Showing papers on "Pyruvate dehydrogenase kinase published in 1991"
TL;DR: Characterization of all combinations of PDC structural gene deletion mutants, which produce different amounts of pyruvate decarboxylase activity, showed that the enzyme is also needed for normal growth in galactose and ethanol medium and in particular for proper growth initiation of spores germinating on ethanol medium.
Abstract: Pyruvate decarboxylase is the key enzyme in alcoholic fermentation in yeast. Two structural genes, PDC1 and PDC5 have been characterized. Deletion of either of these genes has little or no effect on the specific pyruvate decarboxylase activity, but enzyme activity is undetectable in mutants lacking both PDC1 and PDC5 (S. Hohmann and H. Cederberg, Eur. J. Biochem. 188:615-621, 1990). Here I describe PDC6, a gene structurally closely related to PDC1 and PDC5. The product of PDC6 does not seem to be required for wild-type pyruvate decarboxylase activity in glucose medium; delta pdc6 mutants have no reduced specific enzyme activity, and the PDC6 deletion did not change the phenotype or the specific enzyme activity of mutants lacking either or both of the other two structural genes. However, in cells grown in ethanol medium the PDC6 deletion caused a reduction of pyruvate decarboxylase activity. Northern (RNA) blot analysis showed that PDC6 is weakly expressed, and expression seemed to be higher during growth in ethanol medium. This behavior remained obscure since pyruvate decarboxylase catalyzes an irreversible reaction. Characterization of all combinations of PDC structural gene deletion mutants, which produce different amounts of pyruvate decarboxylase activity, showed that the enzyme is also needed for normal growth in galactose and ethanol medium and in particular for proper growth initiation of spores germinating on ethanol medium.
186 citations
TL;DR: The gene encoding D-lactate dehydrogenase of Lactobacillus plantarum has been sequenced, and expressed in Escherichia coli cells with an inducible expression plasmid, in which the 5'-noncoding region of the gene was replaced with the tac promoter, suggesting that the new family consists of D-isomer-stereospecific enzymes.
170 citations
TL;DR: During Pi limitation, PEP carboxylase and PEP phosphatase may function to "bypass" the ADP dependent pyruvate kinase, as well as to recycle Pi for its reassimilation into cellular metabolism.
Abstract: The effects of phosphorus nutrition on several physiological and biochemical parameters of the green alga, Selenastrum minutum, have been examined. Algal cells were cultured in chemostats under conditions of either Pi limitation or nutrient sufficiency. Pi limitation resulted in: (a) a 5-fold lower rate of respiration, (b) a 3-fold decline in rates of photosynthetic carbon dioxide fixation and oxygen evolution, (c) a 3-fold higher rate of dark carbon dioxide fixation, (d) significant increases in activities of phosphoenolpyruvate (PEP) carboxylase and PEP phosphatase (128% and 158% of nutrient sufficient activities, respectively), (e) significant reductions in activities of nonphosphorylating NADP-glyceraldehyde-3-phosphate dehydrogenase and NAD malic enzyme, and (f) no change in levels of ATP:fructose-6-phosphate 1-phosphotransferase, phosphorylating NAD-glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and pyruvate kinase. The intracellular concentrations of Pi, ATP, AMP, soluble protein, and chlorophyll were also significantly reduced in response to Pi limitation. As well, the level of ADP was about 11-fold lower in the Pi-limited cells as compared to the nutrient sufficient controls. It was predicted that because of this low level of ADP, pyruvate kinase catalyzed conversion of PEP to pyruvate may be restricted in Pi-limited cells. During Pi limitation, PEP carboxylase and PEP phosphatase may function to “bypass” the ADP dependent pyruvate kinase, as well as to recycle Pi for its reassimilation into cellular metabolism.
151 citations
TL;DR: Information on the content of both lactate and its redox partner pyruvate is likely to provide more information on the metabolic state of tissue than are measurements of lactate alone.
Abstract: Lactate contents of tissues can vary during normal physiological conditions in mammals from 0.5 to over 5 mM, and higher in pathological states, suitable for measurement by NMR. Lactate itself is a dead-end metabolite, metabolized only by lactate dehydrogenase. The extent and direction of that reaction is determined by the free [NAD+]/[NADH][H+] ratio of cytoplasm with which lactate and pyruvate are in near-equilibrium. Pyruvate is a crossroads of most of the major degradative and synthetic pathways, but present in about one-tenth the amount of lactate. Information on the content of both lactate and its redox partner pyruvate is likely to provide more information on the metabolic state of tissue than are measurements of lactate alone.
83 citations
TL;DR: Modification of the PDX1 gene encoding the protein X component of the mitochondrial pyruvate dehydrogenase (PDH) complex in Saccharomyces cerevisiae did not affect viability of the cells, and extracts of mitochondria from the mutant, in contrast to extracts of wild-type mitochondria, did not catalyze a CoA- and NAD(+)-linked oxidation of pyruVate.
Abstract: Disruption of the PDX1 gene encoding the protein X component of the mitochondrial pyruvate dehydrogenase (PDH) complex in Saccharomyces cerevisiae did not affect viability of the cells. However, extracts of mitochondria from the mutant, in contrast to extracts of wild-type mitochondria, did not catalyze a CoA- and NAD(+)-linked oxidation of pyruvate. The PDH complex isolated from the mutant cells contained pyruvate dehydrogenase (E1 alpha + E1 beta) and dihydrolipoamide acetyltransferase (E2) but lacked protein X and dihydrolipoamide dehydrogenase (E3). Mutant cells transformed with the gene for protein X on a unit-copy plasmid produced a PDH complex that contained protein X and E3, as well as E1 alpha, E1 beta, and E2, and exhibited overall activity similar to that of the wild-type PDH complex. These observations indicate that protein X is not involved in assembly of the E2 core nor is it an integral part of the E2 core. Rather, protein X apparently plays a structural role in the PDH complex; i.e., it binds and positions E3 to the E2 core, and this specific binding is essential for a functional PDH complex. Additional evidence for this conclusion was obtained with deletion mutations. Deletion of most of the lipoyl domain (residues 6-80) of protein X had little effect on the overall activity of the PDH complex. This observation indicates that the lipoyl domain, and its covalently bound lipoyl moiety, is not essential for protein X function. However, deletion of the putative subunit binding domain (residues approximately 144-180) of protein X resulted in loss of high-affinity binding of E3 and concomitant loss of overall activity of the PDH complex.(ABSTRACT TRUNCATED AT 250 WORDS)
76 citations
TL;DR: Observations provide evidence that the C-2 carbanion form of 3-phosphoglycerate is an intermediate in the catalytic sequence of ribulose-bisphosphate carboxylase.
72 citations
TL;DR: Results of metabolic, enzymic, and molecular biologic experiments were each consistent with an intramitochondrial site(s) of down-regulation in islets cultured at low glucose, indicating that regulation by glucose was at transcription of genes coding for some mitochondrial enzymes.
68 citations
Journal Article•
TL;DR: These studies demonstrate that orderly glycolysis in the erythrocyte is regulated by the NAD-to-NADH ratio and also provide a method that makes possible the in vitro study of ery Throatcyte glyCOlysis.
62 citations
TL;DR: Findings show that the insulin receptor tyrosine kinase does not play an obligatory role in the insulin signaling pathway that stimulates pyruvate dehydrogenase activity.
60 citations
TL;DR: It is suggested that sepsis, but not sterile inflammation, induces a stable factor in skeletal muscle mitochondria that increased PDH kinase activity.
Abstract: The effect of sterile inflammation and sepsis on the proportion of active pyruvate dehydrogenase complex (PDH) in mitochondria isolated from skeletal muscle has been investigated. The proportion of active PDH in mitochondria isolated from septic animals was significantly reduced compared with control under all incubation conditions examined, even in the presence of inhibitors of the PDH kinase. There was no significant difference between control and sterile inflammation in any of the incubations examined. The rate constant for ATP-dependent inactivation of the PDH complex in mitochondrial extracts from control animals was -0.42 min-1 (r = 0.993; P less than 0.001) and was not altered in mitochondrial extracts from sterile inflammatory animals (-0.43 min-1; r = 0.999; P less than 0.001). However, rate constants for inactivation in septic animals was significantly increased over twofold to -1.08 min-1 (r = 0.987; P less than 0.001) (P less than 0.001 vs. control or sterile inflammation). In the presence of inhibitors of the PDH kinase reaction (2.5 mM pyruvate or 1 mM dichloroacetate), inactivation of PDH after addition of ATP was significantly greater in mitochondrial extracts from septic than either control or sterile inflammatory animals. These results suggest that sepsis, but not sterile inflammation, induces a stable factor in skeletal muscle mitochondria that increased PDH kinase activity.
57 citations
TL;DR: Applying the summation theorem of the control analysis, it was found that at low Ca2+ and pyruvate concentrations the dehydrogenases shared the control of state 3 respiration with other steps, and the NAD-linked dehydrogenase did not exert any significant control at high Ca2+.
TL;DR: The alpha subunit of the rat heart PDH kinase could be cleaved selectively by chymotrypsin with concomitant loss of kinase activity, as previously shown for the bovine kidney enzyme, suggesting that the catalytic activity of PDH Kinase resides in thealpha subunit.
TL;DR: The mechanism of metronidazole resistance in B. fragilis NCTC 11295 may be due to the high activity of lactate dehydrogenase which compensates for the decreased activity of pyruvate: ferredoxin oxidoreductase in the presence of metronsidazoles.
Abstract: Enzymes acting on pyruvate as a parameter of the ATP regeneration system were studied as a cause of metronidazole resistance in Bacteroides fragilis NCTC 11295. The resistant strain had higher lactate dehydrogenase activity and produced more lactate than susceptible strains, suggesting that the enzyme is more active in lactic acid fermentation. Furthermore, the reaction catalysed by lactate dehydrogenase occurred up to 48 mg/L metronidazole, whereas the reaction catalysed by pyruvate: ferredoxin oxidoreductase reaction stopped at 2 mg/L. The mechanism of metronidazole resistance in B. fragilis NCTC 11295 may be due to the high activity of lactate dehydrogenase which compensates for the decreased activity of pyruvate: ferredoxin oxidoreductase in the presence of metronidazole.
TL;DR: An analogue of cAMP, 8-(4-chlorophenylthio)-cAMP, inhibited the production of L-type pyruvate kinase and glucokinase mRNAs in the presence of glucose plus vanadate.
Abstract: In primary culture of adult rat hepatocytes, vanadate in the presence of glucose stimulates the expression of the liver (L-type) pyruvate kinase gene. Glucose by itself was inactive, and vanadate, like insulin, was also inefficient in the absence of glucose. Similar results were obtained on glucokinase gene expression. An analogue of cAMP, 8-(4-chlorophenylthio)-cAMP, inhibited the production of L-type pyruvate kinase and glucokinase mRNAs in the presence of glucose plus vanadate.
TL;DR: The yeast monocarboxylate (pyruvate) carrier appeared to be less abundant, but more active, than the analogous protein from higher eukaryotes, and was able to catalyse the pyruVate / pyru vate and pyruvates / acetoacetate exchange reactions.
TL;DR: The analysis of three patients with pyruvate dehydrogenase E1α deficiency is reported, one female has a three base pair deletion which affects dephosphorylation of the subunit, and two males analysed, one has a two base pair delete causing a shift in the reading frame.
Abstract: The human pyruvate dehydrogenase complex catalyses the oxidative decarboxylation of pyruvate to acetyl-CoA Defects in several of the seven subunits have been reported, but the majority of mutations affect the E1 component and especially the E1α subunit However, the clinical presentation of patients with pyruvate dehydrogenase E1α deficiency is extremely variable Dependency of the brain on pyruvate dehydrogenase activity and localization of the gene for the somatic form of the pyruvate dehydrogenase E1α subunit to the X chromosome provide the basis for a better understanding of the variation in the clinical manifestations Further understanding of the function and interaction of subunits and the pathophysiology of pyruvate dehydrogenase deficiency necessitates the characterization of mutations in the pyruvate dehydrogenase complex We report the analysis of three patients with pyruvate dehydrogenase E1α deficiency One female has a three base pair deletion which affects dephosphorylation of the subunit Of two males analysed, one has a two base pair deletion causing a shift in the reading frame The other has a base change, resulting in an Arg to His substitution All three mutations are located near the carboxyl terminus of the subunit
TL;DR: It appears that intact protein X and specifically the presence of its cleaved lipoyl domain is not essential for maintenance of an enzymically active pyruvate dehydrogenase complex, however, this protein has an important structural role in promoting the correct association of E3 with the E2 core assembly, an interaction that is required for optimal catalytic efficiency of the complex.
Abstract: Selective proteolysis of the protein X subunit of native bovine heart pyruvate dehydrogenase complex may be accomplished without loss of overall complex activity. Partial loss of function occurs if Mg2+ and thiamin pyrophosphate are not present during proteinase arg C treatment as these cofactors are necessary to prevent cleavage of the E1 alpha subunit. Specific degradation of component X leads to marked alterations in the general enzymic properties of the complex. Lipoamide dehydrogenase (E3) exhibits a decreased affinity for the core assembly and the complex is much more susceptible to inactivation at high ionic strength. The inactive form of the complex is not readily re-activated by removal of salt. It appears that intact protein X and specifically the presence of its cleaved lipoyl domain is not essential for maintenance of an enzymically active pyruvate dehydrogenase complex. However, this protein has an important structural role in promoting the correct association of E3 with the E2 core assembly, an interaction that is required for optimal catalytic efficiency of the complex.
TL;DR: It is suggested that the α and β subunits of the pyruvate and 2-oxoisovalerate dehydrogenases have been derived by the cleavage of the α-ketoglutarate dehydrogenase.
TL;DR: The 11.5-kDa Zn(2+)-binding protein (ZnBP) was covalently linked to Sepharose and a possible function of ZnBP in supramolecular organization of carbohydrate metabolism is proposed.
TL;DR: The ethanol route has significant advantages (minimal acidosis, avoidance of osmotic problems, and endproduct inhibition of the glycolytic chain) but also has disadvantages (relatively low energy yield, loss of carbobydrate carbon).
Abstract: 1. Ethanol is an end product of anaerobic metabolism in a surprisingly large variety of multicellular organisms. These include Angiosperms, Platyhelminthes, Aschelminthes, Acanthocephala, Arthropoda, and Vertebrata. 2. Ethanol formation proceeds in two steps (via pyruvate decarboxylase and alcohol dehydrogenase). 3. Pyruvate decarboxylase of plants is located in the cytosol, whereas that of animals is intramitochondrial and probably part of the pyruvate dehydrogenase complex. 4. Alcohol dehydrogenases of facultative anaerobes are NAD-rather than NADP-dependent, with the enzyme in the parasitic worm Moliniformis dubius as the only known exception. 5. Regulation at the pyruvate branch point in plants seems to involve three different mechanisms: (i) increases of pyruvate decarboxylase and alcohol dehydrogenase activity due to increased gene expression, (ii) control of pyruvate decarboxylase by the intracellular pH and the cytoplasmic NADH/NAD⁺ ratio, and (iii) inhibition of lactate dehydrogenase by ATP at lo...
TL;DR: The observed increase in hepatic production of [3-13C]-aspartate in tumor rats indicates that pyruvate carboxylase activity is significantly enhanced.
TL;DR: In situ activity of the mitochondrial pyruvate dehydrogenase complex can be controlled in part by acetyl-CoA product inhibition, which is indicated by the inhibition by malonate and the apparent lack of coupling.
Abstract: In vitro, the pyruvate dehydrogenase complex is sensitive to product inhibition by NADH and acetyl-coenzyme A (CoA). Based upon Km and Ki relationships, it was suggested that NADH can play a primary role in control of pyruvate dehydrogenase complex activity in vivo (JA Miernyk, DD Randall [1987] Plant Physiol 83:306-310). We have now extended the in vitro studies of product inhibition by assaying pyruvate dehydrogenase complex activity in situ, using purified intact mitochondria from green pea (Pisum sativum) seedlings. In situ activity of the pyruvate dehydrogenase complex is inhibited when mitochondria are incubated with malonate. In some instances, isolated mitochondria show an apparent lack of coupling during pyruvate oxidation. The inhibition by malonate, and the apparent lack of coupling, can both be explained by an accumulation of acetyl-CoA. Inhibition could be alleviated by addition of oxalacetate, high levels of malate, or l-carnitine. The CoA pool in nonrespiring mitochondria was approximately 150 micromolar, but doubled during pyruvate oxidation, when 60 to 95% of the total was in the form of acetyl-CoA. Our results indicate that in situ activity of the mitochondrial pyruvate dehydrogenase complex can be controlled in part by acetyl-CoA product inhibition.
TL;DR: The kinase-activator protein (KAP) of pyruvate dehydrogenase complex (PDC) has been purified and it is concluded that KAP is not a distinct protein, but is kinase which has dissociated from the complex.
Abstract: The kinase-activator protein (KAP) of pyruvate dehydrogenase complex (PDC) has been purified approx. 2250-fold from high-speed supernatants of mitochondrial extracts from the liver of 48 h-starved rats. Purified KAP demonstrates kinase activity towards both the E1 component of PDC and towards a synthetic peptide corresponding to the major phosphorylation site on E1. Furthermore, the activities of KAP and PDC kinase co-fractionate through several stages of purification and have the same apparent mass. We conclude that KAP is not a distinct protein, but is kinase which has dissociated from the complex.
TL;DR: It is concluded that increased duration of exercise can lead to reduced PDH complex activity in rat muscles and it is proposed that increased glycolysis to lactate and increased fatty acid oxidation can simultaneously provide energy for contracting muscle.
Abstract: Muscle glucose uptake is greatly stimulated by moderate exercise, but full oxidation of the glucose to CO2 depends on the activity of the pyruvate dehydrogenase (PDH) complex. Our aim was to determine how PDH complex in different muscle groups responds to varying periods of moderate exercise. Rats were run on a motor-driven treadmill for 5-30 min and muscle PDH complex activity was determined in heart, diaphragm and red quadriceps muscles after isolation of mitochondria in the presence of inhibitors of PDH complex interconversion. In heart and diaphragm muscle, exercise caused an increase in PDH complex activity after 5 min, but this was followed by a significant decrease in activity as exercise progressed. In red quadriceps muscle, PDH complex activity was reduced after 5 min of exercise and was decreased further as exercise continued. We conclude that increased duration of exercise can lead to reduced PDH complex activity in rat muscles. We propose that this is a consequence of elevated fatty acid oxidation, the products of which stimulate PDH kinase. This implies that increased glycolysis to lactate and increased fatty acid oxidation can simultaneously provide energy for contracting muscle.
TL;DR: It is shown that rat liver pyruvate dehydrogenase (PDH) kinase activator protein (KAP) catalyses ATP-dependent inactivation and [32P]phosphorylation of pig heart PDHE1 and of yeast PDH complex devoid of PDH kinase activity.
Abstract: It is shown here that rat liver pyruvate dehydrogenase (PDH) kinase activator protein (KAP) catalyses ATP-dependent inactivation and [32P]phosphorylation of pig heart PDHE1 and of yeast (Saccharomyces cerevisiae) PDH complex devoid of PDH kinase activity, that fluorosulphonylbenzoyladenosine inactivates rat liver KAP and the intrinsic PDH kinase of rat liver PDH complex, and that KAP, like PDH kinase, is inactivated by thiol-reactive reagents. It is concluded that KAP is a free PDH kinase.
TL;DR: The nucleotide sequence of the last gene of the S. aureus pyruvate dehydrogenase operon, pdhD, is reported, which encodes lipoamide dehydrogen enzyme (LPD), which is closely related to other lipoamia dehydrogenases from bacteria and eukaryotes.
TL;DR: Differences in total protein banding patterns from cells grown at pH extremes suggested that synthesis of pyruvate decarboxylase and other enzymes was in part responsible for metabolic regulation of the fermentation products formed.
Abstract: The physiology and biochemistry of Sarcina ventriculi was studied in order to determine adaptations made by the organism to changes in environmental pH. The organism altered carbon and electron flow from acetate, formate and ethanol production at neutral pH, to predominantly ethanol production at pH 3.0. Increased levels of pyruvate dehydrogenase (relative to pyruvate decarboxylase) and acetaldehyde dehydrogenase occurred when the organism was grown at neutral pH, indicating the predominance of carbon flux through the oxidative branch of the pathway for pyruvate metabolism. When the organism was grown at acid pH, there was a significant increase in pyruvate decarboxylase levels and a decrease in acetaldehyde dehydrogenase, causing flux through the non-oxidative branch of the pathway. CO2 reductase and formate dehydrogenase were not regulated as a function of growth pH. Pyruvate dehydrogenase possessed Michaelis-Menten kinetics for pyruvate with an apparent Km of 2.5 mM, whereas pyruvate decarboxylase exhibited sigmoidal kinetics, with a S0.5 of 12.0 mM. Differences in total protein banding patterns from cells grown at pH extremes suggested that synthesis of pyruvate decarboxylase and other enzymes was in part responsible for metabolic regulation of the fermentation products formed.
TL;DR: It is concluded that the structural requirements for the substrate and coenzyme of ϵ-crystallin are similar to those of other dehydrogenases and that the carboxamide carbonyl group of the nicotinamide moiety is important for the coen enzyme activity.
TL;DR: The results strongly support the fractionation of human erythrocytes according to cell age, as occurs with rat ery Throttle, as well as some correlations between enzyme activities.
Abstract: Human and rat erythrocytes were fractionated by counter-current distribution in charge-sensitive dextran/poly(ethylene glycol) two-phase systems. The specific activities of the key glycolytic enzymes (hexokinase, phosphofructokinase and pyruvate kinase) declined along the distribution profiles, although the relative positions of the activity profiles were reversed in the two species. These enzymes maintained their normal response to specific regulatory effectors in all cell fractions. No variations were observed for phosphoglycerate kinase and bisphosphoglycerate mutase activities. Some correlations between enzyme activities (pyruvate kinase/hexokinase, pyruvate kinase/phosphofructokinase, pyruvate kinase/pyruvate kinase plus phosphoglycerate kinase, pyruvate kinase/bisphosphoglycerate mutase and phosphoglycerate kinase/bisphosphoglycerate mutase ratios) were studied in whole erythrocyte populations as well as in cell fractions. These results strongly support the fractionation of human erythrocytes according to cell age, as occurs with rat erythrocytes.
TL;DR: It is proposed that pyruvate carboxylase activity accounts for the generation of 14C-labelled amino acids other than alanine in islets exposed to D-[3,4-14C]glucose and participates to the pyruVate/citrate shuttle for the transport of acetyl-CoA out of the mitochondria in nutrient-stimulated islets.