Showing papers on "Pyruvate kinase published in 1969"

Pyruvate kinase variants of the Alaskan king-crab. Evidence for a temperature-dependent interconversion between two forms having distinct and adaptive kinetic properties

Abstract: 1. Pyruvate kinase of Alaskan king-crab leg muscle exists in two kinetically distinct forms, each of which displays a different temperature-dependence in the Km for phosphoenolpyruvate. 2. A ‘cold’ variant of the enzyme has hyperbolic kinetics and exhibits a minimal Km for substrate at 5°. At physiological concentrations of phosphoenolpyruvate the ‘cold’ enzyme is active only below 10°. A ‘warm’ pyruvate kinase has a minimal Km for substrate at about 12°. This enzyme displays sigmoidal kinetics and is likely to be inactive, at physiological substrate concentrations, at temperatures below 9°. 3. The combined activities of these two pyruvate kinases yield highly temperature-independent rates of catalysis, at physiological substrate concentrations, over the range of habitat temperatures encountered by the organism, namely 4–12°. 4. The two variants of pyruvate kinase do not appear to be isoenzymes in the conventional sense. Electrophoretic and electrofocus analyses revealed only single peaks of activity. 5. The results suggest that the ‘warm’ pyruvate kinase and the ‘cold’ pyruvate kinase are formed by a temperature-dependent interconversion of one protein species. This interconversion has major adaptive significance: as the temperature is lowered the ‘warm’ enzyme is converted into the ‘cold’ enzyme; the opposite situation obtains when the temperature is raised. Temperature changes thus mimic the effects noted for fructose 1,6-diphosphate on certain mammalian pyruvate kinases.

Oxygen-hemoglobulin dissociation curves: effect of inherited enzyme defects of the red cell.

Abstract: The blood of a patient with a deficiency of hexokinase in the red cells and a decreased concentration of 2, 3-diphosphoglycerate in the red cells showed an increased affinity for oxygen, whereas a patient with a deficiency of pyruvate kinase and an elevated concentration of 2, 3-diphosphoglycerate in the red cells had blood with a decreased affinity for oxygen. Defects in red cell glycolysis may alter the oxygen affinity of blood by virtue of their effect on 2, 3-diphosphoglycerate concentrations in red cells.

Pyruvate carboxylase in Rhodopseudomonas spheroides.

Abstract: SUMMARY: Unlike some other photosynthetic bacteria, Rhodopseudomonas spheroides directly carboxylates pyruvate in a reaction catalysed by a pyruvate carboxylase (E.C. 6·4.1·1). A partially purified preparation of the enzyme was acetylCoA-dependent. No phosphoenolpyruvate synthetase or phosphoenolpyruvate carboxylase activity was detected in extracts of R. spheroides. The organisms dependence upon its pyruvate carboxylase was shown by the fact that a mutant strain which lacked this enzyme was unable to grow either anaerobically in the light, or aerobically in the dark, on glucose or pyruvate (with CO2). Convenient spectrophotometric assays for pyruvate carboxylase are reported.

Inhibition of human brain pyruvate kinase and hexokinase by phenylalanine and phenylpyruvate: possible relevance to phenylketonuric brain damage.

Abstract: In phenylketonuria high levels of L-phenylalanine are present along with increased levels of phenylpyruvic acid. The present work shows that L-phenylalanine is a competitive inhibitor of human brain pyruvate kinase and phenylpyruvic acid is an inhibitor of human brain hexokinase. The enzymes have approximately the same Ki for these inhibitors in adult and in fetal human brain. However, in the fetal human brain the absolute activities for both enzymes are less than 10 per cent of those found in the adult. Thus, the fetal brain enzymes may be more vulnerable to inhibition by these compounds in the phenylketonurics. The inhibition of human brain pyruvate kinase and hexokinase by L-phenylalanine and phenylpyruvic acid may have a role in the brain damage in phenylketonurics.

A comparison of the properties of the phosphofructokinases of the fat body and flight muscle of the adult male desert locust

Abstract: 1. The pyruvate kinases of the desert locust fat body and flight muscle were partially purified by ammonium sulphate fractionation. 2. The fat-body enzyme is allosterically activated by very low (1μm) concentrations of fructose 1,6-diphosphate, whereas the flight-muscle enzyme is unaffected by this metabolite at physiological pH. 3. Flight-muscle pyruvate kinase is activated by preincubation at 25° for 5min., whereas the fat-body enzyme is unaffected by such treatment. 4. Both enzymes require 1–2mm-ADP for maximal activity and are inhibited at higher concentrations. With the fat-body enzyme inhibition by ADP is prevented by the presence of fructose 1,6-diphosphate. 5. Both enzymes are inhibited by ATP, half-maximal inhibition occurring at about 5mm-ATP. With the fat-body enzyme ATP inhibition can be reversed by fructose 1,6-diphosphate. 6. The fat-body enzyme exhibits maximal activity at about pH7·2 and the activity decreases rapidly above this pH. This inactivation at high pH is not observed in the presence of fructose 1,6-diphosphate, i.e. maximum stimulating effects of fructose 1,6-diphosphate are observed at high pH. The flight-muscle enzyme exhibits two optima, one at about pH7·2 as with the fat-body enzyme and the other at about pH8·5. Stimulation of the enzyme activity by fructose 1,6-diphosphate was observed at pH8·5 and above.

An enzymatic assay of 2,3-diphosphoglycerate in blood.

Abstract: 2,3-diphosphoglycerate was estimated by an enzymatic method, based on the catalytical effect of 2,3-diphosphoglycerate on the phosphoglycerate mutase reaction, which was coupled with that of lactate dehydrogenase by adding ADP, enolase, pyruvate kinase, and NADH to the system. The activity was estimated from the decrease in NADH-concentration. Samples were obtained by diluting whole blood 100 times with oxygenated water, without any other pretreatment of the blood. The normal range of 2,3-diphosphoglycerate concentration in blood from normal individuals was determined.

Regulation of the Level of Key Enzymes of Glycolysis and Gluconeogenesis in Liver

Abstract: pyruvate carboxykinase were low. A metabolic bifurcation to glycolysis and gluconeogenesis, below and above the trioscphosphates level respectively, has then been shown to be easily maintained in liver. It is suggested that the induction of glucokinase and pyruvate kinase in diabetic animals after insulin administration is a sequential process that involves hormonal induction of glucokinase by insulin and secondary metabolite induction of the L isoenzyme of pyruvate kinase by some glycolytic intermediate. The occurrence of independent mechanisms for the regulation in liver of the activity of enzymes which catalyze irreversible steps of glycolysis and gluconeogenesis affords a valuable metabolic plasticity. Changes in the apparent concentration of a number of enzymes in liver have been observed in relation to the nutritional and hormonal conditions of the animal. Some of these changes could be of considerable significance in the regulation of major metabolic pathways. Glucose-6-phosphatase [l], fructosediphosphatase [a], phosphoenolpyruvate carboxykinase [3] and pyruvate carboxylase [4] had been reported to be increased in gluconeogenic conditions, like fasting, diabetes, or corticosteroids administration. In 1963, an insulin-dependent glucokinase was identified [5-71. After the finding of the induction of glucokinase in rat liver by insulin, Weber et al. [S] postulated the hypothesis that the key steps of glycolysis and gluconeogenesis could depend on two functional genic units, with insulin as inducer of the glycolytic unit and repressor of the gluconeogenic one. Shortly afterwards a number of observations were reported on the behaviour of pyruvate kinase [9- 111 and phosphofructokinase [12], consistent with the hypothesis of induction by insulin of the enzymes that catalyze these two irreversible steps of glycolysis in liver. This paper presents the results of a nutritional approach involving diets rich in fructose and/or

Glyceroneogenesis in Adipose Tissue of Fasted, Diabetic and Triamcinolone Treated Rats

Abstract: Incorporation of [2-14C]pyruvate into adipose tissue triglycerides was measured in fasted, diabetic and triamcinolone treated rats and related to the activity of enzymes active in the elaboration of glyceride glycerol. Incorporation of pyruvate into glyceride glycerol was increased in fasting and diabetes. The activity of phosphoenolpyruvate carboxykinase increased in parallel with the enhanced glyceroneogenesis, whereas the activity of pyruvate carboxylase changed in the opposite direction. After triamcinolone treatment, the activity of both enzymes was decreased as was the incorporation of [2-14C]pyruvate into glyceride glycerol. In all experimental animals glutamate-pyruvate transaminase activity in adipose tissue decreased, whereas the activity of glutamate-oxaloacetate transaminase increased slightly. These results were interpreted as implying that the enhanced glyceroneogenesis in diabetes and fasting is related to the increase in phosphoenolpyruvate carboxykinase activity, whereas pyruvate carboxylase activity seems associated with changes in fatty acid synthesis. In contrast to adipose tissue, in the liver the activity of both enzymes of the dicarboxylic acid shuttle and of transaminases increased in the situations associated with enhanced gluconeogenesis. This finding is discussed in the light of possible differences in the availability of precursors for gluco- and glyceroneogenesis in the respective tissues. The role of glyceroneogenesis is pointed out, as one of the factors maintaining the balance between synthesis and breakdown of triglycerides in adipose tissue, particularly in conditions associated with rapid lipolysis.

The effect of dietary and hormonal conditions on the activities of glycolytic enzymes in rat epididymal adipose tissue.

Abstract: 1. Measurements were made of the activities of nine glycolytic enzymes in epididymal adipose tissues obtained from rats that had undergone one of the following treatments: starvation; starvation followed by re-feeding with bread or high-fat diet; feeding with fat without preliminary starvation; alloxan-diabetes; alloxan-diabetes followed by insulin therapy. 2. In general, the activities of the glycolytic enzymes of adipose tissue, unlike those of liver, were not greatly affected by the above treatments. 3. The ;key' glycolytic enzymes, phosphofructokinase and pyruvate kinase, were generally no more adaptive in response to physiological factors than other glycolytic enzymes such as glucose phosphate isomerase, fructose diphosphate aldolase, triose phosphate isomerase, glycerol 3-phosphate dehydrogenase, phosphoglycerate kinase and lactate dehydrogenase. 4. Adiposetissue pyruvate kinase did not respond to feeding with fat in a manner similar to the liver enzyme. 5. Glyceraldehyde phosphate dehydrogenase had a behaviour pattern unlike the other eight glycolytic enzymes studied in that its activity was depressed by feeding with fat and was not restored to normal by re-feeding with a high-fat diet after starvation. These results are discussed in relation to the requirements of adipose tissue for glycerol phosphate in the esterification of fatty acids. 6. A statistical analysis of the results permitted the writing of linear equations describing the relationships between the activities of eight of the enzymes studied. 7. Evidence is presented for the existence of two constant-proportion groups amongst the enzymes studied, namely (i) glucose phosphate isomerase, phosphoglycerate kinase and lactate dehydrogenase, and (ii) triose phosphate isomerase, fructose diphosphate aldolase and pyruvate kinase. 8. Mechanisms for maintaining the observed relationships between the activities of the enzymes in the tissue are discussed.

Pyruvate kinase: modulation by L -phenylalanine and L -alanine

Abstract: L-Phenylalanine was found to be a competitive inhibitor of pyruvate kinase in the rat prostate, seminal vesicles, uterus, and skeletal muscle. In contrast, L-alanine exerted no appreciable effect o...

Hypertrophied Non-Failing Rat Heart: PARTIAL BIOCHEMICAL CHARACTERIZATION

Abstract: Cardiac hypertrophy was induced in rats by constructing an arteriovenous fistula. Heart weights approximately doubled in 7.5 weeks. The levels of activity, expressed per gram of heart, of a variety of enzymes of the mitochondrial respiratory chain and of the citric acid cycle, as well as the concentrations of cytochrome c and of mitochondrial protein, were the same in the hypertrophied and control hearts. Similarly, the capacity of whole heart homogenate to oxidize pyruvate was unaltered in hypertrophy. There was also no change in the levels of activity of cytochrome oxidase and malate dehydrogenase, or in the concentration of cytochrome c in early hypertrophy (3 to 10 days after construction of an A-V fistula). These results suggest that cardiac mitochondria increase in parallel with the other components of the myocardial cell. The levels of activity, per gram of heart, of creatine phosphokinase and adenylate kinase, and of the rate-limiting enzymes of glycolysis and glycogenolysis, were the same in the ...

Factors affecting the activity of pyruvate kinase of Acetobacter xylinum.

Abstract: 1. Extracts of Acetobacter xylinum were found to contain the glycolytic enzymes involved in the conversion of triose phosphate into pyruvate. Pyruvate kinase had the lowest relative activity. Phosphofructokinase activity was not detected in the extracts. 2. Only slight differences in the activity of pyruvate kinase were observed between cells grown on glucose and those grown on intermediates of the tricarboxylic acid cycle. 3. Pyruvate kinase, partially purified from ultrasonic extracts by ammonium sulphate fractionation, required Mg2+ ions for activity. It was not activated by K+ or NH4+ ions. 4. The plots representing the relationship between initial velocity and phosphoenolpyruvate concentration were sigmoidal, suggesting a co-operative effect for phosphoenolpyruvate. The Hill coefficient (n) for phosphoenolpyruvate was 2. The rate of the reaction changed with increasing ADP concentrations according to normal Michaelis–Menten kinetics. 5. The enzyme was inhibited by ATP (Ki0·9×10−3m). The inhibition was competitive with regard to ADP but not with regard to phosphoenolpyruvate. It was not relieved by excess of Mg2+ ions. 6. The possible relationship of the properties of pyruvate kinase to regulatory mechanisms for controlling gluconeogenesis and carbohydrate oxidation in A. xylinum is discussed.

A low Km phosphoenolpyruvate mutant in the Amish with red cell pyruvate kinase deficiency.

Abstract: Summary. The biochemical heterogeneity of red cell pyruvate kinase (PK)‐deficiency was demonstrated in a study of six patients with this abnormality. Three siblings of an Amish kindred were found to possess a mutant enzyme with a decreased Km phosphoenolpyruvate. In this kindred the Km PEP ranged from 0.018 to 0.024 mM (normal 0.055–0.067 mm). Two deficient patients were found to have an enzyme with a normal Km PEP, while in the sixth patient the Km PEP was increased to 0.120 mM.