Studies on Erythrocyte Glycolysis I. Determination of the Glycolytic Intermediates in Human Erythrocytes*

Minakami, Shigeki; Suzuki, Chiyo; Saito, Toshihito; Yoshikawa, Haruhisa

doi:10.1093/oxfordjournals.jbchem.a128240

Cited by 264 publications

(76 citation statements)

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“…Equilibrium a t the phosphoglucose isomerase step has been studied by many authors in Werent cells or tissues. As seen in Table 8, where data from the literature are collected, a good agreement between massaction ratio and thermodynamic equilibrium constant was found in yeast [26], erythrocytes [27], and rat skeletal muscle [15]. On the other hand, in line with Lowry et al [8] for the mouse brain, with Hohorst et al [9] for the rat liver, with Hess [6] for ascites tumor, with Williamson [28] and Herkel [I41 for the rat heart, and with Ozand and Narahara for the frog sartorius [29] we found a definite shift from equilibrium in other muscle types [16,30].…”

Section: Discussionmentioning

confidence: 83%

Levels of Glycolytic Intermediates in the Musculature of the Chick during Embryonic and Post‐Embryonic Development

Arese

Bosìa

1967

European Journal of Biochemistry

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Levels of glycogen and of glycolytic intermediates were assayed in quickly frozen embryonic mesodermic tissue (decapitated and eviscerated chick embryos) a t the 5th and 7th developmental day and in the hind Limb musculature in subsequent pre-and post-natal stages. Serial determination of dry weight values during embryonic development allows conversion of the levels which are reported on a wet weight basis.While glycogen gradually increases from the 9th day onwards reaching about 'I3 of the adult amount on hatching, no substantial differences between early embryonic and adult levels were noted in the case of other intermediates, such as glucose, dihydroxyacetone-phosphate, glyceraldehyde-3-phosphate, 3-and 2-phosphoglycerate, phosphoenolpyruvate, pyruvate and lactate. Glucose-I-phosphate, fructose-6-phosphate, glucose-6-phosphate and fructose-l,6-diphosphate were low during most of the embryonic development, attaining levels close to the adult ones only on hatching.I n order to study the flow rates, the equilibrium situation a t each single step and the possible control points along the glycolytic pathway, the tissue was stressed by brief periods of ischaemia or by electrical stimulation a t different stages of development.Glycolytic flow rates showed a progressive increase up to hatching, where a lactate accumulation of 1.5 pmole/min/g wet weight was measured. The maximum rate of 12.9 pmoles/min/g wet weight found in electrically stimulated muscle of hatching chicks, is well below the adult value of 52.7 pmoles/min/g wet weight.The approximate stoichiometric correspondence between glucose + glycogen breakdown and lactate formation, found to exist in the early and late embryonic stages, is absent on the 7th and incomplete on the 11th day, where the sharply reduced gluco-and glycogenolysis cannot account for the measured glycolytic rate. The existence of a vicarious anomalous glycolysis, supplying pyruvate and lactate by transamination or by other unknown mechanisms, is possible a t these two stages.The constancy of mass-action ratios under altered glycolytic fluxes and the comparison between the ratios in vivo and the thermodynamic equilibria show that phosphoglucomutase, phosphoglucose isomerase, aldolase, triosephosphate isomerase, phosphoglycerate mutase and enolase were maintained either a t equilibrium or fairly close to equilibrium. Owing to the probable compartmentation of ATP and ADP in muscle, no calculation of kinase-catalyzed reactions was attempted.Activation of glycolysis could not be observed on electrical stimulation of embryonic muscle a t the 14th day, as the constancy of such indicator metabolites as glucose-6-phosphate, fructose-1,6-diphosphate and lactate demonstrates. An activation a t the phosphorylase and phosphofructokinase steps has been indirectly shown on hatching.Non-standard abbreviations. Dihydroxyacetone-phosphate, DAP ; glyceraldehyde-3-phosphate, GAP; fructose-1,Gdiphosphate, FDP.Enzymes. Phosphoglucomutase, or a-D-glucose-1,G-diphosphate : a-D-glucose-1-phosphate phosphotransfer...

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Section: Discussionmentioning

confidence: 83%

Levels of Glycolytic Intermediates in the Musculature of the Chick during Embryonic and Post‐Embryonic Development

Arese

Bosìa

1967

European Journal of Biochemistry

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“…Only ascites tumour cells, incubated with 30 mM glucose contain levels comparable to those of T. brucei [26]. Other tissues, like muscle and liver [17] and erythrocytes [27,28] contain levels which are sometimes one order of magnitude lower. We have previously presented evidence that the glycosomes are identical to microbody-like organelles observed in electron micrographs of thin sections of T. brucei [9] which only represent 7 -8 % of the total cytoplasmic volume [29].…”

Section: Levels Of Glycolytic Intermediatesmentioning

confidence: 99%

“…; triosephosphate isomerase (triose-P isomerase), 22; glyceraldehyde-phosphate dehydrogenase (Gra-P dehydrogenase), 0.08; phosphoglycerate kinase (Grip kinase), 3100; phosphoglyceromutase (Grip mutase), 0.17; enolase, 3; pyruvate kinase, 15 800 [26]. Data used to calculate the displacement graphs for cells other than T. brucei were derived from the literature: muscle [17], ascites cells [26,31], rat liver [17], erythrocytes [28]. For the calculation of the mass-action ratio of the glyceraldehyde-phosphate dehydrogenase reaction in T. brucei the [NADH]/[NAD+] ratio as calculated from the glycerol-3-phosphate dehydrogenase equilibrium ( Table 5 ) was used only slightly displaced from equilibrium.…”

Section: Levels Of Glycolytic Intermediatesmentioning

confidence: 99%

Glycolysis in Trypanosoma brucei

Visser

Opperdoes

1980

European Journal of Biochemistry

122

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The possibility that the glycosomes present in the bloodstream form of Trypanosoma brucei FEBS Lett. 80, 360-3641 constitute a separate pool of glycolytic intermediates within the cell was investigated.In titrations of intact cells with digitonin, a differential activation of glycolytic enzymes was observed. Enolase, pyruvate kinase and the cell-sap marker alanine aminotransferase were activated at 0.05 mg digitonin per mg protein. The nine glycosomal enzymes involved in the conversion of glucose and glycerol into 3-phosphoglycerate were activated only at digitonin concentrations between 0.7 and 9.8 mg/mg protein. In subcellular fractions the activities or the latter enzymes were all latent between 70 and 92 %. Latency was abolished by addition of 0.1 % Triton X-100 or partly by five cycles of freezing and thawing. We conclude that the glycosomal enzymes are surrounded by a membrane, which forms a permeability barrier to intermediates and co-factors of glycolysis.The concentrations of glycolytic intermediates and of adenine nucleotides were measured under aerobic conditions as well as in the presence of 1 mM salicylhydroxamic acid, a respiratory inhibitor. Addition of salicylhydroxamic acid caused the following changes : (a) The levels of almost all glycolytic intermediates measured decreased. Glycerol-3-phosphate, however, increased fourfold. From the high levels of metabolite concentrations found and from comparison of the apparent mass-action ratios calculated for the separate glycolytic reactions with those for other organisms, we conclude that in bloodstream form T. brucei the glycolytic intermediates are present in the glycosomes as well as in the cytosol and that the two pools of intermediates equilibrate with each other, despite the presence of the glycosomal membrane.Bloodstream forms of the African trypanosomes belonging to the brucei group, like Trypanosoma brucei, Trypanosoma rhodesiense and Trypanosoma gambiense, are the causative agents of nagana in domestic animals Abbreviations. Dihydroxyacetone-P, dihydroxyacetone phosphate ; glycerol-3-P, sn-glycerol-3-phosphate; glycerate-3-P, 3-phosphoglycerate; glucose-6-P, glucose-6-phosphate; fructose-6-P, fructose-6-phosphate; fructose-l,h-Pz, fructose-1,6-bisphosphate; glycerate-2-P, 2-phosphoglycerate; glyceraldehyde-3-P, D-glyceraldehyde-3-phosphate.Enzymes. Hexokinase (EC 2.7.1.1); phosphoglucose isomerase (EC 5.3.1.9); 6-phosphofructokinase (EC 2.7.1.11); fructose-bisphosphate aldolase (EC 4.1.2.13); triosephosphate isomerase (EC 5.3.1 .l): sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8); glycerol kinase (EC 2.7.1.30); glyceraldehyde-phosphate dehydrogenase (EC 1.2.1.12); phosphoglycerate kinase (EC 2.7.2.3); phosphoglyceromutase (EC 2.7.5.3); enolase (EC 4.2.1.11); pyruvate kinase (EC 2.7.1.40); alanine aminotransferase (EC 2.6.1.2). and live-stock and of the acute and chronic forms of sleeping sickness in man. For their energy needs, these organisms are entirely dependent on glycolysis which proceeds at an extremely high rate and differs in its...

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“…2,3-DPG concentration was determined according to Ericson and de Verdier [5], whereas the concentrations of other internal phosphates-G-6-P, ATP, and ADP--were measured according to Minakami et al [6]. In order to measure low concentrations reproducibly a sensitive Cary 218 photometer was used.…”

Section: Methodsmentioning

confidence: 99%

Erythrocyte pyruvate kinase deficiency: A kinetic method for differentiation between heterozygosity and compound‐heterozygosity

et al. 1989

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The goal of the present study was to search for criteria that allow one to distinguish between normal individuals and heterozygotes as well as compound heterozygotes for pyruvate kinase (PK) deficiency. As the residual activity of PK with heterozygotes was between 35% and 110% of the normal activity, it was necessary to find other methods to prove heterozygosity. The PK in the hemolysates of 23 patients suffering from PK deficiency, 36 paternal and maternal enzymes as well as the enzymes of five heterozygous and four normal siblings together with those of 20 normal individuals, were studied according to the recommendations of the International Committee for Standardization in Haematology. The following hematological and enzyme kinetic parameters can serve to identify heterozygotes for PK deficiency: 1) a slight reticulocytosis, 2) an up-to-twofold increase of the intracellular concentrations of glucose-6-phosphate in the erythrocyte, 3) a mixed cooperativity of the phosphoenolpyruvate (PEP)-binding process of PK, 4) a decreased nucleotide specificity with guanosine diphosphate and uridine diphosphate, and 5) a lowered affinity for adenosine diphosphate. The most significant criterium found with all heterozygotes was a mixed cooperativity of the PEP-binding process caused by the presence of a mixture of normal and mutant PK.

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Studies on Erythrocyte Glycolysis I. Determination of the Glycolytic Intermediates in Human Erythrocytes*

Cited by 264 publications

References 1 publication

Levels of Glycolytic Intermediates in the Musculature of the Chick during Embryonic and Post‐Embryonic Development

Levels of Glycolytic Intermediates in the Musculature of the Chick during Embryonic and Post‐Embryonic Development

Glycolysis in Trypanosoma brucei

Erythrocyte pyruvate kinase deficiency: A kinetic method for differentiation between heterozygosity and compound‐heterozygosity

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