Showing papers on "Pyruvate dehydrogenase kinase published in 1980"
TL;DR: Evidence that Ca*+ enhances the activity of three key intramitochondrial dehydrogenases and that [Ca”] at 0.1-10 PM is potentially an important regulator of oxidative metabolism within mammalian mitochondria are summarised.
333 citations
TL;DR: Observations tend to support a recent suggestion that there may be a particular form of pyruvate dehydrogenase associated with cholinergic neurones, as shown by the regional distribution of choline acetyltransferase.
235 citations
TL;DR: The greatly improved yield of 2-oxo acid dehydrogenase complexes occasioned by the use of Triton X-100 or Tween-80 as solubilizing agent supports the possibility that the bulk of the pyruvate dehydration complex is associated in some way with the mitochondrial inner membrane and is not free in the mitochondrial matrix space.
Abstract: A new method is described that allows the parallel purification of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes from ox heart without the need for prior isolation of mitochondria. All the assayable activity of the 2-oxo acid dehydrogenase complexes in the disrupted tissue is made soluble by the inclusion of non-ionic detergents such as Triton X-100 or Tween-80 in the buffer used for the initial extraction of the enzyme complexes. The yields of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes are many times greater than those obtained by means of previous methods. In terms of specific catalytic activity, banding pattern on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, sedimentation properties and possession of the regulatory phosphokinase bound to the pyruvate dehydrogenase complex, the 2-oxo acid dehydrogenase complexes prepared by the new method closely resemble those described by previous workers. The greatly improved yield of 2-oxo acid dehydrogenase complexes occasioned by the use of Triton X-100 or Tween-80 as solubilizing agent supports the possibility that the bulk of the pyruvate dehydrogenase complex is associated in some way with the mitochondrial inner membrane and is not free in the mitochondrial matrix space.
191 citations
01 Jan 1980
TL;DR: The chapter describes the control of the tricarboxylate cycle and of pyruvate dehydrogenase in their mitochondrial context and describes the final arbiter of their activity is the respiratory chain.
Abstract: Publisher Summary This chapter discusses the control of oxidative metabolism by adenosine diphosphate (ADP) availability in isolated mitochondria and perfused organs. It focuses on the control of dehydrogenase activity. The enzymes, such as, the pyruvate dehydrogenase complex and some of the enzymes of the tricarboxylate cycle have been considered. The most important characteristics of these enzymes are that (1) they catalyze non-equilibrium reactions; (2) exert a dominant influence over the flux through the pathways that are central to catabolic metabolism; and (3) they are mitochondrial. This localization means that the supply of substrate, and dissipation of product, might be limited by the permeability properties of the inner mitochondrial membrane. Because these enzymes are dehydrogenases, the final arbiter of their activity is the respiratory chain, with its coupling to the phosphorylation of ADP. The chapter describes the control of the tricarboxylate cycle and of pyruvate dehydrogenase in their mitochondrial context
182 citations
TL;DR: The results suggest the occurrence of glycolysis, energy production by substrate level phosphorylation and a flavin, iron-sulfur protein mediated electron transport system as well as the absence of cytochrome mediated oxidative phosphorylated and functional Krebs cycle.
171 citations
TL;DR: The results suggest that a mechanism common to all three agents is responsible for transmitting the stimulation from the plasma membrane to the mitochondrial components of the mixture, which required plasma membranes and could not be produced by treatment of mitochondria alone.
Abstract: The addition of insulin to a mixture of plasma membrane and mitochondrial fractions from rat adipocytes results in a decrease in the phosphorylation of a mitochondrial protein identified as the α subunit of pyruvate dehydrogenase [pyruvate:lipoamide oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1] (Seals, J. R., McDonald, J. M. & Jarett, L. (1979) J. Biol. Chem. 254, 6991-6996). This study confirms the prediction that a corresponding increase in pyruvate dehydrogenase activity can be effected by insulin treatment of this preparation. Incubation of the plasma membrane/mitochondria mixture with ATP inhibited pyruvate dehydrogenase activity as measured in a subsequent enzyme assay. The presence of insulin during this incubation with ATP resulted in a 24.5% stimulation of enzyme activity compared to incubation without insulin (n = 9, P < 0.001). The effect was specific for biologically active insulin and was insulin dose-dependent in the physiological range of insulin. Supermaximal doses of insulin produced reduced effects. An insulin effect of similar magnitude could also be observed when the plasma membrane/mitochondria mixture was incubated without ATP. Two insulin mimickers, concanavalin A and antibody to insulin receptor, stimulated pyruvate dehydrogenase by 30.4% (n = 6, P <0.001) and 28.1% (n = 8, P<0.001), respectively. Both of these agents also produced reduced effects at supermaximal concentrations. The effects of all three agents required plasma membranes and could not be produced by treatment of mitochondria alone. The results suggest that a mechanism common to all three agents is responsible for transmitting the stimulation from the plasma membrane to the mitochondrial components of the mixture.
165 citations
TL;DR: Experiments on brown-adipose-tissue mitochondria incubated in the presence of albumin suggest that it may be possible to use the sensitivity of the dehydrogenases to Ca2- as a means of assessing the distribution of Ca2+ across the mitochondrial inner membrane.
Abstract: 1. Increasing concentrations of both Ca2+ and Sr2+ (generated by using EGTA buffers) resulted in 4-fold increases in the initial activity of pyruvate dehydrogenase within intact uncoupled mitochondria from rat epididymal adipose tissue incubated in the presence of the ionophore A23187, ATP, Mg2+ and oligomycin. The k0.5 values (concentrations required for half-maximal effects) for Ca2+ and Sr2+ were 0.54 and 7.1 microM respectively. In extracts of the mitochondria, pyruvate dehydrogenase phosphate phosphatase activity was stimulated about 4-fold by Ca2+ and Sr2+, with k0.5 values of 1.08 and 6.4 microM respectively. 2. NAD+-isocitrate dehydrogenase and oxoglutarate dehydrogenase appeared to be rate-limiting in the oxidation of threo-Ds-isocitrate and oxoglutarate by uncoupled mitochondria from brown adipose tissue of cold-adapted rats. Ca2+ (and Sr2+) diminished the Km for the oxidation of both threo-Ds-isocitrate and oxoglutarate. The kinetic constants for these oxidations were very similar to those obtained for the activities of NAD+-isocitrate dehydrogenase and oxoglutarate dehydrogenase in extracts of the mitochondria. In particular, the k0.5 values for Ca2+ were all in the range 0.2--1.6 microM and Sr2+ was found to mimic Ca2+, but with k0.5 values about 10 times greater. 3. Overall, the results of this study demonstrate that the activities of pyruvate dehydrogenase, NAD+-isocitrate dehydrogenase and oxoglutarate dehydrogenase may all be increased by Ca2+ and Sr2+ within intact mitochondria. In all cases the k0.5 values are close to 1 and 10 microM respectively, as found for the separated enzymes. Experiments on brown-adipose-tissue mitochondria incubated in the presence of albumin suggest that it may be possible to use the sensitivity of the dehydrogenases to Ca2+ as a means of assessing the distribution of Ca2+ across the mitochondrial inner membrane.
134 citations
TL;DR: It is concluded that pyruvate carboxylase is an important anaplerotic enzyme in skeletal muscle mitochondria and in relation to the regulation of the level of citric acid cycle intermediates in muscle tissues.
Abstract: Oxygen uptake in skeletal muscle mitochondria respiring on pyruvate or on acetylcarnitine plus propionylcarnitine is stimulated 3--4-fold by bicarbonate. The stimulation is highly dependent on ATP. The respiration rate obtained amounts to 1/4-1/3 of the rate obtained with pyruvate-malate in the presence of ADP. With decreasing ATP/ADP ratios in the medium, a decreasing stimulation by bicarbonate is obtained. Similar results were obtained with heart mitochondria. With ATP added, a pyruvate-dependent build up of citric acid cycle intermediates takes place in incubations with skeletal muscle mitochondria amounting to about 0.5 nmol x min-1 x mg protein-1. In 14CO2-fixation experiments, the activity of pyruvate carboxylase (EC 6.4.2.1) amounts to about 3 nmol x min-1 x mg protein-1 under similar conditions. With propionylcarnitine plus acetylcarnitine a similar stimulation of respiration and fixation of bicarbonate is observed. In this case the respiration and the propionyl-CoA carboxylase (EC 6.4.1.3) is less inhibited by ADP. The results are discussed in relation to the regulation of the level of citric acid cycle intermediates in muscle tissues. It is concluded that pyruvate carboxylase is an important anaplerotic enzyme in skeletal muscle mitochondria.
96 citations
87 citations
TL;DR: The results suggest that allosteric effectors, in addition to regulating pyruvate kinase activity directly, may also act indirectly by modulating the phosphorylation state of the enzyme.
86 citations
TL;DR: In this article, a single active fraction was found to stimulate mitochondrial pyruvate dehydrogenase by activating the phosphatase and not by altering the kinase activity, and the involvement of the kinases in this activation mechanism was eliminated by the fact that, in the presence of ATP, NaF completely blocked the stimu ation of the active fraction, and dichloroacetic acid, akinase inhibitor, was add itive to the stimulation caused by the active fractions.
Abstract: Insulin treatment of adipocytes increased the amount or activity of a low molecular weight, acid-stable material which, when isolated from intact adipocytes by heat extraction and subsequent Sephadex G25 chromatography, yielded a single active fraction that stimulated mitochondrial pyruvate dehydrogenase by activating the phosphatase and not by altering the kinase activity. Phosphatase activation was demonstrated by the ability of the active material to increase pyruvate dehydrogenase activity in the absence of ATP and by the ability of NaF, a phosphatase inhibitor, to block this stimulation. Involvement of the kinase in this activation mechanism was eliminated by the fact that, in the presence of ATP, (1) NaF completely blocked the stimu ation of pyruvate dehydrogenase by the active fraction, and (2) the stimulation of pyruvate dehydrogenase by dichloroacetic acid, akinase inhibitor, was add itive to the stimulation caused by the active fraction. This active fraction may contain an intracellular chemical mediator or second messenger for insulin.
TL;DR: It is proposed that band F 2 is the α-subunit of the brain pyruvate dehydrogenase complex, which has similar regulatory properties, subcellular location and electrophoretic mobility, and is suggested to have a potential regulatory role in brain function.
TL;DR: Fructose-1,6-bisphosphatase from rat liver was phosphorylated with cyclic AMP-dependent protein kinase and [gamma-32P]ATP and the partial sequence of the 17-amino acid peptide was found to be Ser-Arg- pro-Ser(P)-Leu-Pro-Lei-Pro-(Ser2, Glx2, Pro2, Leu, Arg2).
TL;DR: Kinetic data suggest that lipid activators of pyruvate oxidase increase the affinity of the enzyme for pyruVate and its analogues, as well as the binding of methyl acetylphosphonate, results in ligand-induced changes in the near ultraviolet circular dichorism spectrum of the oxidase.
TL;DR: It is suggested that the branched chain 2-oxo acid dehydrogenase undergoes a stable modification that requires the splitting of the ATP gamma-phosphate group and may be physiologically regulated via a covalent mechanism.
Abstract: The branched chain 2-oxo acid dehydrogenase from rat skeletal muscle, heart, kidney and liver mitochondria can undergo a reversible activation-inactivation cycle in vitro. Similar results were obtained with the enzyme from kidney mitochondria of pig and cow. The dehydrogenase is markedly inhibited by ATP and the inhibition is not reversed by removing the nucleotide. The non-metabolizable ATP analogue adenosine 5'-[beta gamma-imido] triphosphate can block the effect of ATP when added with the nucleotide, but has no effect by itself, nor can it reverse the inhibition in mitochondria preincubated with ATP. These findings suggest that the branched chain 2-oxo acid dehydrogenase undergoes a stable modification that requires the splitting of the ATP gamma-phosphate group. In skeletal muscle mitochondria the rate of inhibition by ATP is decreased by oxo acid substrates and enhanced by NADH. The dehydrogenase can be reactivated 10-20 fold by incubation at pH 7.8 in a buffer containing Mg2+ and cofactors. Reactivation is blocked by NaF (25 mM). The initial activity of dehydrogenase extracted from various tissues of fed rats varies considerably. Activity is near maximal in kidney and liver whereas the dehydrogenase in heart and skeletal muscle is almost completely inactivated. These studies emphasize that comparisons of branched chain 2-oxo acid dehydrogenase activity under various physiological conditions or in different tissues must take into account its state of activation. Thus the possibility exists that the branched chain 2-oxo acid dehydrogenase may be physiologically regulated via a covalent mechanism.
TL;DR: Results showed that livers from diabetic rats had approximately the same amount of immunoprecipitable enzyme protein as the control animals, but a larger proportion of the enzyme was in its inactive state, and the rates of synthesis and degradation of pyruvate dehydrogenase were not affected significantly by diabetes.
Abstract: Immunochemical techniques were used to study the effect of streptozotocin-induced diabetes on the amounts of pyruvate carboxylase and pyruvate dehydrogenase and on their rates of synthesis and degradation. Livers from diabetic rats had twice the pyruvate carboxylase activity of livers from normal rats when expressed in terms of DNA or body weight. The changes in catalytic activity closely paralleled changes in immunoprecipitable enzyme protein. Relative rates of synthesis determined by pulse-labelling studies showed that the ratio of synthesis of pyruvate carboxylase to that of average mitochondrial protein was increased 2.0-2.5 times in diabetic animals over that of control animals. Other radioisotopic studies indicated that the rate of degradation of this enzyme was not altered significantly in diabetic rats, suggesting that the increase in this enzyme was due to an increased rate of synthesis. Similar experiments with pyruvate dehydrogenase, the first component of the pyruvate dehydrogenase complex, showed that livers from diabetic rats had approximately the same amount of immunoprecipitable enzyme protein as the control animals, but a larger proportion of the enzyme was in its inactive state. The rates of synthesis and degradation of pyruvate dehydrogenase were not affected significantly by diabetes.
TL;DR: Isozymes of pyruvate kinase (PK) have been isolated from developing castor bean endosperm and the properties of the two isozymes are similar to those of their counterparts in green tissue.
Abstract: Isozymes of pyruvate kinase (PK) have been isolated from developing castor bean endosperm. One isozyme, PK(c), is localized in the cytosol, and the other, PK(p), is in the plastid. Both isozymes need monovalent and divalent cations for activity, requirements which can be filled by K(+) and Mg(2+). Both isozymes are inhibited by citrate, pyruvate, and ATP. PK(c) has a much broader pH profile than PK(p) and is also more stable. Both have the same K(m) (0.05 millimolar) for PEP, but PK(p) has a 10-fold higher K(m) (0.3 millimolar) for ADP than PK(c) (0.03 millimolar). PK(c) also has a higher affinity for alternate nucleotide substrates than PK(p). The two isozymes have different kinetic mechanisms. Both have an ordered sequential mechanism and bind phosphoenolpyruvate before ADP. However, the plastid isozyme releases ATP first, whereas pyruvate is the first product released from the cytosolic enzyme. The properties of the two isozymes are similar to those of their counterparts in green tissue.
01 Jan 1980
TL;DR: Insulin treatment of adipocytes increased the amount or activity of a low molecular weight, acid-stable material which, when isolated from intact adipocytes by heat extraction and subsequent Sephadex G25 chromatography, yielded a single active fraction that stimulated mitochondrial pyruvate dehydrogenase by activating the phosphatase and not by altering the kinase activity.
Abstract: SUMMARY Insulin treatment of adipocytes increased the amount or activity of a low molecular weight, acid-stable material which, when isolated from intact adipocytes by heat extraction and subsequent Sephadex G25 chromatography, yielded a single active fraction that stimulated mitochondrial pyruvate dehydrogenase by activating the phosphatase and not by altering the kinase activity. Phosphatase activation was demonstrated by the ability of the active material to increase pyruvate dehydrogenase activity in the absence of ATP and by the ability of NaF, a phosphatase inhibitor, to block this stimulation. Involvement of the kinase in this activation mechanism was eliminated by the fact that, in the presence of ATP, (1) NaF completely blocked the stimulation of pyruvate dehydrogenase by the active fraction, and (2) the stimulation of pyruvate dehydrogenase by dichloroacetic acid, a kinase inhibitor, was additive to the stimulation caused by the active fraction. This active fraction may contain an intracellular chemical mediator or second messenger for insulin. DIABETES 29:852-855, October 1980.
TL;DR: The increase in the initial activity of pyruvate dehydrogenase that follows brief exposure of fat-cells to insulin was shown to be associated with a decrease in the steady-state incorporation of 32P into the alpha-subunits of pyRuvate dehydrationase, and the relative phosphorylation of all three serine residues appeared to be similar in 32P-labelled alpha- subunits in both control and insulin-treated fat- cells.
Abstract: 1. Intact rat epididymal fat-cells were incubated with 32Pi, and the intracellular proteins were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. One of the separated bands of phosphorylated proteins had an apparent subunit mol.wt. of 42 000, which is the same as that of the alpha-subunit of the pyruvate dehydrogenase complex. By using a combination of subcellular fractionation, immunoprecipitation with antiserum raised against pyruvate dehydrogenase complex and two-dimensional electrophoresis it was apparent that the incorporation into alpha-subunits accounted for 35--45% of the total incorporation into this band of phosphoproteins. 2. The increase in the initial activity of pyruvate dehydrogenase that follows brief exposure of fat-cells to insulin was shown to be associated with a decrease in the steady-state incorporation of 32P into the alpha-subunits of pyruvate dehydrogenase. 3. Tryptic peptide analysis of pyruvate dehydrogenase [32P]phosphate, labelled in intact fat-cells, indicated that three serine residues on the alpha-subunit were phosphorylated, corresponding to the three sites phosphorylated when purified pig heart pyruvate dehydrogenase was incubated with [gamma-32P]ATP. The relative phosphorylation of all three serine residues appeared to be similar in 32P-labelled alpha-subunits in both control and insulin-treated fat-cells.
TL;DR: The rapidity with which the activity was restored and the insensitivity of the process to inhibitors of protein synthesis strongly suggest that the apoenzyme of pyruvate carboxylase accumulates during differentiation in the presence of avidin.
Abstract: The specific activity of pyruvate carboxylase [pyruvate:carbon-dioxide ligase (ADP-forming); EC 6.4.1.1] in 3T3-L1 cells increases approximately 20-fold when these cells differentiate to an adipocyte-like form [Mackall, J. C. & Lane, M. D. (1977) Biochem. Biophys. Res. Commun. 79, 720-725]. A specific antibody to the purified rat liver enzyme quantitatively precipitated pyruvate carboxylase from 3T3-L1 crude homogenates. Use of this immunological technique permitted us to demonstrate that the increase in pyruvate carboxylase activity is due to an increase in the intracellular concentration of the enzyme. The content of pyruvate carboxylase in differentiated 3T3-L1 cells is sufficiently high (1-2% of total protein) that the increase in this large protein (subunit Mr = 130,000) can be visualized when 3T3-L1 crude extracts are subjected to electrophoresis on sodium dodecyl sulfate/polyacrylamide gels. When 3T3-L1 cells differentiated in the presence of avidin, they contained less than 5% of the pyruvate carboxylase activity of cells that differentiated in the absence of avidin. However, the immunoprecipitable pyruvate carboxylase content of the avidin-treated cells was essentially the same as that of cells that differentiated without avidin. Full activity of the enzyme was rapidly restored in the avidin-treated cells upon the addition of excess biotin. The recovery of activity was closely correlated with the incorporation of [14C]biotin into immunoprecipitable pyruvate carboxylase. The rapidity with which the activity was restored and the insensitivity of the process to inhibitors of protein synthesis strongly suggest that the apoenzyme of pyruvate carboxylase accumulates during differentiation in the presence of avidin.
TL;DR: The results of the present study suggest distinct systems for the regulation of the activity of the two multienzyme complexes of interest, and argue neither for nor against an inactivation of the branched chain alpha-keto acid dehydrogenase complex by a protein kinase.
TL;DR: Covalent reaction of pyruvate kinase with 5'-p-fluorosulfonylbenzoyl adenosine thus occurs at two distinct sites, suggesting that both amino acids are within the two nucleotide sites.
TL;DR: The phosphorylation/dephosphorylation system and direct feedback-inhibitory effects on the enzyme complex both play a significant role in rapidly adapting pyruvate decarboxylation to changes in the requirements for mitochondrial energy production.
Abstract: Studies were performed to elucidate factors involved in the regulation of pyruvate dehydrogenase activity in rat brain synaptosomes during membrane depolarization. Addition of 24 mM-KCl to synaptosomes resulted in increases in rates of O2 consumption (90%) and [1-(14)C]pyruvate decarboxylation (85%) and in the active/total ratio of extractable pyruvate dehydrogenase (90--100%) within 10 s. Neither pyruvate (10 mM) nor dichloroacetate (10 mM) affected the activation state of the enzyme complex. Also, the activation state of pyruvate dehydrogenase was unaffected by addition of 1 mM-octanoate, L-(--)-carnitine, 3-hydroxybutyrate, glutamate, citrate, lactate, L-malate, acetate, acetaldehyde or ethanol. Removal of Ca2+ by using EGTA lowered the active/total ratio to about 70%, although the rate of O2 consumption and pyruvate decarboxylation was unaffected. Rates of pyruvate decarboxylation in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence and absence of NaF and EGTA demonstrated a linear correlation with changes in the activity of the enzyme complex. This observation indicated that a change in the activation state of pyruvate dehydrogenase from 90 to 100% active could result in a 27% increase in the rate of pyruvate decarboxylation. It is suggested that the pyruvate dehydrogenase complex is an important site for the regulation of substrate utilization in rat brain synaptosomes. Further, the phosphorylation/dephosphorylation system and direct feedback-inhibitory effects on the enzyme complex both play a significant role in rapidly adapting pyruvate decarboxylation to changes in the requirements for mitochondrial energy production.
TL;DR: It is concluded that the inactivation and reactivation process of pyruvate dehydrogenase complex from spinach leaf mitochondria is due to phosphorylation and dephosphorylation.
TL;DR: In gel chromatography experiments a significant peak of citrate synthase activity co-migrated with the pyruvate dehydrogenase complex peak, which suggests the possible association of two enzymes.
TL;DR: Purified enzyme showed allosteric properties at low concentration of malate and this characteristic can be modified by succinate and fumarate which do not affect the maximum velocity of the reaction.
TL;DR: Rabbit spermatozoa have a metabolic strategy which is very similar to muscle cells, which suggests that the major use of the sperm cell's metabolic machinery is maintenance of energy for the contractile work of motility and that only minor amounts of metabolic energy appear to be consumed in other reactions, including those involved in fertilization.
Abstract: Rabbit sperm pyruvate kinase remains bound to the cell structure of hypotonically treated mature rabbit epididymal spermatozoa (HTRES). It displays kinetic behavior very similar to that of rabbit muscle pyruvate kinase with regard to KM values for substrates, activation by monovalent and divalent cations, inhibition by phenylalanine which is reversed by alanine, and lack of activation by fructose-1,6-biphosphate. The flagellar ATPase also remains bound to the cell structure of HTRES, whose motility may be reactivated by a source of ATP. It requires Mg+2 for activity; the KM for both ATP and MG+2 is 0.2 mM, implying that MgATP is the substrate. The ATPase activity is not inhibited by ouabain, oligomycin, or vanadate, which also do not affect reconstituted motility, and is not affected by cyclic AMP in the presence of an inhibitor of phosphodiesterase. The activities of pyruvate kinase and the flagellar ATPase in a given preparation of HTRES are comparable. Rabbit spermatozoa have a metabolic strategy which is very similar to muscle cells. This suggests that the major use of the sperm cell's metabolic machinery is maintenance of energy for the contractile work of motility and that only minor amounts of metabolic energy appear to be consumed in other reactions, including those involved in fertilization.