Effect of calcium ions on pyruvate carboxylase from pigeon liver.

Pigeon liver pyruvate carboxylase (pyruvate: CO2 ligase (ADP forming), EC 6.4.1.1) shows allosteric properties similar to those of chicken or rat liver enzyme. Kinetic methods have been used to determine the effect of Ca2+ on this enzyme. The Ca2+ activation effect is absolutely dependent on the Mg2+ concentration; in the absence of Mg2+, pyruvate carboxylase has no catalytic activity. Furthermore, Ca2+ cannot replace Mg2+ and also shows a paradoxical effect on the liver enzyme activity. It is an activator at low pyruvate or Mg2+ concentrations; at increased pyruvate concentrations, however, it becomes an inhibitor. At low levels of ATP a pronounced activation of pigeon liver pyruvate carboxylase by Ca2+ has been demonstrated. The results of this communication demonstrate pigeon liver pyruvate carboxylase to be different from pyruvate carboxylase from other sources.     

Regulation of gluconeogenesis and lipogenesis. The regulation of mitochondrial pyruvate metabolism in guinea-pig liver synthesizing precursors for gluconeogenesis

1. The carboxylation of pyruvate to oxaloacetate by pyruvate carboxylase in guinea-pig liver mitochondria was determined by measuring the amount of (14)C from H(14)CO(3) (-) fixed into organic acids in the presence of pyruvate, ATP, Mg(2+) and P(i). The main products of pyruvate carboxylation were malate, fumarate and citrate. Pyruvate utilization, metabolite formation and incorporation of (14)C from H(14)CO(3) (-) into these metabolites in the presence and the absence of ATP were examined. The synthesis of phosphoenolpyruvate from pyruvate and bicarbonate is minimal during continued oxidation of pyruvate. Larger amounts of phosphoenolpyruvate are formed from alpha-oxoglutarate than from pyruvate. Addition of glutamate, alpha-oxoglutarate or fumarate did not appreciably increase formation of phosphoenolpyruvate when pyruvate was used as substrate. With alpha-oxoglutarate as substrate addition of fumarate resulted in increased formation of phosphoenolpyruvate, whereas addition of succinate inhibited phosphoenolpyruvate formation. In the presence of added oxaloacetate guinea-pig liver mitochondria synthesized phosphoenolpyruvate in amount sufficiently high to play an appreciable role in gluconeogenesis. 2. Addition of fatty acids of increasing carbon chain length caused a strong inhibition of pyruvate oxidation and phosphoenolpyruvate formation, and greatly promoted carbon dioxide fixation and malate, citrate and acetoacetate accumulation. The incorporation of (14)C from H(14)CO(3) (-), [1-(14)C]pyruvate and [2-(14)C]pyruvate into organic acids formed was examined. 3. It is concluded that guinea-pig liver pyruvate carboxylase contributes significantly to gluconeogenesis and that fatty acids and metabolites play an important role in its regulation.     

Metabolism of pyruvate by isolated rat mesenteric lymphocytes, lymphocyte mitochondria and isolated mouse macrophages.

1. The activities of pyruvate dehydrogenase in rat lymphocytes and mouse macrophages are much lower than those of the key enzymes of glycolysis and glutaminolysis. However, the rates of utilization of pyruvate (at 2 mM), from the incubation medium, are not markedly lower than the rate of utilization of glucose by incubated lymphocytes or that of glutamine by incubated macrophages. This suggests that the low rate of oxidation of pyruvate produced from either glucose or glutamine in these cells is due to the high capacity of lactate dehydrogenase, which competes with pyruvate dehydrogenase for pyruvate. 2. Incubation of either macrophages or lymphocytes with dichloroacetate had no effect on the activity of subsequently isolated pyruvate dehydrogenase; incubation of mitochondria isolated from lymphocytes with dichloroacetate had no effect on the rate of conversion of [1-14C]pyruvate into 14CO2, and the double-reciprocal plot of [1-14C]pyruvate concentration against rate of 14CO2 production was linear. In contrast, ADP or an uncoupling agent increased the rate of 14CO2 production from [1-14C]pyruvate by isolated lymphocyte mitochondria. These data suggest either that pyruvate dehydrogenase is primarily in the a form or that pyruvate dehydrogenase in these cells is not controlled by an interconversion cycle, but by end-product inhibition by NADH and/or acetyl-CoA. 3. The rate of conversion of [3-14C]pyruvate into CO2 was about 15% of that from [1-14C]pyruvate in isolated lymphocytes, but was only 1% in isolated lymphocyte mitochondria. The inhibitor of mitochondrial pyruvate transport, alpha-cyano-4-hydroxycinnamate, inhibited both [1-14C]- and [3-14C]-pyruvate conversion into 14CO2 to the same extent, and by more than 80%. 4. Incubations of rat lymphocytes with concanavalin A had no effect on the rate of conversion of [1-14C]pyruvate into 14CO2, but increased the rate of conversion of [3-14C]pyruvate into 14CO2 by about 50%. This suggests that this mitogen causes a stimulation of the activity of pyruvate carboxylase.     

Regulation of purified rat liver acetyl CoA carboxylase by phosphorylation

Acetyl CoA carboxylase was purified from liver of fasted-refed rats to near homogeneity, based on electrophoretic analysis and biotin content. These preparations contained an endogenous protein kinase that catalyzed the transfer of radioactive phosphate from [gamma-32P]ATP to acetyl CoA carboxylase, accompanied by a decrease in acetyl CoA carboxylase activity. Phosphate incorporated into acetyl CoA carboxylase was removed when the preparation was incubated with partially purified phosphorylase phosphatase catalytic subunit with regain of enzymatic activity. This endogenous protein kinase was shown not to be affected by either cyclic-AMP-dependent protein kinase inhibitor, EGTA, or trifluoperazine. The addition of either cyclic-AMP or purified cyclic-AMP-dependent protein kinase catalytic subunit to the purified acetyl CoA carboxylase preparation increased protein phosphorylation but had no further effect on acetyl CoA carboxylase activity. Purified acetyl CoA carboxylase was shown to act as an ATPase during the phosphorylation reaction.     

Studies on the regulation of pyruvate dehydrogenase in isolated beef heart mitochondria.

The regulation of the pyruvate dehydrogenase multienzyme complex of isolated beef heart mitochondria by a phosphorylation-dephosphorylation mechanism was investigated. From mitochondria incubated under conditions favoring either a protein kinasemediated inactivation or a phosphatase-mediated reactivation, the pyruvate dehydrogenase complex was extracted and partially purified. Incorporation of ³²P from [γ-³²P]ATP into the pyruvate dehydrogenase complex corresponded to the loss of enzymatic activity. Upon incubation of the mitochondria that were preincubated with [γ-³²P]ATP under metabolic conditions favoring the phosphatase reaction, the amount of radioactivity in the ³²P-labeled fraction decreased significantly with a concomitant increase in the pyruvate dehydrogenase activity. The estimated molecular weight of the ³²P-labeled fraction derived from the mitochondrial incubation was 41,000, corresponding to the reported molecular weight of the α-subunit of the pyruvate dehydrogenase portion of the multienzyme complex.     

Effects of Ammonia and β-Methylene-dl-Aspartate on the Oxidation of Glucose and Pyruvate by Neurons and Astrocytes in Primary Culture

Both ammonia and beta-methylene-DL-aspartate (beta-MA), an irreversible inhibitor of aspartate aminotransferase activity and thus of the malate-aspartate shuttle, were found previously to decrease oxidative metabolism in cerebral cortex slices. In the present work, the possibility that ammonia and beta-MA affect energy metabolism by a common mechanism (i.e., via inhibition of the malate-aspartate shuttle) was investigated using primary cultures of neurons and astrocytes. Incubation of astrocytes for 30 min with 5 mM beta-MA resulted in a decreased production of 14CO2 from [U-14C]glucose, but did not affect 14CO2 production from [2-14C]pyruvate. Conversely, incubation of astrocytes with 3 mM ammonium chloride resulted in decreased 14CO2 production from [2-14C]pyruvate, but 14CO2 production from [U-14C]glucose was not significantly affected. Ammonium chloride had no significant effect on 14CO2 production from either [U-14C]glucose or [2-14]pyruvate by neurons. However, incubation of neurons with beta-MA or beta-MA plus ammonium chloride resulted in a approximately 45% decrease of 14CO2 production from both [U-14C]glucose and [2-14C]pyruvate. A 2-h incubation of astrocytes with beta-MA resulted in no change in ATP levels, but a 35% decrease in phosphocreatine. Similar treatment of neurons resulted in greater than 50% decrease in ATP, but had little effect on phosphocreatine. beta-MA also caused a decrease in glutamate and aspartate content of neurons, but not of astrocytes. The different metabolic responses of neurons and astrocytes towards beta-MA were probably not due to a differential inhibition of aspartate aminotransferase which was inhibited by approximately 45% in astrocytes and by approximately 55% in neurons.     

Acute control of fatty acid synthesis by cyclic AMP in the chick liver cell: possible site of inhibition of citrate formation.

Glucagon and N,(6)O(2)-dibutyryl cyclic adenosine 3',5'-cyclic monophosphate (Bt(2)cAMP) inhibit fatty acid synthesis from acetate by more than 90% and prevent citrate formation in chick hepatocytes metabolizing glucose. With substrates that enter glycolysis at or below triose-phosphates, e.g., fructose, lactate, or pyruvate, Bt(2)cAMP has no effect on the citrate level and its inhibitory effect on fatty acid synthesis is substantially reversed. Because acetyl-CoA carboxylase requires a tricarboxylic acid activator for activity, it is proposed that regulation of fatty acid synthesis by Bt(2)cAMP is due, in part, to changes in the citrate level. Reduced citrate formation appears to result from a cAMP-induced inhibition of glycolysis. Bt(2)cAMP inhibits (14)CO(2) production from [1-(14)C]-, [6-(14)C]-, and [U-(14)C]glucose and has little effect on (14)CO(2) formation from [1-(14)C]- or [2-(14)C]pyruvate or from [1-(14)C]fructose. [(14)C]Lactate formation from glucose is depressed 50% by Bt(2)cAMP. In the presence of an inhibitor of mitochondrial pyruvate transport lactate accumulation is enhanced, but continues to be lowered 50% by Bt(2)cAMP. The activity of phosphofructokinase is greatly decreased in Bt(2)cAMP-treated cells while the activities of pyruvate kinase and acetyl-CoA carboxylase are unaffected. It appears that decreased glycolytic flux and decreased citrate formation result from depressed phosphofructokinase activity. Fatty acid synthesis from [(14)C]acetate is partially inhibited by Bt(2)cAMP in the presence of fructose, lactate, and pyruvate despite a high citrate level. Incorporation of [(14)C]fructose, [(14)C]pyruvate, or [(14)C]lactate into fatty acids is similarly depressed by Bt(2)cAMP. Synthesis of cholesterol from [(14)C]acetate or [2-(14)C]pyruvate is unaffected by Bt(2)cAMP. These results implicate a second site of inhibition of fatty acid synthesis by Bt(2)cAMP that involves the utilization, but not the production, of cytoplasmic acetyl-CoA.-Clarke, S. D., P. A. Watkins, and M. D. Lane. Acute control of fatty acid synthesis by cyclic AMP in the chick liver cell: possible site of inhibition of citrate formation.     

The effects of alpha-adrenergic stimulation on the regulation of the pyruvate dehydrogenase complex in the perfused rat liver

The regulation of the pyruvate dehydrogenase multienzyme complex was investigated during alpha-adrenergic stimulation with phenylephrine in the isolated perfused rat liver. The metabolic flux through the pyruvate dehydrogenase reaction was monitored by measuring the production of 14CO2 from infused [1-14C] pyruvate. In livers from fed animals perfused with a low concentration of pyruvate (0.05 mM), phenylephrine infusion significantly inhibited the rate of pyruvate decarboxylation without affecting the amount of pyruvate dehydrogenase in its active form. Also, phenylephrine caused no significant effect on tissue NADH/NAD+ and acetyl-CoA/CoASH ratios or on the kinetics of pyruvate decarboxylation in 14CO2 washout experiments. Phenylephrine inhibition of [1-14C]pyruvate decarboxylation was, however, closely associated with a decrease in the specific radioactivity of perfusate lactate, suggesting that the pyruvate decarboxylation response simply reflected dilution of the labeled pyruvate pool due to phenylephrine-stimulated glycogenolysis. This suggestion was confirmed in additional experiments which showed that the alpha-adrenergic-mediated inhibitory effect on pyruvate decarboxylation was reduced in livers perfused with a high concentration of pyruvate (1 mM) and was absent in livers from starved rats. Thus, alpha-adrenergic agonists do not exert short term regulatory effects on pyruvate dehydrogenase in the liver. Furthermore, the results suggest either that the rat liver pyruvate dehydrogenase complex is insensitive to changes in mitochondrial calcium or that changes in intramitochondrial calcium levels as a result of alpha-adrenergic stimulation are considerably less than suggested by others.     

Pig liver pyruvate carboxylase. The reaction pathway for the carboxylation of pyruvate

1. The reaction pathway for the carboxylation of pyruvate, catalysed by pig liver pyruvate carboxylase, was studied in the presence of saturating concentrations of K(+) and acetyl-CoA. 2. Free Mg(2+) binds to the enzyme in an equilibrium fashion and remains bound during all further catalytic cycles. MgATP(2-) binds next, followed by HCO(3) (-) and then pyruvate. Oxaloacetate is released before the random release, at equilibrium, of P(i) and MgADP(-). 3. This reaction pathway is compared with the double displacement (Ping Pong) mechanisms that have previously been described for pyruvate carboxylases from other sources. The reaction pathway proposed for the pig liver enzyme is superior in that it shows no kinetic inconsistencies and satisfactorily explains the low rate of the ATP[unk][(32)P]P(i) equilibrium exchange reaction. 4. Values are presented for the stability constants of the magnesium complexes of ATP, ADP, acetyl-CoA, P(i), pyruvate and oxaloacetate.     

Phosphorylation of rat kidney pyruvate kinase type L by cyclic 3',5'-AMP-dependent protein kinase.

Pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) type L was partly purified from rat kidney. During the last two purification steps, the incorporation of [32P]phosphate into protein on incubation with [32P]ATP and cyclic 3',5'-AMP-dependent protein kinase was found to parallel the pyruvate kinase activity. After phosphorylation of the enzyme, a major radioactive band with a molecular weight of 57 000 was found on polyacrylamide gel electrophoresis [32P]Phosphorylserine was isolated from the kidney pyruvate kinase. Immunological identity was found between the liver and kidney pyruvate kinases type L. By autoradiography of high-voltage electropherograms after partial acid hydrolysis of the phosphorylated rat liver and kidney pyruvate kinases type L, identical results were obtained. The affinity for phosphoenolpyruvate was found to be decreased by phosphorylation of the enzyme with a change in the apparent Km from 0.15 mM to 0.35 mM. After incubation of the phosphorylated kidney pyruvate kinase with phosphatase the phosphoenolpyruvate saturation curve was found to be identical to that for the unphosphorylated enzyme. Thus, the activity of the rat kidney pyruvate kinase type L is with all probability regulated by a reversible phosphorylation-dephosphorylation reaction, thereby indicating that hormonal regulation of gluconeogenesis via cyclic AMP may be of importance in the renal cortex.     

Control of pyruvate dehydrogenase activity in intact cardiac mitochondria. Regulation of the inactivation and activation of the dehydrogenase.

The control of pyruvate dehydrogenase activity by inactivation and activation was studied in intact mitochondria isolated from rabbit heart. Pyruvate dehydrogenase could be completely inactivated by incubating mitochondria with ATP, oligomycin, and NaF. This loss in dehydrogenase activity was correlated with the incorporation of 32P from [gamma-32P]ATP into mitochondrial protein(s) and with a decrease in the mitochondrial oxidation of pyruvate. ATP may be supplied exogenously, generated from endogenous ADP during oxidative phosphorylation, or formed from exogenous ADP in carbonyl cyanid p-trifluoromethoxyphenylhydrazone-uncoupled mitochondria. With coupled mitochondria the concentration of added ATP required to half-inactivate the dehydrogenase was 0.24 mM. With uncoupled mitochondria the apparent Km was decreased to 60 muM ATP. Inactivation of pyruvate dehydrogenase by exogenous ATP was sensitive to atractyloside, suggesting that pyruvate dehydrogenase kinase acts internally to the atractyloside-sensitive barrier. The divalent cation ionophore, A23187, enhanced the loss of dehydrogenase activity. Pyruvate dehydrogenase activity is regulated additionally by pyruvate, inorganic phosphate, and ADP. Pyruvate, in the presence of rotenone, strongly inhibited inactivation. This suggests that pyruvate facilitates its own oxidation and that increases in pyruvate dehydrogenase activity by substrate may provide a modulating influence on the utilization of pyruvate via the tricarboxylate cycle. Inorganic phosphate protected the dehydrogenase from inactivation by ATP. ADP added to the incubation mixture together with ATP inhibited the inactivation of pyruvate dehydrogenase. This protection may result from a direct action on pyruvate dehydrogenase kinase, as ADP competes with ATP, and an indirect action, in that ADP competes with ATP for the translocase. It is suggested that the intramitochondrial [ATP]:[ADP] ratio effects the kinase activity directly, whereas the cytosolic [ATP]:[ADP] ratio acts indirectly. Mg2+ enhances the rate of reactivation of the inactivated pyruvate dehydrogenase presumably by accelerating the rate of dephosphorylation of the enzyme. Maximal activation is obtained with the addition of 0.5 mM Mg2+..     

Defective intramitochondrial NADH oxidation in skin fibroblasts from an infant with fatal neonatal lacticacidemia.

A small-for-gestational-age female infant born at term developed severe lactic acidosis and died on day 13 of life. Two previous sibs had also died of overwhelming lactic acidosis in the neonatal period. The lactate-to-pyruvate and 3-hydroxybutyrate-to-acetoacetate ratios were elevated at 136 and 42 to one, respectively. The activities of the pyruvate dehydrogenase complex and pyruvate carboxylase in cultured skin fibroblasts were normal but a defect in respiration was indicated by the low rates of conversion of 1-[14C]pyruvate, glutamate, and lactate to 14CO2 in these cells. Skin fibroblast cultures also displayed an elevated lactate-to-pyruvate ratio (72:1) when incubated with glucose as substrate compared to control cell cultures (20:1). When mitochondrial preparations of skin fibroblasts (prepared by digitonin extraction) were tested for their ability to synthesize ATP from a variety of substrates, it was found that those of the patient made adequate amounts of ATP with either succinate or ascorbate/tetramethyl-phenylenediamine as substrate but not with the NAD-linked substrates pyruvate, isocitrate, and palmitoyl carnitine. We propose that this is indicative of a defect in the respiratory chain between NADH and coenzyme Q, for the first time demonstrable in cultured skin fibroblasts.     

Determination of the roles of active sites in F1-ATPase by controlled affinity labeling

The affinity reagents 3'-O-(5-fluoro-2,4-dinitrophenyl) [alpha-32P]ATP (FDNP-[alpha-32P]ATP) and 3'-O-(5-fluoro-2,4-dinitrophenyl) [8-14C]ATP (FDNP-[14C]ATP) were synthesized and used to characterize the structure and function of the three active sites in F1-ATPase. FDNP-[alpha-32P]ATP was found to bind covalently to F1 up to two DNP-[alpha-32P]ATP labels per F1 in the absence of Mg2+ without decreasing the ATPase activity. However, when MgCl2 was subsequently added to the reaction mixture, the enzyme could be further labeled with concomitant decrease in ATPase activity that is consistent with the complete inactivation of one enzyme molecule by an affinity label at the third ATP-binding site. Partial hydrolysis of the FDNP-[14C]ATP-labeled enzyme and sequencing of the isolated peptide indicated that the affinity label was attached to Lys-beta 301 at all three active sites. Samples of F1 with covalent affinity label on Lys-beta 301 were also used to reconstitute F1-deficient submitochondrial particles. The reconstituted particles were assayed for ATPase and oxidative phosphorylation activities. These results show that the catalytic hydrolysis of ATP either by F1 in solution or by F0F1 complex attached to inner mitochondrial membrane takes place essentially at only one active site, but is promoted by the binding of ATP at the other two active sites, and that ATP synthesis during oxidative phosphorylation takes place at all three active sites [corrected].     

Effect of insulin on the metabolic distribution of carbons 1, 2, and 3 of pyruvate

It has long been known that the carbons of pyruvate are converted to CO2 at different points in the metabolic process. This report deals with the observation that insulin affects the oxidation of carbons 2 and 3 primarily and has little effect on the oxidation of the carboxyl carbon. Oxidation of different carbons of pyruvate and their incorporation into various metabolic components was studied in isolated rat hepatocytes. Insulin stimulated the 14CO2 production from [2-14C]- and [3-14C]pyruvate and from [U-14C]alanine. However, it had little or no effect on the activity of the pyruvate dehydrogenase complex as measured by the evolution of 14CO2 from [1-14C]pyruvate or [1-14C] alanine. Insulin also stimulated the incorporation of carbons 2 and 3 of pyruvate into protein but had no effect on the incorporation of carbon 1. Incorporation of [1-14C]- and [U-14C]alanine into protein was differentially enhanced by insulin in a manner similar to that of the pyruvate carbons. The fact that insulin stimulates the incorporation of [1-14C]alanine into protein but not [1-14C]pyruvate suggests the possibility of a compartmentation of pyruvate metabolism in the isolated hepatocytes. These studies show that the stimulation of [2-14C]- and [3-14C]pyruvate incorporation into protein involves the stimulatory effect of insulin on the activity of the Krebs cycle which is evident from the fact that insulin did not stimulate the pyruvate carbons to enter protein via alanine but the incorporation via glutamate was increased by about 40%.     

Ammonium ions enhance the flow through the pyruvate dehydrogenase in Ehrlich ascites tumor cells

The flow through pyruvate dehydrogenase was assayed in glycolysing cells by the evolution of 14CO2 from [1-14C] pyruvate. Parallel incubations were carried out in high bicarbonate buffer (25 mM) and in bicarbonate-free buffer. The activation of the complex by NH+4 was only observed in high bicarbonate buffer, because the dilution of labelled CO2 in the presence of an excess of bicarbonate enables the quantitative determination of labelled CO2 evolved from pyruvate in the decarboxylase step. In the bicarbonate-free buffer the activation of the complex was not observed, because the 14CO2 evolved from pyruvate was consumed by biosynthetic processes inside the cell. On the contrary in isolated hepatocytes the NH+4 activation of the pyruvate dehydrogenase was observed in both buffers. In Ehrlich ascites cells, in common with other mammalian tissues, pyruvate dehydrogenase activity was found to be inversely correlated to the intramitochondrial ATP/ADP ratio.     

Pyruvate dehydrogenase and 3-fluoropyruvate: chemical competence of 2-acetylthiamin pyrophosphate as an acetyl group donor to dihydrolipoamide

The pyruvate dehydrogenase component (E1) of the pyruvate dehydrogenase complex catalyzes the decomposition of 3-fluoropyruvate to CO2, fluoride anion, and acetate. Acetylthiamin pyrophosphate (acetyl-TPP) is an intermediate in this reaction. Incubation of the pyruvate dehydrogenase complex with 3-fluoro[1,2-14C]pyruvate, TPP, coenzyme A (CoASH), and either NADH or pyruvate as reducing systems leads to the formation of [14C]acetyl-CoA. In this reaction the acetyl group of acetyl-TPP is partitioned by transfer to both CoASH (87 +/- 2%) and water (13 +/- 2%). When the E1 component is incubated with 3-fluoro[1,2-14C]pyruvate, TPP, and dihydrolipoamide, [14C]acetyldihydrolipoamide is produced. The formation of [14C]acetyldihydrolipoamide was examined as a function of dihydrolipoamide concentration (0.25-16 mM). A plot of the extent of acetyl group partitioning to dihydrolipoamide as a function of 1/[dihydrolipoamide] showed 95 +/- 2% acetyl group transfer to dihydrolipoamide when dihydrolipoamide concentration was extrapolated to infinity. It is concluded that acetyl-TPP is chemically competent as an intermediate for the pyruvate dehydrogenase complex catalyzed oxidative decarboxylation of pyruvate.     

Ligand-induced conformational transitions and secondary-structure composition of chicken liver pyruvate carboxylase

Apparent conformational transitions induced in chicken liver pyruvate carboxylase by substrates, KHCO(3) and MgATP, and the allosteric effector, acetyl-CoA, were studied by using the fluorescent probe, 8-anilinonaphthalene-1-sulphonic acid and c.d. Fluorescence measurements were made with both conventional and stopped-flow spectrophotometers. Additions of acetyl-CoA and/or ATP to the enzyme-probe solutions quenched fluorescence of the probe by the following cumulative amounts regardless of the sequence of additions: acetyl-CoA, 10-13%; ATP, 21-24%; acetyl-CoA plus ATP, about 35%. Additions of KHCO(3) had no effect on the fluorescence. The rates of quenching by acetyl-CoA and MgATP (in the presence of acetyl-CoA) were too rapid to measure by stopped-flow kinetic methods, but kinetics of the MgATP effect (in the absence of acetyl-CoA) indicate three unimolecular transitions after the association step. The negligible effect of the probe on enzyme catalytic activity, a preservation of the near-u.v. c.d. effect of MgATP and acetyl-CoA in the presence of the probe and no observable unimolecular transitions after binding of the probe to the enzyme indicate that the probe had no deleterious effect on the enzyme. In contrast with results with 8-anilinonaphthalene-1-sulphonic acid, fluorescence of the epsilon-derivative of acetyl-CoA or ATP [fluorescent analogues; Secrist, Barrio, Leonard & Weber (1972) Biochemistry11, 3499-3506] was not changed when either one was added to the enzyme. Secondary-structure composition of chicken liver pyruvate carboxylase estimated from the far-u.v. c.d. spectrum of the enzyme is 27% helix, 7% beta-pleated sheet and 66% other structural types.     

Regulation of hepatic acetyl coenzyme A carboxylase by phosphorylation and dephosphorylation

Acetyl CoA carboxylase, in a partially purified preparation, was inactivated by ATP in a time- and temperature-dependent reaction. Adenosine 3′,5′-monophosphate did not affect the inactivation. Further purification separated the carboxylase from a protein fraction which could greatly enhance the inactivation of the enzyme.Inactivation of the enzyme with [γ-³²P]ATP resulted in the incorporation of ³²P which copurified with the enzyme. No label was incorporated when [U-¹⁴C]ATP was used. When carboxylase inactivated by exposure to [γ-³²P]ATP was precipitated with antibody, isotope incorporation into the precipitate paralleled enzyme inactivation. The phosphate was bound to serine and threonine residues by an ester linkage.Sodium fluoride completely inhibited the activation of partially purified enzyme by magnesium ions. Activation by magnesium, accompanied by the release of protein-bound ³²P, was antagonistic to inactivation of the enzyme by ATP.The data presented in this communication are consistent with a mechanism for controlling acetyl CoA carboxylase activity by interconversion between phosphorylated and dephosphorylated forms. Phosphorylation of the enzyme by a portein kinase decreases enzyme activity, whereas dephosphorylation by a protein phosphatase reactivates the enzyme.     

Effect of thiamine on the activity of the pyruvate dehydrogenase complex in rat liver following stimulation of lipogenesis

It is shown that thiamine administration to rats (250 micrograms per 100 g of mass) who were given high-carbohydrate diet (lipogenesis intensification) after fasting inhibits an increase in the pyruvate dehydrogenase activity in the liver homogenate and mitochondria usual under these conditions. This is observed when determining total activity of the pyruvate dehydrogenase complex and activity of its first component--pyruvate dehydrogenase estimated from the ferricyanide reduction and [1-14C] CO2 formation from [1-14C] pyruvate. Fasting animals and animals whom thiamine was administered against a background of lipogenesis intensification revealed a higher ability of the liver tissue to synthesize acetoin as compared with the control group and animals with the intensified lipogenesis without thiamine administration.