phosphatase and NADPH cytochrome c reductase are absent. It appears that functionally related enzyme from UDP-glucose biosynthesis are aggregated onto specific patches of the membrane, most likely from Golgi apparatus.

Animals↗

PubMed1995

Steady-state content of glycolytic/tricarboxylic acid-cycle intermediates, adenine nucleotide pools and the cellular redox-status in the infective (L3) larvae of (homogonic) Strongyloides ratti.

Infective (L3) larvae of Strongyloides ratti (homogonic strain) were freeze-clamped (-196 degrees C) and the steady-state content of the glycolytic, Krebs tricarboxylic acid (KTA)-cycle intermediates and adenine nucleotides analysed. Comparison of the mass-action ratios (MARs) of the glycolytic enzymes with their apparent equilibrium constants (K9eq) indicate that phosphoglucomutase, glucosephosphate isomerase, triosephosphate isomerase, phosphoglyceromutase and phosphopyruvate hydratase reactions were all at or near equilibrium, whilst hexokinase, phosphofructokinase and pyruvate kinase were displaced from equilibrium. The S. ratti aldolase and myokinase appear to be somewhat displaced from equilibrium and thus may have pseudoregulatory roles. The adenylate energy charge (AEC), ATP/ADP ratio and the available adenylate energy (AAE) indices were 0.9 +/- 0.04, 8.76 +/- 1.5 and 397 +/- 43, respectively. The free [NAD+]/[NADH+H+] ratio of the cytoplasmic compartment of S. ratti L3 larvae calculated employing the steady-state content of the oxidised and reduced substrates of lactate dehydrogenase (E.C. 1.1.1.27) and the combined glyceraldehyde 3-phosphate dehydrogenase (E.C. 1.2.1.12)/3-phosphoglycerate kinase (E.C. 2.7.2.3) system were ca. 523 and 1200, respectively. The free[NAD+]/[NADH+H+] ratio in the mitochondrial compartment of S. ratti L3 larvae calculated using the malate dehydrogenase (E.C. 1.1.1.37) equilibrium was found to be 1962:1. The data is discussed with respect to the predominantly aerobic nature of the energy metabolism of the L3 larvae.

Adenine Nucleotides↗

PubMed1988

Histochemical features of tubular aggregates in diseased human skeletal muscle fibres.

This communication presents the results obtained in tubular aggregates of 24 enzyme histochemical techniques for demonstrating activity of oxidoreductases, transferases, hydrolases and isomerases. The activity characteristics of the tubular aggregates in m. gluteus medius of 18 patients with diseases of the neuromuscular system were almost identical. A high activity of the mitochondrial enzymes, NADPH: tetrazolium oxidoreductase, NADH:tetrazolium oxidoreductase and cytochrome c oxidase, could be shown in the pathological structures, whereas the activity of the mitochondrial enzymes, glycerol-3-phosphate:menadione oxidoreductase, succinate:PMS oxidoreductase, malate:NAD+ oxidoreductase and isocitrate:NAD+ oxidoreductase, and the partial mitochondrial enzymes, malate:NADP+ oxidoreductase and isocitrate:NADP+ oxidoreductase, was very slight or even absent. There was a moderate to strong activity of the glycolytic enzymes lactate:NAD+ oxidoreductase, glyceraldehyde-3-phosphate:NAD+ oxidoreductase, phosphofructokinase, phosphoglucomutase and glucose phosphate isomerase. In contrast, the activity of alpha-glucan phosphorylase was slight. The activity of phosphogluconate:NADP+ oxidoreductase, glucose-6-phosphate:NADP+ oxidoreductase and 5'-nucleotidase was slight, whereas there was no activity of myosin ATPase and mitochondrial ATPase, acid phosphatase or alkaline phosphatase. The high activity of AMP-deaminase was very striking. The activity of peroxidase was moderate. Results obtained with adsorption studies point to adsorption of some of the enzymes studied to the tubular aggregates in vivo and this phenomenon very probably determined the histochemical characteristics of these structures.

Histocytochemistry↗

PubMed1987

Effect of Duchenne muscular dystrophy on enzymes of energy metabolism in individual muscle fibers.

Individual muscle fibers from patients with Duchenne muscular dystrophy at an early stage in their disease, and from apparently normal boys of similar age, were analyzed for 13 enzymes of energy metabolism. This approach avoided the serious problems with muscle homogenate assays from increases in nonparenchymal components and permitted assessment of disease changes in different fiber types. Some enzymes of glycogenolysis (phosphorylase, phosphoglucomutase, and pyruvate kinase) were decreased in dystrophic fibers of all types. Phosphofructokinase was decreased in presumptive type II fibers. Lactate dehydrogenase was increased in type I fibers and essentially unchanged in type II. Phosphoglucoisomerase was near normal. Two enzymes of glucose metabolism not involved in glycogenolysis, hexokinase and glycogen synthase, were near normal, but a third, fructose bisphosphatase, was sharply reduced. Two enzymes of oxidative metabolism, citrate synthase, and beta-hydroxyacyl CoA dehydrogenase, were unchanged or increased. Two enzymes of high energy phosphate transfer, creatine kinase and adenylokinase, were only marginally affected. The net result is to leave the type II fibers, which normally exert the greatest force, with a severe deficit in the glycogenolytic enzyme machinery to maintain that force.

Adenylate Kinase↗

PubMed1978

The detection of mutation in human diploid fibroblasts after mutagen treatment using non-selective cloning and enzyme electrophoresis.

In an attempt to study mutagenesis in human diploid fibroblasts, clones derived from mass cultures treated with mutagen have been examined by starch-gel electrophoresis for 43 different enzyme loci. A technique of mutagen treatment was devised which facilitated the cloning and which enabled the cells to be exposed to very high doses of EMS and MNNG. Two alterations in phenotype, presumably the result of mutation, were observed, one involving peptidase D (PEPD) and the other phosphoglucomutase (PGM1).

Clone Cells↗

PubMed1977

The identification by isoenzyme patterns of two distinct strain-groups of Trypanosoma cruzi, circulating independently in a rural area of Brazil.

Culture forms of 17 Trypanosoma cruzi stocks, primarily isolated from a rural area of endemic Chagas disease at São Felipe, Bahia, Brazil, were compared by the electrophoretic patterns of six enzymes: aspartate aminotransferase, alanine aminotransferase, glucose-6-phosphate dehydrogenase, malate dehydrogenase (decarb-oxylating) (NADP+), glucosephosphate isomerase and phosphoglucomutase. Two markedly distinct combinations of isoenzyme patterns were seen, justifying the arrangement of the 17 stocks into two strain-groups, each of which was enzymically homogeneous. One combination was characteristic of the 11 domestic stocks of T. cruzi derived from both human infections and domiciliated animals; the second was characteristic of the six sylvatic stocks derived from opossums and a sylvatic triatomine species. The enzyme patterns were independent of the original host and the type of culture medium used. Distinction of the two strain-groups accords with epidemiological evidence that the domestic and sylvatic transmission cycles in São Felipe do not overlap. It is suggested that the diverse enzyme characters of the two strain-groups circulating in São Felipe reflect diverse origins; the domestic form of T. cruzi probably invaded the area from the south of Brazil with the domestic triatomine vector, Panstrongylus megistus.

Animals↗

PubMed1980

The epidemiology of Entamoeba histolytica in Mexico City. A pilot survey I.

Stocks of intestinal amoebae isolated from hospital patients in Mexico City and grown in monoxenic culture were compared among themselves and with those already described (SARGEAUNT & WILLIAMS, 1979), using the electrophoretic patterns of four enzymes: glucose phosphate isomerase (GPI), phosphoglucomutase (PGM), L-malate:NADP+ oxido-reductase (oxalacetate-decarboxylating) (ME) and hexokinase (HK). New isoenzyme groups (SARGEAUNT & WILLIAMS, 1979) of all the amoebae, including Entamoeba histolytica have been demonstrated. Amongst these have been found seven more groups of E. histolytica, two new groups of E. hartmanni, one new group of Dientamoeba fragilis and one new group of E. coli. Of the seven new groups of E. histolytica three are known to originate from patients with clinical amoebiasis whilst the remainder are from asymptomatic subjects. Only 11.2% of the 125 isolations were associated with clinical amoebiasis, and these are clearly distinguished from the isolations from asymptomatic patients by their electrophoretic isoenzyme pattern.

Amoeba↗