Showing papers on "Pyruvate dehydrogenase kinase published in 1969"
TL;DR: The discovery that the activity of the multienzyme pyruvate dehydrogenase complex from beef kidney mitochondria is regulated by a phosphorylation-dephosphorylation reaction sequence is reported.
Abstract: This paper reports the discovery that the activity of the multienzyme pyruvate dehydrogenase complex from beef kidney mitochondria is regulated by a phosphorylation-dephosphorylation reaction sequence. The site of this regulation is the pyruvate dehydrogenase component of the complex. Phosphorylation and concomitant inactivation of pyruvate dehydrogenase are catalyzed by an ATP-specific kinase (i.e., a pyruvate dehydrogenase kinase), and dephosphorylation and concomitant reactivation are catalyzed by a phosphatase (i.e., a pyruvate dehydrogenase phosphatase). The kinase and the phosphatase appear to be regulatory subunits of the pyruvate dehydrogenase complex.
594 citations
TL;DR: The activity of the multienzyme pyruvate dehydrogenase complexes, isolated from mitochondria of beef kidney, beef heart, and pork liver, is regulated by phosphorylation and dephosphorylation.
Abstract: The activity of the multienzyme pyruvate dehydrogenase complexes, isolated from mitochondria of beef kidney, beef heart, and pork liver, is regulated by phosphorylation and dephosphorylation. Phosphorylation and concomitant inactivation of each of the three complexes are catalyzed by an ATP-specific kinase, and dephosphorylation and concomitant reactivation are catalyzed by a phosphatase. The phosphatase has been separated from the other component enzymes of each pyruvate dehydrogenase complex, and the three phosphatases are functionally interchangeable. The kinase has been isolated from the beef kidney complex, and it is functional with the beef heart and pork liver complexes. ADP is competitive with ATP, and the ADP effect is more pronounced with the kidney kinase than with the liver and heart kinases. Pyruvate protects strongly the heart and liver pruvate dehydrogenase complexes and, to a lesser extent, the kidney complex against inactivation by ATP. Pyruvate apparently exerts its effect on the pyruvate dehydrogenase component of the complex, rather than on the kinase.
312 citations
TL;DR: Oleate increased the state of reduction of the pyridine nucleotide systems in both mitochondrial and cytosolic spaces as shown by increases in the ratios of lactate to pyruvate, β-hydroxybutyrate to acetoacetate, and malate to oxalacetate.
282 citations
TL;DR: Data is reported on the effect of oleate on tissue levels of coenzyme A derivatives, ketone bodies, citric acid cycle intermediates, and adenine nucleotides in rat livers perfused with alanine, lactate, or pyruvate to conclude that citrate accumulation is regulated by the mitochondrial oxalacetate concentration, which itself is controlled by the pyruVate carboxylase activity and the NAD oxidation-reduction potential.
210 citations
TL;DR: It is shown that pyruvate and α-ketobutyrate are oxidized by the same enzyme (pyruvates dehydrogenase), while α- ketovalerate is oxidizedBy a different enzyme (α-ketovalerate), which behaves mainly as a competitive inhibitor to NAD.
Abstract: 1
Studies with intact mitochondria and with soluble pyruvate dehydrogenase indicate that pyruvate and α-ketobutyrate are oxidized by the same enzyme (pyruvate dehydrogenase), while α-ketovalerate is oxidized by a different enzyme.
2
Pyruvate and α-ketobutyrate have about the same affinity for the enzyme, but pyruvate is oxidized at a much higher rate.
3
Acetyl-CoA and propionyl-CoA both behave as competitive inhibitors to CoA. The Ki for both is slightly higher than the Km for CoA. Accordingly the enzyme is only moderately inhibited by a high acetyl-CoA/CoA ratio.
4
NADH behaves mainly as a competitive inhibitor to NAD. The Ki is significantly lower than the Km for NAD. Accordingly the enzyme is strongly inhibited by a high NADH/NAD ratio.
5
The significance of these properties of the enzyme for the regulation of the activity of pyruvate dehydrogenase in vivo is discussed.
180 citations
TL;DR: The rate of pyruvate transfer into mitochondria is of primary importance in the control of mitochondrial CO2 fixation, and the primary role of mitochondrial pyruVate uptake was confirmed.
146 citations
TL;DR: Some highly purified glycolytic enzymes have been subjected to isoelectric focusing and found to contain a number of enzymatically active species and suggest random combinations of two closely related subunits into tetramers and dimers.
Abstract: Some highly purified glycolytic enzymes have been subjected to isoelectric focusing and found to contain a number of enzymatically active species. Crystalline aldolase A and glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle were resolved into five components, crystalline aldolase from yeast was resolved into three components, pyruvate kinase from rabbit muscle yielded four components, and yeast enolase was resolved into two components. Rabbit muscle lactate dehydrogenase (M4) gave one major peak of protein and enzymatic activity. The profiles of aldolase, glyceraldehyde-3-phosphate dehydrogenase, and yeast aldolases suggest random combinations of two closely related subunits into tetramers and dimers, respectively. The molecular heterogeneity of the other enzymes is not so easily related to subunit structure.
113 citations
TL;DR: In this article, the overall activity and distribution of pyruvate carboxylase and PPI carboxykinase in rat liver was studied by applying an improved technique for the fractional extraction of hepatic enzymes.
Abstract: The overall activity and distribution of pyruvate carboxylase and of phosphoenolpyruvate carboxykinase in rat liver was studied by applying an improved technique for the fractional extraction of hepatic enzymes. In the rat the mean activity of pyruvate carboxylase amounted to 10 units/g fresh liver (1 unit = 1 μmole pyruvate carboxylated/min at 30°). In the human liver the corresponding value was about 1 unit/g fresh wt. For phosphoenolpyruvate carboxy-kinase 2.5 and 10 units/g fresh wt. were measured in rat and human liver, respectively. During fractional extraction only negligible quantities of pyruvate carboxylase appeared in the soluble cytoplasmatic fraction. It is concluded that the bulk of enzyme activity is located within the mitochondria. By digitonin treatment of isolated rat liver mitochondria pyruvate carboxylase could be localized within the matrix space. Phosphoenolpyruvate carboxykinase was found to be located mainly in the soluble cytoplasmatic fraction. Our results strongly support the view that in rat liver gluconeogenesis pyruvate has to enter the mitochondria prior to its carboxy-lation to oxaloacetate.
105 citations
97 citations
TL;DR: The ability to dissimilate pyruvate was found to arise only and specifically from an interaction between enzyme I, ferrous ion-activated enzyme II and adenosylmethionine, in presence of either of the above auxiliary systems.
Abstract: Fractionation of the cell extract led to partial isolation of four protein fractions that are involved in the dissimilation of pyruvate into acetyl-CoA and formate: Enzyme I, molecular weight about 140000; enzyme II, molecular weight about 30000, which requires an activation by ferrous ion and dithiols; enzyme III, molecular weight 25000, which is a flavorprotein with flavin-monoucleotide as coenzyme; and a hitherto less characterized fraction IV. The system in addition comprises the cofactors S-adenosylmethionine and thiamine diphosphate.
Enzyme III, fraction IV, and thiamine diphosphate could be replaced by the reagents cobaltous ion and thiols which form complexes of high reducing power. This part of the enzyme system obviously plays an auxiliary role, most likely by providing an appropriate redox potential via pyruvate as electron donor.
The ability to dissimilate pyruvate was found to arise only and specifically from an interaction between enzyme I, ferrous ion-activated enzyme II and adenosylmethionine, in presence of either of the above auxiliary systems. Enzyme I is thereby converted into an active form, possibly by reduction, which alone is responsible for the catalysis of the pyruvate dissimilation reaction. The identification of this pyruvate formate-lyase was accomplished by sedimentation experiments in anaerobic sucrose gradients.
94 citations
TL;DR: 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 aerobic in the dark, on glucose or pyruVate (with CO2).
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.
TL;DR: The results presented in this paper strongly indicate that the pyruvate dehydrogenase complex from pig heart muscle is similarly regulated by ATP-dependent phosphorylation (kinase reaction) and by hydrolytic dephosphorylated (phosphatase Reaction) of the enzyme protein.
TL;DR: Although temporally related to the passage of the embryos from the oviducts into the uterine horns, the changes in enzyme activity do not result from this change in embryonic environment, and specific degradative processes beginning on day 2 of embryonic development are postulated.
Abstract: The activities of four enzymes were determined during the first four days of mouse embryogenesis. Two enzymes, fructose 1,6-diphosphate aldolase and malate dehydrogenase, increase about 30% in activity, and this increase is attributed to slow but continued enzyme synthesis. The other two enzymes, glucose 6-phosphate dehydrogenase (X-linked) and lactate dehydrogenase, remain constant for the first two days and then decline exponentially with half-times of 19 and 17 hr, respectively. These declines in activity cannot be explained by the appearance of soluble inactivators or by the disappearance of soluble activators. Likewise, although temporally related to the passage of the embryos from the oviducts into the uterine horns, the changes in enzyme activity do not result from this change in embryonic environment, and specific degradative processes beginning on day 2 of embryonic development are postulated.
TL;DR: It is suggested that the inhibition of human adult and fetal brain pyruvate kinase by l -phenylalanine may have a role in the metabolic damage in phenylketonuria.
TL;DR: Calcium inhibited 14C-carbon dioxide production from carboxyl-labeled pyruvate by isolated rat liver mitochondria and the possibility that calcium may play an important role in regulating the initial steps of gluconeogenesis is discussed.
TL;DR: Lactic dehydrogenase X, an isozyme found thus far only in mature sperm of mammals and birds, has been purified to homogeneity from rat tissue and exhibits substantial activity for the similar interconversions of α-ketoglutarate and α-hydroxyglutarate, a property not present in other isozymes of lactic dehydration.
TL;DR: Pyruvate carboxylase was partially purified from Aspergillus niger and the properties were studied, and the enzyme was shown to be a biotin-containing enzyme by its in activation by avidin and protection against such inactivation by biotin.
Abstract: Pyruvate carboxylase was partially purified from Aspergillus niger and the properties were studied. The enzyme was found to be cold-labile and protected by 25% glycerol. The pH optimum was determin...
TL;DR: It was found that the pig heart pyruvate dehydrogenase complex is capable of oxidatively decarboxylating α-ketobutyrate at about the rate of 62% of that of pyruVate in the over-all reaction assay and about 79% in the dehydrogen enzyme assay.
TL;DR: Human-mouse somatic cell hybrids have been isolated and examined for enzyme and chromosome constitution and evidence predicated on the absence of chromosomal rearrangements is provided for the lack of genetic linkage in the human genome for these four enzymes.
Abstract: Human-mouse somatic cell hybrids have been isolated and examined for enzyme and chromosome constitution. The enzymes assayed were lactate dehydrogenase (LDH), isocitrate dehydrogenase (IDH), NADP-dependent malate dehydrogenase (MDH), glucose 6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), phosphoglucomutase (PGM), and several esterases. Coexpression of mouse and human genomes and formation of heteropolymeric enzymes were observed in seven different hybrid populations for the enzymes LDH, IDH, MDH, and G6PD. Evidence predicated on the absence of chromosomal rearrangements is provided for the lack of genetic linkage in the human genome for these four enzymes, as well as for thymidine kinase.
TL;DR: Acyl-CoA inhibition of the pyruvate and α-oxoglutarate dehydrogenase complexes was studied in order to gain some insight into possible factors which might regulate these two enzymes.
TL;DR: The simplest and most rapid check on the purity of pyruvate or of α-ketoglutarate is to assay using lactate debydrogcnase or glutamate dehydrogenase, based on relatively common procedures.
Abstract: Publisher Summary The chapter discusses the purity and stability of pyruvate, of radioactive pyruvate. Pyruvate forms a dimer, γ-methyl-γ,-hydroxy-α-ketoglutarate, or parapyruvate, as one of the products. This dimer is an inhibitor of α-ketoglutarate dehydrogenase and interrupts Krebs cycle oxidations. Pyruvate oxidation by rat heart mitochondria in the presence of parapyruvate is blocked, unless a dicarboxylic acid is present in which case α-ketoglutarate accumulates. Pyruvate oxidation by rabbit heart mitochondria in the presence of parapyruvate yields acetate as the major oxidation product even when dicarboxylic acid is present. The simplest and most rapid check on the purity of pyruvate or of α-ketoglutarate is to assay using lactate debydrogcnase or glutamate dehydrogenase. These assays, when possible, should be compared with calculated values based on dry weight or neutralization equivalent. The assays may be conducted in a matter of minutes and are based on relatively common procedures.
TL;DR: The reconstitution of active pyruvate Carboxylase shows a virtually complete dependence on acetyl-coenzyme A and is inhibited by L-aspartate, which may represent a novel mode of regulating the formation of an active enzyme.
Abstract: Pyruvate carboxylase is formed by enzyme-catalysed attachment of biotin to an inactive protein precursor. Acetyl-CoA and aspartate allosterically influence this reaction.
TL;DR: Samples of pyruvate are prepared which give no excess substrate inhibition and suggestive evidence for the chemical nature of the inhibitor present in the specimens of pyRuvate normally used for investigations on lactate dehydrogenase is obtained.
TL;DR: This type of flavin is mostly, if not exclusively, connected with succinate dehydrogenase flavoprotein from aerobic cells (“SD-flavin”) and differs from acid-extractable flavin of the same tissue by the following main characteristics: pH-dependence of the fluorescence, optical spectra and alkaline photolysis.
TL;DR: The results suggest that the interactions between L-phenylalanine and L-alanine are important to the regulation of pyruvate kinase activity in tissues containing the M-type isozyme.
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...
TL;DR: It appears that pyruvate apocarboxylase and the holoenzyme synthetase were present in the deficient liver and once biotin was available, invivo synthesis of pyruVate holocar boxylase occurred.