Showing papers on "Pyruvate kinase published in 1984"
TL;DR: Damage to the plasma membrane of rabbit epididymal spermatozoa during spontaneous lipid peroxidation was examined by means of trypan blue uptake and expression of activity of the intracellular enzymes, lactate dehydrogenase and pyruvate kinase, and there is a linear correlation between increase in expressed enzyme activities and malondialdehyde production.
Abstract: Damage to the plasma membrane of rabbit epididymal spermatozoa during spontaneous lipid peroxidation was examined by means of trypan blue uptake and expression of activity of the intracellular enzymes, lactate dehydrogenase and pyruvate kinase. Both the dye uptake and the expression of enzyme activity probe cell damage from lipid peroxidation as loss of integrity of the plasma membrane. A linear correlation was obtained between trypan blue staining of the cells and malondialdehyde production, a quantifiable measure of the extent of lipid peroxidation. At the point of trypan blue staining of all cells, 0.5 nmol malondialdehyde/10(8) cells was produced. This is the same amount produced at the point of complete loss of motility and superoxide dismutase activity. We have defined this as the "lipoperoxidative lethal end point." Expression of lactate dehydrogenase and pyruvate kinase activities increased with time of aerobic incubation. In the high Na+ medium, NTP, in which lipid peroxidation is slow, there is a linear correlation between increase in expressed enzyme activities and malondialdehyde production. But in the high K+ medium, KTP, in which lipid peroxidation is rapid, there is an initial rapid rise in expressed enzyme activity over 3 h, followed by a slower increase. Activities of rabbit sperm lactate dehydrogenase, pyruvate kinase, and flagellar ATPase were unaffected by aerobic incubations for up to 48 h, double the incubation period used for the assay of enzymatic activities for the first two. The activity of glyceraldehyde-3-phosphate dehydrogenase decreased during aerobic incubation, the time course matching the loss of motility. The subcellular distribution of lactate dehydrogenase in rabbit spermatozoa was determined: 4% in the mitochondrial matrix, 10% in the plasma membrane and 85% in the cytosolic compartment.
163 citations
TL;DR: The findings suggest that the females have a significantly lower overall capacity for aerobic oxidation and for anaerobic glycolysis than the males and the females has a greater capacity for beta-oxidation relative to the capacity of the citric acid cycle.
138 citations
TL;DR: The following result suggest that retention of the phosphoenolpyruvate pool by starved cells is a consequence of Pi-mediated inhibition of pyruVate kinase, and the increase in the phosphate pool (and Pi) preceded depletion of fructose 1,6-bisphosphate, and reduction in intracellular Pi caused the restoration of pyRuvate Kinase activity in starved cells.
Abstract: High-resolution 31P nuclear magnetic resonance spectroscopy and 14C fluorography have been used to identify and quantitate intermediates of the Embden-Meyerhof pathway in intact cells and cell extracts of Streptococcus lactis. Glycolysing cells contained high levels of fructose 1,6-bisphosphate (a positive effector of pyruvate kinase) but comparatively low concentrations of other glycolytic metabolites. By contrast, starved organisms contained only high levels of 3-phosphoglycerate, 2-phosphoglycerate, and phosphoenolpyruvate. The concentration of Pi (a negative effector of pyruvate kinase) in starved cells was fourfold greater than that maintained by glycolysing cells. The following result suggest that retention of the phosphoenolpyruvate pool by starved cells is a consequence of Pi-mediated inhibition of pyruvate kinase: the increase in the phosphoenolpyruvate pool (and Pi) preceded depletion of fructose 1,6-bisphosphate, and reduction in intracellular Pi (by a maltose-plus-arginine phosphate trap) caused the restoration of pyruvate kinase activity in starved cells. Time course studies showed that Pi was conserved by formation of fructose 1,6-bisphosphate during glycolysis. Conversely, during starvation high levels of Pi were generated concomitant with depletion of intracellular fructose 1,6-bisphosphate. The concentrations of Pi and fructose 1,6-bisphosphate present in starved and glycolysing cells of S. lactis varied inversely. The activity of pyruvate kinase in the growing cell may be modulated by the relative concentrations of the two antagonistic effectors.
119 citations
TL;DR: Aluminum inhibited both the cytosolic and mitochondrial hexokinase activities in rat brain and may be one of the mechanisms by which aluminum can act as a neurotoxin.
Abstract: Aluminum inhibited both the cytosolic and mitochondrial hexokinase activities in rat brain. The IC50 values were between 4 and 9 microM. Aluminum was effective at mildly acidic (pH 6.8) or slightly alkaline (pH 7.2-7.5) pH, in the presence of a physiological level of magnesium (0.5 mM). However, saturating (8 mM) magnesium antagonized the effect of aluminum on both forms of hexokinase activity. Other enzymes examined were considerably less sensitive to inhibition by aluminum. The IC50 of aluminum for phosphofructokinase was 1.8 mM and for lactate dehydrogenase 0.4 mM. At 10-600 microM, aluminum actually stimulated pyruvate kinase. Aluminum also inhibited lactate production by rat brain extracts: this effect was much more marked with glucose as substrate than with glucose-6-phosphate. However, the IC50 for inhibiting lactate production using glucose as substrate was 280 microM, higher than that required to inhibit hexokinase. This concentration of aluminum is comparable to those reportedly found in the brains of patients who had died with dialysis dementia and in the brains of some of the patients who had died with Alzheimer disease. Inhibition of carbohydrate utilization may be one of the mechanisms by which aluminum can act as a neurotoxin.
113 citations
Journal Article•
TL;DR: The results indicate that, in animals receiving this carcinogenic diet, isozyme alterations associated with neoplasia result from the proliferation of a new cell population which contains these enzymes and not from "dedifferentiation" of mature hepatocytes, and suggest that this new cellpopulation may include a liver stem cell compartment containing cells in transitional states of differentiation.
Abstract: The fetal liver isozymes aldolase A and pyruvate kinase K increase in livers of adult rats fed a choline deficient-diet containing 01% ethionine Oval cells isolated by centrifugal elutriation from preneoplastic livers of animals receiving the carcinogenic diet contained these fetal forms as well as fetal-adult isozyme hybrids In contrast, parenchymal cells isolated from the livers of these animals had only aldolase B and pyruvate kinase L, the same isozymes present in parenchymal cells of normal adult rats Liver homogenates from rats receiving the carcinogenic diet contain lactate dehydrogenase (LDH) 1, LDH 2, and LDH 3 in addition to LDH 4 and LDH 5, which are the forms detected in normal liver homogenates LDH 1, LDH 2, and LDH 3 are present in oval cells of preneoplastic livers and in biliary epithelial cells of normal livers, but not in parenchymal cells isolated from normal and preneoplastic livers Cells of biliary epithelium from normal livers also contain aldolase A and pyruvate kinase K, but not the fetal-adult isozymes present in oval cell populations The results indicate that, in animals receiving this carcinogenic diet, isozyme alterations associated with neoplasia result from the proliferation of a new cell population which contains these enzymes and not from "dedifferentiation" of mature hepatocytes Furthermore, the data suggest that this new cell population may include a liver stem cell compartment containing cells in transitional states of differentiation
94 citations
TL;DR: Comparison of the extent of phosphorylation with activity showed that a 50% decrease in the ratio of kinase activity to bisphosphate activity occurred when only 0.25 mol of phosphate was incorporated per mol of enzyme subunit, and maximal changes occurred with 0.7 mol incorporated.
87 citations
TL;DR: The rate of immediate myofibrillar MgADP rephosphorylation in the endogenous creatine-kinase reaction was observed to be governed by the concentration of phosphocreatine in accordance with the kinetics of this enzyme.
83 citations
TL;DR: The dissimilarity in kinetic properties of PK-aerobic and PK-anoxic indicate that red muscle PK activity is probably strongly depressed in vivo during anoxia stress.
Abstract: Aerobic and anoxic variants of radular retractor muscle pyruvate kinase (PK-aerobic and Pk-anoxic) from the gastropod mollusc, Busycotypus canaliculatum, were purified to homogeneity and respective specific activities of 368 and 186 mumol of product min-1 mg protein-1. Both PK variants were apparent homotetramers with native molecular masses of about 235 kDa, but differed in several other physical characteristics including pI (5.81 +/- 0.06 for PK-aerobic, 5.42 +/- 0.03 for PK-anoxic) and chromatographic behavior on several columns used during their respective purifications. The two enzymes differed greatly in several kinetic properties. Affinity for phosphoenolpyruvate was more than tenfold greater for PK-aerobic (K0.5 = 0.067 +/- 0.002 mM; h = 0.99 +/- 0.10), whereas the cooperative effect for phosphoenolpyruvate binding was greatly enhanced for PK-anoxic (K0.5 = 0.85 +/- 0.02 mM, h = 2.57 +/- 0.01). Although the affinities for the second substrate, ADP, were identical for both enzyme forms (apparent Km = 0.25 mM) pK-anoxic showed greater substrate inhibition by high concentrations of ADP. Likewise, affinities for K+ and Mg2+ were similar but PK-anoxic showed a greater degree of cooperativity with Mg2+ (h = 2.50 +/- 0.02) than did PK-aerobic (h = 1.70 +/- 0.06). Saturating concentrations of fructose 1,6-bisphosphate (50 microM) activated PK-anoxic resulting in an enzyme with properties similar to fructose-1,6-bisphosphate-activated PK-aerobic, with K0.5 values for phosphoenolpyruvate of about 0.04 mM and Hill coefficients of 1.1. PK-anoxic showed much stronger regulation by the allosteric inhibitors MgATP, phenylalanine, proline and alanine. Fructose 1,6-bisphosphate partially relieved the inhibitions by ADP, MgATP, alanine, proline and arginine phosphate of both enzyme forms. However, at 0.1 mM phosphoenolpyruvate PK-aerobic was much more sensitive to activation by fructose 1,6-bisphosphate, Ka values being 0.05 +/- 0.01 microM for PK-aerobic and 1.3 +/- 0.1 microM for PK-anoxic. In the presence of 1.0 mM alanine and 1.5 mM MgATP much higher concentrations of fructose 1,6-bisphosphate were required for activation of PK-anoxic (Ka = 5.2 +/- 0.4 microM) than for PK-aerobic (Ka = 0.02 +/- 0.01 microM). Variations in pH over the range likely occurring in vivo during anaerobiosis caused no significant additional kinetic differences between the two enzyme forms. The dissimilarity in kinetic properties of PK-aerobic and PK-anoxic indicate that red muscle PK activity is probably strongly depressed in vivo during anoxia stress.
82 citations
TL;DR: It appears now that glucagon, beside regulating the enzyme activity by phosphorylation mechanisms, may also modulate L-type pyruvate kinase synthesis at a pre-translational level.
76 citations
TL;DR: It is suggested that the altered metabolic demands of ischaemia are satisfied by changes in glycolytic enzyme organisation as the enzymes shift from the soluble to the particulate phase of cardiac muscle.
Abstract: Perfused rat hearts show a markedly increased binding of phosphofructokinase and fructose-bisphosphate aldolase as a consequence of ischaemia, but little change in binding of pyruvate kinase, lactate dehydrogenase or glyceraldehyde-3-phosphate dehydrogenase. After 10 min ischaemia over one quarter of the phosphofructokinase and three quarters of the aldolase are bound. The effect of anoxia is less well marked in its influence on binding with only aldolase showing a significant increase in binding. These results suggest that one factor involved in the increased binding during ischaemia is the fall in pH of the heart. Binding studies with isolated myofibrils confirm that the affinity and stoichiometry of aldolase binding are considerably increased as the pH is lowered over a range comparable to that which occurs in ischaemic heart. The low level of binding of glyceraldehyde-3-phosphate dehydrogenase in perfused rat hearts correlates with the relatively low affinity of this enzyme for binding to rat or rabbit cardiac myofibrils. There are species differences in the enzyme binding response to ischaemia. Sheep hearts show rapid and large increases in the binding of glyceraldehyde-3-phosphate dehydrogenase in addition to changes in aldolase and phosphofructokinase binding. The greater binding of glyceraldehyde-3-phosphate dehydrogenase reflects the greater affinity of sheep cardiac myofibrils. It is suggested that the altered metabolic demands of ischaemia are satisfied by changes in glycolytic enzyme organisation as the enzymes shift from the soluble to the particulate phase of cardiac muscle.
71 citations
TL;DR: Comparison of the electrophoretic mobilities of products derived from acid hydrolysis of purified 32P-labelled PK-anoxic with authentic substances suggest the presence of an O-phospho-L-threonine residue in the protein.
Abstract: That red muscle pyruvate kinase from anoxic Busycotypus canaliculatum (PK-anoxic) is a phosphoprotein was demonstrated by the anoxia-dependent, in vivo, covalent incorporation of injected [32P]orthophosphate into the enzyme molecule. Specificity in labelling of PK-anoxic was strongly suggested by: (a) coincidental elution of pyruvate kinase activity and radioactivity following chromatography of purified PK-anoxic on Sepharose CL-6B, and (b) comigration of the area containing [32P]phosphate and Coomassie-Blue-staining protein following SDS-polyacrylamide gel electrophoresis of homogenous PK-anoxic. The [32P]phosphate content of the enzyme was calculated to be 7.3 mol phosphate/mol enzyme (233 kDa, 180 units/mg protein). Evidence for the reversibility of this phosphorylation was provided by the consistent kinetic similarities between purified red muscle pyruvate kinase from aerobic animals (PK-aerobic) and homogenous, unlabelled, alkaline phosphatase treated PK-anoxic. Comparison of the electrophoretic mobilities of products derived from acid hydrolysis of purified 32P-labelled PK-anoxic with authentic substances suggest the presence of an O-phospho-L-threonine residue in the protein. That this residue plays a probable role in an interconversion mechanism was suggested by the lack of phosphate exchange of homogenous 32P-labelled PK-anoxic in the presence of all substrates. A possible role of protein phosphorylation as a mechanism for the overall control of molluscan anaerobic metabolism is suggested.
TL;DR: The results suggest that glucose may, in part, regulate aflatoxin synthesis via a carbon catabolite repression of NADPH-generating and tricarboxylic acid cycle enzymes.
Abstract: Catabolism of carbohydrates has been implicated in the regulation of aflatoxin synthesis To characterize this effect further, the activities of various enzymes associated with glucose catabolism were determined in Aspergillus parasiticus organisms that were initially cultured in peptone-mineral salts medium and then transferred to glucose-mineral salts and peptone-mineral salts media After an initial increase in activity, the levels of glucose 6-phosphate dehydrogenase, mannitol dehydrogenase, and malate dehydrogenase were lowered in the presence of glucose Phosphofructokinase activity was greater in the peptone-grown mycelium, but fructose diphosphatase was largely unaffected by carbon source Likewise, carbon source had relatively little effect on the activities of pyruvate kinase, malic enzyme, isocitrate-NADP dehydrogenase, and isocitrate-NAD dehydrogenase The results suggest that glucose may, in part, regulate aflatoxin synthesis via a carbon catabolite repression of NADPH-generating and tricarboxylic acid cycle enzymes
TL;DR: No single enzyme is likely to be responsible for the regulation of the metabolic flux through any pathway, as first expressed by Kacser and Burns 4.
TL;DR: With increasing population doubling in vitro, human diploid fibroblasts exhibited a highly significant increase in glucose uptake from the growth medium and a corresponding increase in lactate production.
Abstract: With increasing population doubling in vitro, human diploid fibroblasts exhibited a highly significant increase in glucose uptake from the growth medium and a corresponding increase in lactate production. The switch to glycolysis occurred prior to the onset of changes in intracellular glucose and lactate concentrations or in the specific activity of the glycolytic regulatory enzyme, pyruvate kinase. It also preceded the morphological alterations held to be characteristic of cellular senescence.
TL;DR: It is suggested that the activity of the first isoform of pyruvate kinase from spinach is regulated by respiratory metabolism and may be regulated by the demand for carbon skeletons for use in ammonia assimilation.
Abstract: Pyruvate kinase from spinach (Spinacea oleracea L.) leaves consists of two isoforms, separable by blue agarose chromatography. Both isoforms share similar pH profiles and substrate and alternate nucleotide Km values. In addition, both isoforms are inhibited by oxalate and ATP and activated by AMP. The isoforms differ in their response to three key metabolites; citrate, aspartate, and glutamate. The first isoform is similar to previously reported plant pyruvate kinases in its sensitivity to citrate inhibition. The Ki for this inhibition is 1.2 millimolar citrate. The second isoform is not affected by citrate but is regulated by aspartate and glutamate. Aspartate is an activator with a Ka of 0.05 millimolar, and glutamate is an inhibitor with a Ki of 0.68 millimolar. A pyruvate kinase with these properties has not been previously reported. Based on these considerations, we suggest that the activity of the first isoform is regulated by respiratory metabolism. The second isoform, in contrast, may be regulated by the demand for carbon skeletons for use in ammonia assimilation.
TL;DR: The results seem to indicate that the mechanisms responsible for carbohydrate induction of L-type pyruvate kinase and aldolase B are different, and dietary control of each enzyme is also different in the various tissues which synthesize them.
TL;DR: Antibody prepared against the catalytic subunit of protein phosphatase-2A from rabbit skeletal muscle, could completely inhibit this enzyme, but did not significantly affect the activities ofprotein phosphatases-1, 2B and 2C.
Abstract: Antibody prepared against the catalytic subunit of protein phosphatase-2A from rabbit skeletal muscle, could completely inhibit this enzyme, but did not significantly affect the activities of protein phosphatases-1, 2B and 2C. The antibody was used to establish the following points. The three forms of protein phosphatase-2A that can be resolved by ion-exchange chromatography, termed 2A0, 2A1, and 2A2, share the same catalytic subunit. The antigenic sites on the catalytic subunit of protein phosphatase-2A remain accessible to the antibody, when the catalytic subunit is complexed with the other subunits of protein phosphatases-2A0, 2A1 and 2A2. The catalytic subunits of protein phosphatase-2A from rabbit skeletal muscle and rabbit liver are very similar, as judged by immunotitration experiments. Protein phosphatase-1 and protein phosphatase-2A account for virtually all the phosphorylase phosphatase activity in dilute tissue extracts prepared from skeletal muscle, liver, heart, brain and kidney, and for essentially all the glycogen synthase phosphatase activity in dilute skeletal muscle and liver extracts. Protein phosphatase-2A is almost absent from the protein-glycogen complex prepared from skeletal muscle or liver extracts. Protein phosphatase-2A accounts for a major proportion of the phosphatase activity in dilute liver extracts towards 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, 6-phosphofructo-1-kinase, fructose 1,6-bisphosphatase, pyruvate kinase and phenylalanine hydroxylase, the major phosphorylated enzymes involved in the hormonal control of hepatic glycolysis and gluconeogenesis.
TL;DR: After onset of anaerobic conditions Arenicola Marina does not switch immediately to typical anaerobiosis, the adaptation occurs in three steps and the energy supply occurs exclusively via the known pathways typical for long-term anaerOBiosis.
Abstract: After onset of anaerobic conditions Arenicola Marina does not switch immediately to typical anaerobiosis. The adaptation occurs in three steps. 1. 1. In the first 3 hr the phosphagen stores are mobilized, glycogen is degraded to alanine, aspartate is metabolized to succinate and volatile fatty acids ( transition period ). 2. 2. Starting approximately after 3 hr, the route of glycogen degradation at the phosphoenolpyruvate-branchpoint is changed gradually from metabolizing via pyruvate kinase to phosphoenolpyruvate carboxykinase. At the same time the metabolic rate is reduced significantly ( switching period ). 3. 3. After more than 12 hr the energy supply occurs exclusively via the known pathways typical for long-term anaerobiosis.
TL;DR: It is demonstrated here that the phosphorylated metabolites of the glycolytic pathway experience a dramatic downshift upon addition of 2-ketobutyrate (or its analogues).
Abstract: We have previously proposed that 2-ketobutyrate is an alarmone in Escherichia coli. Circumstantial evidence suggested that the target of 2-ketobutyrate was the phosphoenol pyruvate: glycose phosphotransferase system (PTS). We demonstrate here that the phosphorylated metabolites of the glycolytic pathway experience a dramatic downshift upon addition of 2-ketobutyrate (or its analogues). In particular, fructose-1,6-diphosphate, glucose-6-phosphate, fructose-6-phosphate and acetyl-CoA concentrations drop by a factor of 10, 3, 4, and 5 respectively. This result is consistent with (i) an inhibition of the PTS by 2-ketobutyrate, (ii) a control of metabolism by fructose-1,6-diphosphate. Since fructose-1,6-diphosphate is an activator of phosphoenol pyruvate carboxylase and of pyruvate kinase, the concentration of their common substrate, phosphoenol pyruvate, does not decrease in parallel.
TL;DR: Chronic ethanol consumption for 12 weeks did not cause histological changes but decreased GPT activity, increased glutamate dehydrogenase and pyruvate kinase activities and did not alter their pp/pv distribution.
TL;DR: The inhibition of gluconeogenesis and enhancement of glycolysis from gluconeogenic precursors in hepatocytes treated with 2,5-anhydromannitol can be explained by an inhibition of fructose 1,6-bisphosphatase, an activation of pyruvate kinase, and an abolition of the influence of phosphorylation on pyruve kinase.
TL;DR: The phosphate esters of 2,5-anhydro-D-mannitol would, therefore, be expected to inhibit glycogenolysis and gluconeogenesis and stimulate glycolysis in liver.
TL;DR: The results showed that PK remains tetrameric both in the presence and in the absence of Phe; however, Phe induces a small decrease in the sedimentation coefficient of the enzyme; hence, it suggests a loosening of the protein structure.
Abstract: The mechanism of allosteric regulation of rabbit muscle pyruvate kinase (PK) was examined in the presence of the allosteric inhibitor phenylalanine (Phe). Steady-state kinetic, equilibrium binding, and structural studies were conducted to provide a broad data base to establish a reasonable model for the interactions. Phe was shown to induce apparent cooperativity in the steady-state kinetic measurements at pH 7.5 and 23 degrees C. The apparent Km for phosphoenolpyruvate was shown to increase with increasing Phe concentrations. These results imply that Phe reduces the affinity of PK for phosphoenolpyruvate. This conclusion was substantiated by equilibrium binding studies which yielded association constants of phosphoenolpyruvate as a function of Phe concentration. The binding constant of Phe was also determined at pH 7.0 and 23 degrees C. The effect of ligands on the hydrodynamic properties of PK was monitored by difference sedimentation velocity, sedimentation velocity, and equilibrium experiments. The results showed that PK remains tetrameric both in the presence and in the absence of Phe. However, Phe induces a small decrease in the sedimentation coefficient of the enzyme; hence, it suggests a loosening of the protein structure. The accessibility of the sulfhydryl residues of the enzyme also increases in the presence of Phe. Furthermore, the Phe-induced conformational change was approximately 90% complete when only 25% of the binding sites were saturated. This result suggested that the regulatory behavior of PK might satisfactorily be described by the two-state model of Monod-Wyman-Changeux [Monod, J., Wyman, J., & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88-118].
TL;DR: It appears likely that caffeine inhibits aflatoxin synthesis by restricting the uptake of carbohydrates which are ultimately used by the mold to synthesize this family of mycotoxins.
Abstract: Aflatoxin production by pregrown cultures of Aspergillus parasiticus was completely inhibited by incorporation of 2 mg of caffeine per ml into the medium. This was accompanied by a decrease in glucose utilization and an inhibition of oxygen uptake and carbon dioxide evolution. Enzyme analyses indicated no significant differences in specific activities on glucose-6-phosphate dehydrogenase, mannitol dehydrogenase, phosphofructokinase, fructose 1,6-diphosphatase, pyruvate kinase, or malate dehydrogenase. Glucose uptake kinetics indicated a linear dose-related inhibition of glucose uptake. It appears likely that caffeine inhibits aflatoxin synthesis by restricting the uptake of carbohydrates which are ultimately used by the mold to synthesize this family of mycotoxins.
TL;DR: The effect of hypoxia was studied in cold (15°C and warm (30°C) acclimated goldfish as mentioned in this paper, and the hypoxic thresholds, defined as the lowest sustainablePO2 were found to be 1.6 and 4.0 kPa O2 at, respectively, 15°C, and 30°C. At these levels the fish did not loose either weight or appetite over a 2-months period.
Abstract: The effect of hypoxia was studied in cold (15°C) and warm (30°C) acclimated goldfish. The hypoxic thresholds, defined as the lowest sustainablePO2 were found to be 1.6 and 4.0 kPa O2 at, respectively, 15°C and 30°C. At these levels the fish did not loose either weight or appetite over a 2-months period. While during starvation under normonic conditions a significant weight loss and breakdown of lactate dehydrogenase (90%) was observed, no such changes were found in fed hypoxic animals. In red lateral muscle, white epaxial muscle and liver of goldfish from 4 differently acclimated groups the maximal activities were measured of: glycogen phosphorylase, hexokinase, malate dehydrogenase, glycerol-3-P dehydrogenase, glucose-6-P dehydrogenase, malic enzyme, succinate oxidase, pyruvate carboxylase, phosphoenol-pyruvate carboxykinase, fructose-bisphosphatase and glucose-6-phosphatase. Thermal compensation, according to Precht's typology, was predominantly observed in red muscle and to a lesser extent in white muscle. The liver glucose-6-P dehydrogenase showed a strong inverse response, which points to enhanced synthetic activity at the higher temperature. Hypoxia acclimation exerted weaker responses at 15°C than at 30°C. Changes in liver enzyme activities suggest depressed protein synthesis and enhanced gluconeogenesis in hypoxic animals. In muscle of 30°C-acclimated goldfish hypoxia induces a significant increase of succinate oxidase activity, indicating adaptation of the aerobic energy metabolism. The occurrence of pyruvate carboxylase, never before observed in vertebrate muscle, probably plays an important role in pyruvate catabolism. Because its action produces oxalo-acetate, the enzyme may stimulate pyruvate oxidation and thus prevent early lactate accumulation. Since all gluconeogenic enzymes were shown to be active in goldfish muscle, the possible occurrence of gluconeogenesis in muscle (albeit at low rate) must be accepted. Enzyme activities in goldfish muscle were compared with literature data for a number of other fish species. This comparison indicates that maximal glycolytic flux in goldfish muscle tissue is rather low, although muscular glycogen levels are very high. It is suggested that this is part of the gold-fish's strategy to cope with hypoxia.
TL;DR: The data indicate that the effect of adenosine to decrease the concentration of fructose 2,6-bisphosphate is mediated by the stimulation of adenylate cyclase, secondary to the binding of adenoine to membranous receptors.
Abstract: The presence of adenosine (25-250 microM) or of 2-chloroadenosine (2.5-100 microM) in the incubation medium caused a marked decrease in the concentration of fructose 2,6-bisphosphate in isolated hepatocytes. This effect was accompanied by an increase in the concentration of cyclic AMP, an activation of phosphorylase and of fructose 2,6-bisphosphatase, and an inactivation of pyruvate kinase and of 6-phosphofructo-2-kinase. As a rule, the changes in the fructose 2,6-bisphosphate-modifying system were slower but more persistent than those in the activities of phosphorylase and pyruvate kinase. The effect of the nucleoside to decrease the concentration of fructose 2,6-bisphosphate was not affected by an inhibitor of adenosine transport and could not be obtained in a liver high-speed supernatant. These data indicate that the effect of adenosine to decrease the concentration of fructose 2,6-bisphosphate is mediated by the stimulation of adenylate cyclase, secondary to the binding of adenosine to membranous receptors. Like glucagon, 2-chloroadenosine stimulated gluconeogenesis in isolated hepatocytes, whereas adenosine had an opposite effect.
TL;DR: N-Acetylglucosamine kinase was found as a single enzyme form in both hepatocytes and non-parenchymal cells, with higher activities in the former, confirming that they are good cellular markers.
Abstract: The cellular distribution of hexokinase isoenzymes, N-acetylglucosamine Kinase and pyruvate kinases in rat liver was studied. Hepatocytes and non-parenchymal cells with high viability and almost no cross-contamination were obtained by perfusion in situ of the liver with collagenase, with the use of an enriched cell-culture medium in all steps of cell isolation. Separation of hexokinase isoenzymes was done by DEAE-cellulose chromatography, and enzyme activities were measured by a specific radioassay. Cytosol from isolated hepatocytes contained high-affinity hexokinases A, B and C, in addition to hexokinase D. The last-mentioned represented about 95% of total glucose-phosphorylating activity. Only hexokinase A was found associated t the particulate fraction. Isolated non-parenchymal cells contained only hexokinases A, B and C. N-Acetylglucosamine kinase was measured with a specific radioassay and was found as a single enzyme form in both hepatocytes and non-parenchymal cells, with higher activities in the former. Pyruvate kinase isoenzyme L was present only in the hepatocytes and isoenzyme K only in the non-parenchymal liver cells, confirming that they are good cellular markers.
TL;DR: Late committed progenitor cells of erythropoiesis, CFU-E (colony-forming unit--erythroid), were isolated from mouse spleens to near homogeneity by a three-step enrichment procedure, and marked declining activities of several enzymes, including hexokinase, phosphofructokinases, aldolase, enolase and pyruvate kinase, were observed during in vitro differentiation.
TL;DR: It is suggested that adrenalectomy impaired the gluconeogenic action of glucagon in livers of fasted rats at the level of regulation of 6-phosphofructo-1-kinase and/or fructose 2,6-bisphosphate content.
TL;DR: It is concluded that the enzyme purified in the presence of fructose-1,6-diphosphate (FDP) probably has FDP bound to the protein in vivo and the significance of this hypothesis to the regulation of glycolysis in A. niger is discussed.
Abstract: Pyruvate kinase from the filamentous, citric acid producing fungus Aspergillus niger was purified about 100-fold by ammonium sulfate precipitation, DEAE-cellulose chromatography, and gel filtration. The addition of fructose-1,6-diphosphate was necessary to prevent loss of activity during purification. The enzyme purified in the presence of fructose-1,6-diphosphate (FDP) exhibits hyperbolic kinetics with respect to phosphoenolpyruvate (PEP) and ADP. Monovalent cations activated the enzyme (K+, NH4+). FDP neither activated nor inhibited the enzymatic activity from extracts freshly prepared in the absence of exogenous FDP; ATP showed a weak activation. In contrast the enzyme from crude extracts which had been stored in the presence of glycerol for 3 days showed activation by FDP or a metabolite thereof and inhibition by ATP. In the absence of FDP sigmoidal kinetics were obtained with respect to PEP, which became hyperbolic kinetics after addition of FDP. ATP inhibition turned into slight ATP activation in th...