Showing papers on "Pyruvate kinase published in 1973"

Assay of yeast enzymes in situ. A potential tool in regulation studies.

Abstract: A permeabilization method which allows the assay of several intracellular enzymes within the boundaries of the yeast cell wall is described. The kinetic properties of hexokinase and pyruvate kinase examined in the permeabilized cells, including the allosteric activation of the latter by fructose bisphosphate, are essentially the same as in cell-free extracts.

Pyruvate kinase. Classes of regulatory isoenzymes in mammalian tissues.

Abstract: El nombre actual de la revista es FEBS Journal: Open Access para articulos anteriores al 2006.-- Autores pertenecientes al extinto Instituto de Enzimologia del CSIC.

Activities of Pentose Shunt and Glycolytic Enzymes in Lungs of Ozone-Exposed Rats

Abstract: After seven days of exposure of rats to 0.8 ppm ozone, the activities of pentose shunt and glycolytic enzymes of the lungs were significantly increased, α-Tocopherol partially retarded the elevation of glucose-6-phosphate dehydrogenase (G-6-PD), 6-phosphogluconate dehydrogenase (6-P-GD), and malic enzyme, but not of others. In a separate exposure of rats to 0.75 ppm ozone, the activities of glutathione (GSH) peroxidase, GSH reductase, G-6-PD, 6-P-GD, and pyruvate kinase decreased initially and then increased above their respective controls. The results can be partially attributed to increased needs of reductive detoxification of lipid peroxide. Increased activities may be related to the development of adaptation by the animals.

Relationship between Intracellular Distribution of Phosphoenolpyruvate Carboxykinase, Regulation of Gluconeogenesis, and Energy Cost of Glucose Formation

Abstract: Gluconeogenesis was studied in isolated perfused livers from pigeons, guinea pigs and rats in order to evaluate the role of intramitochondrial formation of phosphoenolpyruvate and the rate and importance of “futile cycling” of carbon for the regulation of glucose formation. The net formation of glucose from lactate (20 mM) by pigeon liver is about twice as high as in guinea pig liver and about 3 to 4 times higher than in rat liver, although the intracellular ATP/ADP ratio in pigeon liver is extremely low (< 1) in the presence and absence of gluconeogenesis. In contrast to experiments with rat livers, but in accordance with those with guinea pig livers, oleate (2 mM) failed to stimulate gluconeogenesis from lactate in pigeon liver. With pyruvate (20 mM) there is no net formation of glucose by pigeon livers in the absence or presence of hexanoate, oleate or xylitol. In the presence of ethanol the rate of glucose formation from pyruvate increased but did not exceed 30% of the rate observed with lactate as precursor. In contrast to the results obtained in rat liver experiments the transaminase inhibitor (aminooxy)acetate did not inhibit gluconeogenesis from lactate in pigeon liver although soluble and mitochondrial glutamate oxaloacetate aminotransferase from pigeon liver were strongly inhibited by (aminooxy)acetate. On the other hand, n-butylmalonate (5 mM) inhibited strongly gluconeogenesis from lactate in isolated perfused rat and pigeon livers. This inhibition was accompanied by an inhibition of oxygen uptake in pigeon but not in rat liver. Benzene 1,2,3-tricarboxylate, an inhibitor of the tricarboxylate translocator, had no effect on gluconeogenesis from lactate in isolated perfused rat and pigeon livers. Gluconeogenesis from propionate (10 mM) decreased in the order guinea pig-rat-pigeon. The activity of phosphoenolpyruvate carboxykinase as well as the phosphoenolpyruvate carboxykinase/pyruvate kinase ratio in pigeon liver were significantly higher than in rat and guinea pig liver. The activities of phosphoenolpyruvate carboxykinase as well as pyruvate carboxylase in pigeon liver were almost completely located intramitochondrially. Malic enzyme activity in pigeon liver was about 33 times higher than in rat liver and about 260 times higher than in guinea pig liver. However, the sum of the activities of the 4 NADPH-generating enzymes, malic enzyme + isocitrate dehydrogenase (NADP) + glucose-6-phosphate dehydrogenase + 6-phosphogluconate dehydrogenase was similar in pigeon and guinea pig liver, but somewhat lower in rat liver. Measurement of oxygen uptake during gluconeogenesis from intraportally infused lactate+pyruvate (10/1) was performed with isolated perfused livers from pigeon, rats and guinea pigs. The ratio oxygen used/glucose formed was 2.47 in pigeon liver, 2.42 in rat liver, and 2.35 in guinea pig liver. These results are very close to the theoretically expected value of 2. The following conclusions have been made: In pigeon liver phosphoenolpyruvate formed intramitochondrially is used for gluconeogenesis. There is no correlation between the overall ATP/ADP ratio and the rate of glucose formation. The regulation of gluconeogenesis is strongly species-dependent as can be demonstrated for instance by the inability of pigeon liver to form glucose from pyruvate. The rate of “futile cycling” of carbon between phosphoenolpyruvate and pyruvate cannot exceed 40% of the rate of pyruvate carboxylation. The significant species difference in the maximum capacity for glucose formation from lactate cannot be explained on the basis of different degrees of “futile cycling”.

A kinetic study of rabbit muscle pyruvate kinase.

Abstract: The paper reports a study of the kinetics of the reaction between phosphoenolpyruvate, ADP and Mg(2+) catalysed by rabbit muscle pyruvate kinase. The experimental results indicate that the reaction mechanism is equilibrium random-order in type, that the substrates and products are phosphoenolpyruvate, ADP, Mg(2+), pyruvate and MgATP, and that dead-end complexes, between pyruvate, ADP and Mg(2+), form randomly and exist in equilibrium with themselves and other substrate complexes. Values were determined for the Michaelis, dissociation and inhibition constants of the reaction and are compared with values ascertained by previous workers.

11 Pyruvate Kinase

Abstract: Publisher Summary This chapter describes pyruvate kinase. The enzyme has a number of attributes that make it of particular interest to various investigators. It was the first and perhaps the most clearly apparent example of an enzyme with an absolute requirement for a monovalent cation for its catalytic activity. The skeletal muscle enzyme was quite easily obtained in good yields of high purity and was stable over a period of a year. It was also recognized quite early to be a multisite subunit containing protein. In contrast, the enzyme from other sources has been somewhat difficult to stabilize both during and after purification. Skeletal muscle pyruvate kinase is most commonly prepared from rabbit muscle by the method of Tietz and Ochoa. The basic procedure utilized no chromatographic steps and can be conveniently scaled up to produce gram quantities of enzyme in a few days starting with commercially available frozen muscle. In this chapter, molecular properties of pyruvate kinase are described. Enzyme purification, composition, structure, chemical modification, and conformational change are explained. Catalytic properties are also described in the chapter.

Isoenzyme patterns in parenchymal and non-parenchymal cells isolated from regenerating and regenerated rat liver.

Abstract: Parenchymal and non-parenchymal cells were isolated from adult rat liver that had been fully regenerated after a 70% partial hepatectomy. The characteristics of the parenchymal cell preparations from regenerated rat liver indicated that they were a homogeneous population and comparable with parenchymal cells isolated from intact liver. The parenchymal cells from regenerated adult rat liver contain glucokinase, hexokinase, pyruvate kinase type I and aldolase B. The non-parenchymal cells contain hexokinase, pyruvate kinase type III and aldolase B. When cells were isolated at different times of the day from rats on controlled feeding schedules, variation of tyrosine aminotransferase activity and liver glycogen content were observed in the parenchymal cells in keeping with the reported diurnal oscillations found in whole liver extracts. When parenchymal cells were isolated from rats 48 and 72h after partial hepatectomy, different isoenzyme patterns were observed. These cells appeared to synthesize pyruvate kinase type III, a function that was assigned previously to non-parenchymal cells or to foetal rat liver hepatocytes.

Studies on Certain Cytoplasmic Enzymes and Specific Estrogen Receptors in Human Breast Cancer and in Nonmalignant Diseases of the Breast

Abstract: Summary Samples of normal breast tissue, fibrocystic disease, fibroadenomas, and infiltrating ductal carcinomas were examined for several cytoplasmic enzymes and specific estrogen-binding capacity, as well as for the levels of RNA, DNA, and protein. Compared with normal breast tissue, the activities per mg DNA of pyruvate kinase, glucose 6-phosphate dehydrogenase, isocitrate dehydrogenase, hexokinase, glucose phosphate isomerase, and aspartate aminotransferase were significantly elevated in carcinomas. Furthermore, the activities of these enzymes were also significantly higher in the carcinomas, compared with those of samples of fibrocystic disease and fibroadenoma. Overall, there appeared to be little difference in the biochemical parameters measured in this comparison of normal breast tissue, fibrocystic disease, and fibroadenoma when the data were normalized by expression of results per mg DNA. A notable exception was the decrease in activity in α-glycerol phosphate dehydrogenase, which was highest in normal breast tissue and significantly decreased in all the breast diseases examined. Neither the age of the patient at the time the tumor was removed nor the presence of metastatic lesions was apparently related to the biochemical characteristics, although the carcinomas from women 70 years or older showed somewhat higher activities of glucose phosphate isomerase, lactate dehydrogenase, and glucose 6-phosphate dehydrogenase. Carcinomas that possessed specific estrogen-binding capacity (>7 fmoles/mg protein), as estimated by sucrose gradient centrifugation of tumor cytosols, had lower activities of glucose 6-phosphate dehydrogenase, isocitrate dehydrogenase, and pyruvate kinase and slightly higher levels of DNA. The data reported here indicate that infiltrating ductal carcinoma of the breast presents with certain metabolic alterations that are not shared by nonmalignant breast diseases. Carcinomas that are potentially hormone responsive demonstrate a different enzyme activity profile from that of tumors that are potentially hormone independent.

Pyruvate Kinase, a Possible Regulatory Enzyme in Higher Plants

Abstract: A number of plant species were examined for the presence of pyruvate kinase (pyruvate-ATP phosphotransferase, EC 2.7.1.40), and of a phosphatase activity which hydrolyzes phosphoenolpyruvate. Of those examined, only cotton (Gossypium sp. L.) seeds were found to be sufficiently free of the phosphatase to permit a kinetic study of pyruvate kinase.During germination of cotton seeds, pyruvate kinase activity rises for the first 3 days, after which it falls back to its original level. This developmental pattern is characteristic of enzymes involved in the conversion of fat into carbohydrate in fatstoring seeds. The phosphatase also rose rapidly during germination, which precluded the use of extracts from seedlings in the study of pyruvate kinase. No evidence was found for the presence of more than one pyruvate kinase in cotton seedlings.In crude extracts from ungerminated seeds, the enzyme shows slight deviations from normal kinetics with respect to phosphoenolpyruvate, magnesium, and to a lesser extent, ADP. After partial purification of the enzyme by ion exchange chromatography, the enzyme shows normal kinetics. The enzyme is activated by AMP, and inhibited by both ATP and citrate, in both crude and partially purified preparations. It is suggested that cotton seed pyruvate kinase is a regulatory enzyme.

Mitochondria-Lipid-Glycogen (MLG) Disease of Muscle: A Morphologically Regressive Congenital Myopathy

Abstract: Muscle biopsies of a 7-week-old girl with profound weakness of all but the ocular muscles, combined with hypotonia, hyporeflexia, hepatomegaly, macroglossia, myopathic electromyogram, and slight elevation of serum enzyme levels revealed mild glycogen and marked lipid and mitochondrial excess Glycogen structure and anaerobic glycolysis were normal Aldolase and pyruvate kinase levels were relatively low and the lactic dehydrogenase isoenzyme pattern resembled that of heart muscle Subsequently the patient had normal intellectual and delayed motor development and her macroglossia disappeared At 22 months pathologic alterations in muscle were strikingly improved and glycolytic enzyme levels and oxidation of labeled oleic acid and Krebs cycle intermediates were normal Although still unidentified, the basic metabolic lesion is one that (1) affects mitochondria! morphology, (2) alters lipid and glycogen metabolism, (3) may improve with age, and (4) is compatible with a benign clinical course after early infancy

Glycolytic isoenzymes and glycogen metabolism in regenerating liver from rats on controlled feeding schedules

Abstract: Intact rats trained on a controlled feeding and lighting schedule designated ;8+16' exhibited diurnal oscillations in liver weight, glucokinase activity and liver glycogen content. Glucokinase activity expressed as units/g of liver decreased to 30% of that from unoperated controls during the first 48h after partial hepatectomy and returned to near normal values in 2 weeks. When the glucokinase activity was expressed as units/liver per 100g body wt., a decrease to 50% of control activity was observed between 24 and 48h after the operation. A similar pattern was found for pyruvate kinase type I. In contrast, pyruvate kinase type III activity increased after partial hepatectomy. It is suggested that the newly divided cells after partial hepatectomy do not synthesize glucokinase and pyruvate kinase I but do synthesize pyruvate kinase III. Glycogen was found to accumulate as early as 24h after partial hepatectomy, and normal concentrations were reached after 48h if the operation was performed at times other than during the feeding periods.

Studies on glycolysis in the muscle tissue of Ascaris lumbricoides (nematoda)

Abstract: 1. 1. The activities of the glycolytic and associated enzymes have been investigated in the muscle tissue of Ascaris lumbricoides and the steady-state content of the phosphorylated glycolytic intermediates measured in freeze clamped tissue. 2. 2. A comparison of the mass action ratios of the glycolytic enzymes with their equilibrium constants has shown that in Ascaris muscle, phosphoglucomutase, glucosephosphate isomerase, aldolase, triosephosphate isomerase, phosphoglyceromutase and phosphopyruvate hydratase are all at or near equilibrium, whilst phosphofructokinase and pyruvate kinase are displaced from equilibrium. 3. 3. The results are discussed in relation to the energy metabolism of the parasite.

Assay for glutamine synthetase activity (Short Communication)

Abstract: The use of phosphoenolpyruvate plus pyruvate kinase as an ATP-generating system in the assay for glutamine synthetase activity via the formation of γ-glutamylhydroxamate from glutamate and hydroxylamine with crude tissue preparations is shown to give values far in excess of the true glutamine synthetase activity of the tissue. This is due to the generation of pyruvate, which reacts with hydroxylamine to give a compound that is chromogenic with the ferric chloride reagent used for measuring γ-glutamylhydroxamate.