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PubMed1999

The effect of aging and acetyl-L-carnitine on the pyruvate transport and oxidation in rat heart mitochondria.

The effect of aging and acute treatment with acetyl-L-carnitine on the pyruvate transport and oxidation in rat heart mitochondria was studied. The activity of the pyruvate carrier as well as the rates of pyruvate-supported respiration were both depressed (around 40%) in heart mitochondria from aged rats, the major decrease occurring during the second year of life. Administration of acetyl-L-carnitine to aged rats almost completely restored the rates of these metabolic functions to the level of young control rats. This effect of acetyl-L-carnitine was not due to changes in the content of pyruvate carrier molecules. The heart mitochondrial content of cardiolipin, a key phospholipid necessary for mitochondrial substrate transport, was markedly reduced (approximately 40%) in aged rats. Treatment of aged rats with acetyl-L-carnitine reversed the age-associated decline in cardiolipin content. As the changes in cardiolipin content were correlated with changes in rates of pyruvate transport and oxidation, it is suggested that acetyl-L-carnitine reverses the age-related decrement in the mitochondrial pyruvate metabolism by restoring the normal cardiolipin content.

Acetylcarnitine↗
PubMed1999

A hypothetical model of the influence of inorganic phosphate on the kinetics of pyruvate kinase.

This paper presents a simple solution to the problem of approximating the calculated curve of reaction progress to the measured curve which is usually disturbed by initial oscillation of auxiliary lactate dehydrogenase (LDH) reaction. The experiments leading to the determination of the apparent Km for phosphoenolpyruvate (PEP) and Vm were performed. For precise estimation of kinetic parameters (Km and Vm) of the M1 isozyme of pyruvate kinase (PK), measured by coupling it to LDH reaction, the sequence of Michaelis-Menten for pyruvate kinase and second-order kinetics for lactate dehydrogenase reaction as well as a non-zero initial concentration of lactate was assumed. The functions of apparent Km and Vm of pyruvate kinase with respect to phosphate concentration, computed by an analysis of the total reaction progress curves, indicate that the reaction mixture contains an uncompetitive inhibitor of pyruvate kinase, and that the phosphate binds this inhibitor. The proposed simple mathematical model of pyruvate kinase Km and Vm increase by inorganic phosphate assumes that the pyridine nucleotides (NAD-derivatives) are kinase inhibitors. An approximate dissociation constant for pyridine nucleotides-phosphate complex and true Km of pyruvate kinase for PEP were estimated. The proposed model fits exactly the entire measured reaction process.

Adenosine Diphosphate↗
PubMed2003

The pyruvate dehydrogenase complex of Mycoplasma hyopneumoniae contains a novel lipoyl domain arrangement.

The genes encoding the pyruvate dehydrogenase (PDH) complex (pdhA, pdhB, pdhC and pdhD) from Mycoplasma hyopneumoniae have been cloned and sequenced. The genes are arranged into two operons, designated pdhAB and pdhCD, which are not found together in the chromosome. The pdhA, pdhB, pdhC and pdhD genes encode proteins of predicted molecular masses of 44.2 kDa (pyruvate dehydrogenase major subunit; E1alpha), 36.6 kDa (pyruvate dehydrogenase minor subunit; E1beta), 33.1 kDa (dihydrolipoyl acetyltransferase; E2) and 66.3 kDa (dihydrolipoyl dehydrogenase; E3), respectively. Sequence analysis of the pdhCD operon revealed the presence of a lipoyl-binding domain in pdhD but not in pdhC. The lipoyl domain is believed to act as a "swinging arm" that spans the gaps between the catalytic domains of each of the subunits. Portions of the N-terminal regions of pdhA and pdhD were expressed as 6xHis-tag fusion proteins in Escherichia coli and purified by nickel affinity chromatography. The purified proteins were used to raise antibodies in rabbits, and Western blot analysis was performed with the polyclonal rabbit antiserum. Both the pdhA and pdhD genes were expressed among various strains of M. hyopneumoniae as well as the porcine mycoplasmas, Mycoplasma hyorhinis and Mycoplasma flocculare. Southern hybridisation analysis using probes from pdhA and pdhD detected one copy of each gene in the chromosome of M. hyopneumoniae. Since previous studies have shown pyruvate dehydrogenase activity in M. hyopneumoniae [J. Gen. Microbiol. 134 (1988) 791], it appears likely that a functional lipoyl-binding domain in the N terminus of PdhC is not an absolute prerequisite for pyruvate dehydrogenase enzyme activity. We hypothesise that the lipoyl-binding domain of PdhD is performing the enzymatic function normally attributed to the PdhC lipoyl-binding domain in other organisms. Searches of pyruvate dehydrogenase gene sequences derived from other Mycoplasma species showed that a putative lipoyl domain was absent in the pdhC gene from Mycoplasma pulmonis. However, like other bacterial species, pdhC gene sequences from Mycoplasma capricolum, Mycoplasma genitalium and Mycoplasma pneumoniae contain a putative lipoyl domain.

Amino Acid Sequence↗
PubMed2002

Toxicity by pyruvate in HepG2 cells depleted of glutathione: role of mitochondria.

Several studies have shown that pyruvate can scavenge H(2)O(2) and protect from H(2)O(2)-mediated cell injury. Mitochondria are critical participants in the control of apoptotic and necrotic cell death. Mitochondrial GSH plays an important role in the maintenance of cell functions and viability by metabolism of oxygen free radicals generated by the respiratory chain. Since loss of GSH, especially mitochondrial GSH, is associated with increased production of reactive oxygen species and cell toxicity, the ability of pyruvate to protect against these actions was evaluated. Adding pyruvate to HepG2 cells depleted of GSH by treatment with l-buthionine sulfoximine (BSO) surprisingly caused loss of viability after 24 and 48 h of incubation. Anoxia, treatment with antioxidants, and infection with cytosolic catalase, and interestingly, catalase expressed in the mitochondrial compartment were able to rescue the HepG2 cells from this pyruvate plus BSO injury, suggesting a key role for H(2)O(2), and lipid peroxides as mediators in the cytotoxicity. This toxicity and cell death observed was linked to damage to the mitochondria as evidenced by the increased lipid peroxidation in total homogenate and mitochondrial fraction, loss of mitochondrial membrane potential, and a decrease in protein-sulfhydryl groups. The type of cell death observed under these conditions was a mixture of apoptosis and necrosis. These results suggest that the protective ability of pyruvate against oxidant damage requires a functional GSH pool, especially in the mitochondrial compartment, and that in the absence of GSH, pyruvate increases cell injury by damaging the mitochondria, presumably as a consequence of enhanced electron flow and reactive oxygen production by the respiratory chain.

Antioxidants↗
PubMed1999

The effects of pyruvate supplementation on body composition in overweight individuals.

A 6-wk, double-blinded, placebo-controlled study was done to ascertain the effects of pyruvate supplementation (6 g/d) on body weight, body composition, and vigor and fatigue levels in healthy overweight Caucasian men and women. Twenty-six individuals were randomly assigned to a placebo group (seven men, seven women) and a pyruvate-supplemented group (three men, nine women). In addition, all subjects participated in a 3 d/wk exercise program, which consisted of a 45-60 min aerobic/anaerobic routine. After 6 wk of treatment, there was a statistically significant decrease in body weight (-1.2 kg, P<0.001), body fat (-2.5 kg, P<0.001), and percent body fat (23.0% pre versus 20.3% 6 wk post) in the pyruvate group. Furthermore, Profile of Mood States fatigue and vigor scores improved significantly for the pyruvate group (P<0.05) at 6 wk (vigor) and 4 and 6 wk (fatigue). There was no significant change in total lean body mass in the pyruvate group. The placebo group demonstrated a significant increase (P<0.05) for POMS vigor at 2 and 4 wk with no changes occurring in any of the remaining parameters measured. Thus, the ingestion of 6 g of pyruvate for 6 wk, in conjunction with mild physical activity, resulted in a significant decrease in body weight and fat mass.

Adult↗
PubMed1998

In vitro correlation between two colorimetric assays and the pyruvic acid consumption by fibroblasts cultured to determine the sodium laurylsulfate cytotoxicity.

The target of this research was to determine the cytotoxicity of sodium laurylsulfate on single-layer cultures of human fibroblasts, using two colorimetric methods (neutral red and MTT tests) and the evaluation of the pyruvic acid consumption by the cells. For the determination of the cytotoxicity by colorimetric tests, we have determined the absorbance at 540 nm using a spectrophotometer. Pyruvic acid, present in the culture medium, is the mitochondria's C3 energetic metabolite. So, a measure of the cell's consumption of pyruvic acid was developed. The reaction is as follows: Pyruvic acid + NADH --> Lactic acid + NAD+ and the enzyme employed is the LDH (lactate dehydrogenase). This method can be used to measure cytotoxicity, proliferation, and the cell's activation. The method is rapid, precise, and lacks any toxic byproduct. The absorbance was measured using a spectrophotometer at 340 nm. The consumption of pyruvic acid follows upon the fibroblast's growth. Sodium laurylsulfate cytotoxicity test after 24 h shows that the NR colorimetric test and the pyruvic acid consumption are correctly correlated (r = 0.91, alpha = 0.05). This dosage can be used to study the barrier properties of the corneocyte layer without destroying the artificial skin.

Cells, Cultured↗
PubMedDate not supplied

Substitution of glutamine by pyruvate to reduce ammonia formation and growth inhibition of mammalian cells.

In mammalian cell culture technology glutamine is required for biomass synthesis and as a major energy source together with glucose. Different pathways for glutamine metabolism are possible, resulting in different energy output and ammonia release. The accumulation of ammonia in the medium can limit cell growth and product formation. Therefore, numerous ideas to reduce ammonia concentration in cultivation broths have been developed. Here we present new aspects on the energy metabolism of mammalian cells. The replacement of glutamine (2 mM) by pyruvate (10 mM) supported cell growth without adaptation for at least 19 passages without reduction in growth rate of different adherent commercial cell lines (MDCK, BHK21, CHO-K1) in serum-containing and serum-free media. The changes in metabolism of MDCK cells due to pyruvate uptake instead of glutamine were investigated in detail (on the amino acid level) for an influenza vaccine production process in large-scale microcarrier culture. In addition, metabolite profiles from variations of this new medium formulation (1-10 mM pyruvate) were compared for MDCK cell growth in roller bottles. Even at very low levels of pyruvate (1 mM) MDCK cells grew to confluency without glutamine and accumulation of ammonia. Also glucose uptake was reduced, which resulted in lower lactate production. However, pyruvate and glutamine were both metabolized when present together. Amino acid profiles from the cell growth phase for pyruvate medium showed a reduced uptake of serine, cysteine, and methionine, an increased uptake of leucine and isoleucine and a higher release of glycine compared to glutamine medium. After virus infection completely different profiles were found for essential and nonessential amino acids.

Ammonia↗
PubMed2003

Pyruvic acid and acetaldehyde production by different strains of Saccharomyces cerevisiae: relationship with Vitisin A and B formation in red wines.

The production of pyruvate and acetaldehyde by 10 strains of Saccharomyces cerevisiae was monitored during the fermentation of Vitis vinifera L. variety Tempranillo grape must to determine how these compounds might influence the formation of the pyroanthocyanins vitisin A and B (malvidin-3-O-glucoside-pyruvate acid and malvidin-3-O-glucoside-4 vinyl, respectively). Pyruvate and acetaldehyde production patterns were determined for each strain. Pyruvate production reached a maximum on day four of fermentation, while acetaldehyde production was at its peak in the final stages. The correlation between pyruvate production and vitisin A formation was especially strong (R (2) = 0.80) on day 4, when the greatest quantity of pyruvate was found in the medium. The correlation between acetaldehyde production and the formation of vitisin B was strongest (R (2) = 0.81) at the end of fermentation when the acetaldehyde content of the medium was at its highest. Identification and quantification experiments were performed by HPLC-DAD. The identification of the vitisins was confirmed by LC/ESI-MS.

Acetaldehyde↗
PubMed1999

Interactions between grape anthocyanins and pyruvic acid, with effect of pH and acid concentration on anthocyanin composition and color in model solutions.

The formation of vitisin A, an anthocyanin formed naturally in small quantities in maturing port wines, was studied in model wine solutions at a range of pH values (2.0-4.5) and pyruvate concentrations [molar ratios of pyruvic acid to total anthocyanins (PA/TA) ranging from 12.20 to 172.40]. Additionally, the effect of vitisin A formation on the color changes of these model wines was evaluated. Vitisin A was formed through the interaction between malvidin 3-glucoside and pyruvic acid, and vitisin A in acylated forms, having the 6-position of the sugar acylated with acetic acid (3-acetylvitisin A) and p-coumaric acid (3-p-coumarylvitisin A), formed through the interaction between pyruvic acid and malvidin 3-acetylglucoside and malvidin 3-p-coumarylglucoside, respectively; their identities were confirmed by spectral analysis and FABMS. The maximum formation of these new anthocyanin derivatives was at pH 2. 7-3.0, at the higher pyruvic acid concentration (PA/TA of 172.40 units). The vitisins A caused changes in the color of the solution and expressed about 11 times (pH 3) to 14 times (pH 2) more color than the normal anthocyanins. On aging, the model solutions changed from a bluish red, attributable to the main anthocyanins present, to a slightly more orange red, attributable to the vitisin compounds. The aged models containing vitisins A were all much redder than the more red-brown color of the models aged without pyruvic acid.

Anthocyanins↗
PubMedDate not supplied

Studies on the nitroso-glyoxylate reaction. Relative hydroxamic acid production by glyoxylate, pyruvate, and formaldehyde in reactions with 4-nitrosobiphenyl.

The pH rate profiles for the reactions of 4-nitrosobiphenyl with three carbonyl substrates in aqueous buffers were determined by use of chromatographic and spectrophotometric methods. Glyoxylate and formaldehyde caused the conversion of 4-nitrosobiphenyl to N-(4-biphenyl)-formohydroxamic acid, while pyruvate resulted in the production of N-(4-biphenyl)acetohydroxamic acid. The dramatic effect of pH on the kinetics of these reactions provided considerable information concerning the nature of these reactions. The reactions with pyruvate and formaldehyde displayed similar pH rate profiles and were significant only at acidic pH. Glyoxylate displayed a pH rate profile that differed markedly from those of pyruvate and formaldehyde as the pH was increased beyond 2.0. The ability of glyoxylate to convert 4-nitrosobiphenyl to the hydroxamic acid increased rapidly in the pH range 2.0-4.0, above which the pH dependency was constant. This biphasic appearance of the pH rate profile was unique to glyoxylate, since the reactions of pyruvate and formaldehyde became extremely slow as solution neutrality was approached. A second substrate, 4-chloronitrosobenzene, displayed similar pH rate profiles in its reactions with these carbonyl substrates. For 4-nitrosobiphenyl, hydroxamic acid formation by glyoxylate was 10(4) times faster than that by pyruvate at neutral pH, but only about 3-fold faster at pH 1.0. The appearance of the pH rate profile for glyoxylate suggested that this alpha-oxo acid reacts with nitrosoarenes at neutrality via a pathway that is insignificant for pyruvate or formaldehyde. Thus, the nitroso-glyoxylate reaction is unique to this alpha-oxo acid under physiological pH conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Biphenyl Compounds↗
PubMed1999

Evolution of pyruvate carboxylase and other biotin containing enzymes in developing rat liver and kidney.

The evolution of pyruvate carboxylase has been studied in rat liver and kidney during perinatal development. The pyruvate carboxylase activity, amount of enzyme and mRNA levels have been assayed from 2 days before delivery to weaning. In liver, there is a peak of activity and amount of enzyme 24 h before delivery and 2 peaks, at 12 h and 6 days, after parturition. The transcription of the enzyme gene followed a similar pattern, with mRNA peaks preceding those of activity and amount of enzyme. However, in kidney, pyruvate carboxylase activity, amount and mRNA remain low until weaning. These results confirm the limited role of renal gluconeogenesis during the perinatal development. Since all carboxylases contain biotin as prosthetic group, the biotinylation of pyruvate carboxylase during the perinatal period was investigated by western-blot using streptavidin-biotin peroxidase. In the mitochondrial samples from liver and kidney, all the pyruvate carboxylase detected was fully biotinylated, indicating an early development of the holocarboxylase synthetase activity in the perinatal period. This Western-blot technique also allowed us the detection of other biotin-enzymes based on their molecular weight. In liver, during the perinatal development propionyl-coA and 3-methyl-crotonyl-coA carboxylases followed a pattern of induction similar to pyruvate carboxylase. In kidney, the expression of mitochondrial carboxylases was lower compared to liver and propionyl-coA carboxylase was not detected during the studied period.

Animals↗
PubMed1988

Crystal structure of enolase indicates that enolase and pyruvate kinase evolved from a common ancestor.

Enolase or 2-phospho-D-glycerate hydrolase catalyses the dehydration of 2-phosphoglycerate to phosphoenolpyruvate, which in turn is converted by pyruvate kinase to pyruvate. We describe here the crystallographic determination of the structure of yeast enolase at high resolution (2.25 A) and an analysis of the structural homology between enolase, pyruvate kinase and triose phosphate isomerase. Each of the two subunits of enolase forms two distinctive domains. The larger domain (residues 143-420) is a regular 8-fold beta/alpha-barrel, as first found in triose phosphate isomerase, and later in pyruvate kinase and 11 other functionally different enzymes. An analysis of the molecular geometries of enolase and pyruvate kinase based on the roughly 8-fold symmetry of the barrel showed a structural homology better than expected for proteins related by convergent evolution. We argue that enolase and pyruvate kinase have evolved from a common ancestral multifunctional enzyme which could process phosphoenolpyruvate in both directions along the glycolytic pathway. There is structural and sequence evidence that muconate lactonizing enzyme later evolved from enolase.

Biological Evolution↗
PubMed2006

A bioluminescence technique for quantitative and structure-associated imaging of pyruvate.

A novel bioluminescence assay has been developed for measuring pyruvate within sections of snap-frozen tissue in a quantitative manner as well as with a spatial resolution on a microscopical level. The assay was verified via HPLC and two independent photometric tests. The novel assay makes it possible to determine pyruvate concentrations in cryosections in the range of 0-5.0 micromol/g tissue (dry weight). Based on the analysis of samples of given pyruvate concentrations, the assay exhibits a recovery with a deviation < or =15%. The minimal detectable amount was 0.02 pmol based on a 20 microm thick tissue section with an area of 1 cm(2). Combination of the already established imaging bioluminescence techniques for ATP, glucose, and lactate with the novel pyruvate assay allows for a comprehensive characterization of the metabolic profile of individual tumors. As the redox state of cancer cells can be critical for the efficiency of irradiation and a number of chemotherapeutics, and as pyruvate and lactate are known to have radical scavenger functions, we hypothesize that the novel bioluminescence assay may be used for measuring the pretherapeutic lactate-to-pyruvate ratio which may predict the radiosensitivity of individual malignancies.

Biological Assay↗
PubMed1968

A direct pathway for the conversion of propionate into pyruvate in Moraxella lwoffi.

1. The identity of the organism previously known as Vibrio O1 (N.C.I.B. 8250) with a species of Moraxella is established. 2. The ability of cells to oxidize propionate is present only in cells with an endogenous respiration and this ability is increased 80-fold when the organism is grown with propionate. 3. Isocitrate lyase activity in extracts from propionate-grown cells is the same as that in extracts from lactate-grown cells, about tenfold greater than that in extracts from succinate-grown cells and slightly greater than half the activity in extracts from acetate-grown cells. 4. With arsenite as an inhibitor conditions were found in which the organism would catalyse the quantitative oxidation of propionate to pyruvate. When propionate was completely utilized pyruvate was metabolized further to 2-oxoglutarate. 5. The oxidation of propionate by cells was incomplete both in a ;closed system' with alkali to trap respiratory carbon dioxide and in an ;open system' with an atmosphere of oxygen+carbon dioxide (95:5). Acetate accumulated. Under these conditions [2-(14)C]- and [3-(14)C]-propionate gave rise to [(14)C]acetate. The rate of conversion of [2-(14)C]propionate into (14)CO(2), although much less than the rate of conversion of [1-(14)C]propionate into (14)CO(2), was slightly greater than the rate of conversion of [3-(14)C]propionate into (14)CO(2). 6. The oxidation of propionate by cells was complete in an ;open system' with an atmosphere of either oxygen or air. Under these conditions very little [1-(14)C]propionate was converted into (14)C-labelled cell material. The conversion of [2-(14)C]- and [3-(14)C]-propionate into (14)C-labelled cell material occurred at an appreciable rate, the rate for the incorporation of [3-(14)C]propionate being slightly more rapid. In the absence of a utilizable nitrogen source part of the [(14)C]propionate was incorporated into some reserve material, which was oxidized when added substrate had been completely utilized. 7. [(14)C]-Pyruvate produced from [(14)C]propionate was chemically degraded. The C((1)) of propionate was found only in C((1)) of pyruvate. At least 86% of C((2)) of pyruvate was derived from C((2)) of propionate and at least 92% of C((3)) of pyruvate from C((3)) of propionate. 8. These results are incompatible with the operation of any of the previously described pathways for propionate metabolism except the direct one, perhaps via an activated acrylate.

Carbon Dioxide↗
PubMed1969

Pyruvate carboxylase in lactating rat and rabbit mammary gland.

1. Pyruvate carboxylase [pyruvate-carbon dioxide ligase (ADP), EC 6.4.1.1] was found in cell-free preparations of lactating rat and rabbit mammary glands, and optimum assay conditions for this enzyme were determined. 2. Subcellular-fractionation studies with marker enzymes showed pyruvate carboxylase to be distributed between the mitochondrial and soluble fractions of lactating rat mammary gland. Evidence is presented that the soluble enzyme is not an artifact due to mitochondrial damage. 3. In contrast, pyruvate carboxylase in lactating rabbit mammary gland is confined to the mitochondrial fraction. 4. The final product of pyruvate carboxylase action in the mitochondrial and particle-free supernatant fractions of lactating rat mammary gland was shown to be citrate. 5. The effects of freeze-drying, ultrasonic treatment and freezing-and-thawing on the specific activity of mitochondrial pyruvate carboxylase were investigated.

Animals↗
PubMed1970

The role of the cytoplasmic redox potential in the control of fatty acid synthesis from glucose, pyruvate and lactate in white adipose tissue.

The metabolism of lactate, pyruvate and glucose was studied in epididymal adipose tissue of starved, normally fed and starved-re-fed rats. Lactate conversion into fatty acid occurred at an appreciable rate only in the adipocyte of starved-re-fed animals. NNN'N'-Tetramethyl-p-phenylenediamine, an agent that transports reducing power from the cytoplasm to the mitochondria, caused large increments of fatty acid synthesis from lactate and a smaller one from glucose but a decrease in that from pyruvate. Glucose (1.0mm) increased fatty acid synthesis from lactate 4.3-fold but only 1.67-fold from pyruvate in adipocytes from normally fed animals. 2-Deoxyglucose decreased fatty acid synthesis from lactate to a greater degree (threefold) compared to that from pyruvate in adipocytes from starved-re-fed animals. l-Glycerol 3-phosphate contents were approximately equal in epididymal fat-pads, incubated in the presence of lactate or pyruvate, from normally fed animals, whereas the addition of 1mm-glucose resulted in a tenfold increase in l-glycerol 3-phosphate content only in the presence of lactate. The l-glycerol 3-phosphate content was tenfold higher in adipose tissue from starved-re-fed animals incubated in the presence of lactate than in the presence of pyruvate. 2-Deoxyglucose caused these values to be slightly lowered in the presence of lactate. We suggest that lactate metabolism is limited by the rate of NADH removal from the cytoplasm. In the starved-re-fed state, this occurs by reduction of dihydroxyacetone phosphate formed from glycogen to produce l-glycerol 3-phosphate, thus permitting lactate conversion into fatty acid. When glucose is the substrate, and rates of transport are not limiting, the rate of removal of cytoplasmic NADH limits glucose conversion into fatty acid.

Adipose Tissue↗
PubMed1978

Stimulation of pyruvate transport in metabolizing mitochondria through changes in the transmembrane pH gradient induced by glucagon treatment of rats.

Glucagon treatment of rats allowed the isolation of liver mitochondria with enhanced rates of pyruvate metabolism measured in either sucrose or KCl media. No change in the activity of the pyruvate carrier itself was apparent, but under metabolizing conditions, use of the inhibitor of pyruvate transport, alpha-cyano-4-hydroxycinnamate, demonstrated that pyruvate transport limited the rate of pyruvate metabolism. The maximum rate of transport under metabolizing conditions was enhanced by glucagon treatment. Problems involved in measuring the transmembrane pH gradient under metabolizing conditions are discussed and a variety of techniques are used to estimate the matrix pH. From the distribution of methylamine, ammonia and D-lactate and the Ki for inhibition by alpha-cyano-4-hydroxycinnamate it is concluded that the matrix is more acid than the medium and that the pH of the matrix rises after glucagon treatment. The increase in matrix pH stimulates pyruvate transport. The membrane potential, ATP concentration and O2 uptake were also increased under metabolizing conditions in glucagon-treated mitochondria. These changes were correlated with a stimulation of the respiratory chain which can be observed in uncoupled mitochondria [Yamazaki (1975) J. Biol. Chem. 250, 7924--7930]. The mitochondrial Mg2+ content (mean +/- S.E.M.) was increased from 38.8 +/- 1.2 (n = 26) to 47.5 +/- 2.0 (n = 26) ng-atoms/mg by glucagon and the K+ content from 126.7 +/- 10.3 (n = 19) ng-atoms/mg. This may represent a change in membrane potential induced by glucagon in vivo. The physiological significance of these results in the control of gluconeogenesis is discussed.

Adenosine Triphosphate↗
PubMed1979

Studies on the biosynthesis of hepatic pyruvate kinase and its correlation with enhanced hepatic lipogenesis in meal-trained rats.

Metabolic and enzymic changes were measured in meal-trained rats fed on high-carbohydrate diet. Rates of hepatic fatty acid synthesis are probably greater than rates of gluconeogenesis throughout the 24 h day provided that animals are fed. The daily enhancement of fatty acid synthesis on meal feeding coincided with the maximum activation of hepatic pyruvate kinase. Maximum activation of this enzyme was reflected in increased total catalytic activity (Vmax.), increased activity at 0.5 MM-phosphoenolpyruvate (V0.5), decreased Vmax./V0.5 ratio and a decrease in co-operativity of phosphoenolpyruvate binding as measured by the Hill coefficient (h). The latter changes are consistent with a decrease in enzyme phosphorylation during activation of the enzyme. To estimate changes in enzyme protein, quantitative enzyme precipitation with rabbit antisera was used. Giving a high-carbohydrate diet to meal-trained animals induced enzyme synthesis within a few hours. Adaptations in diet that enhanced fatty acid synthesis (chow to high carbohydrate; starved to high carbohydrate) led to an increased steady-state concentration of pyruvate kinase protein. An approximate estimate of the half-life of hepatic pyruvate kinase was 56 h. Whenever pyruvate kinase specific activity was measured in liver tissue extracts it was always considerably less (20--100 mumol/min per mg of protein, depending on dietary status) than the specific activity of pure pyruvate kinase (200 mumol/min per mg of protein). Antigenically active, catalytically inactive protein was removed during enzyme purification from cytosol at the stage of (NH4)2SO4 fractionation. The fraction precipitated by 30--45%-satd. (NH4)2SO4 was enzymically active, antigenically reacting protein was identified in the remaining (NH4)2SO4 fractions (0--30%- and 45--85%-satd.) and this contained no enzyme activity. These may correspond to inactive proteolytic fragments of pyruvate kinase. The rate-determining step in adjusting enzyme concentration seems to be proteolysis.

Animals↗