Mitochondrial Phosphoenolpyruvate Carboxykinase Research Articles

Fetal to neonatal development of gluconeogenic enzymes in the guinea-pig kidney

1. 1. The activities of pyruvate carboxylase and phosphoenolpyruvate carboxykinase were barely detectable in foetal guinea-pig kidney. 2. 2. From mid-gestation to term, glucose-6-phosphatase was present at about 50% of the adult activity whereas fructose-l,6-diphosphatase activity equalled that in the adult. 3. 3. In the neonatal period no appreciable changes were noted for both glucose-6-phosphatase and fructose-1,6-diphosphatase in the first 24 hr of life. 4. 4. In this period pyruvate carboxylase activity increased 2-fold, and the activities of cytosolic and mitochondrial phosphoenolpyruvate carboxykinases increased 20- and 6-fold, respectively. 5. 5. These changes suggest that, like the liver, the kidney may also be a potentially important organ for glucose synthesis in the newborn.

Effects of oleate, palmitate, and octanoate on gluconeogenesis in isolated rabbit liver cells

The effect of oleate, palmitate, and octanoate on glucose formation was studied with lactate or pyruvate as substrate. Octanoate was much more quickly oxidized and utilized for ketone body production than were oleate and palmitate. Among fatty acids studied, only octanoate resulted in a marked increase of the 3-hydroxybutyrate/acetoacetate (3- OHB AcAc ) ratio. Each of the fatty acids studied stimulated glucose synthesis from pyruvate. The enhancement of gluconeogenesis by long-chain fatty acids was abolished after the addition of ammonia. As concluded from the “crossover” plot, the stimulatory effect of fatty acids was due to: (i) a stimulation of pyruvate carboxylation, (ii) a provision of reducing equivalents for glyceraldehyde phosphate dehydrogenase, and (iii) an acceleration of flux through hexose diphosphatase. Moreover, palmitate and oleate resulted in an increased generation of mitochondrial phosphpenolpyruvate, while in the presence of octanoate, the activity of mitochondrial phosphoenolpyruvate carboxykinase was diminished. When lactate was used as the glucose precursor, palmitate and oleate increased glucose production by about 50% but did not affect the contribution of mitochondrial phosphoenolpyruvate carboxykinase to gluconeogenesis. In contrast, in spite of the stimulation of both pyruvate carboxylase and hexose diphosphatase, as judged from the crossover plot, the addition of octanoate resulted in a marked inhibition of both glucose formation and mitochondrial generation of phosphoenolpyruvate. The inhibitory effect of octanoate was reversed by ammonia. Results indicate that fatty acids and ammonia are potent regulatory factors of both the rate of glucose formation and the contribution of mitochondrial phosphoenolpyruvate carboxykinase to gluconeogenesis in hepatocytes of the fasted rabbit.

The appearance of gluconeogenesis at birth in sheep. Activation of the pathway associated with blood oxygenation

1. Measurements of pyruvate carboxylase, mitochondrial phosphoenolpyruvate carboxykinase (GTP), hexose bisphosphatase and glucose 6-phosphatase in developing sheep liver showed substantial activities of all enzymes in the foetus, especially towards the end of gestation. Cytosol phosphoenolpyruvate carboxykinase (GTP) in livers of mid-term foetuses was only 10% of the activity at birth. 2. All enzymes except pyruvate carboxylase showed 1.5-2-fold increases after birth. 3. Gluconeogenesis form [14C]actate could not be detected in chronically cannulated sheep foetuses at any developmental stage and was not initiated by the infusion of adrenaline or glucagon. 4. An active pathway of gluconeogenesis was evident in vivo within 2 min after natural birth or within 4 min after Caesarian delivery of term lambs, and was delayed in prematurely delivered lambs until breathing was established and the blood fully oxygenated. 5. It is proposed that oxygen availability initiates gluconeogenesis in the newborn lamb.

Nucleotide specificity of pyruvate kinase and phospho enolpyruvate carboxykinase

Various analogues of adenosine 5′-diphosphate with modifications in the heterocyclic base residue were tested as substrates of rabbit muscle pyruvate kinase (ATP:pyruvate 2-O- phosphotransferase , EC. 2.7.1.40) and guinea pig liver mitochondrial phospho enolpyruvate carboxykinase (GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32). The significance of different structural elements for the enzyme-substrate interaction is discussed. While pyruvate kinase shows a rather broad specificity for these analogues, phospho enolpyruvate carboxykinase has a more stringent requirement for nucleotides, the intact keto and NH groups at C 6 and N 1 of the pyrimidine ring representing essential sites for the phospho enolpyruvate carboxykinase substrate interaction. The biological significance of the different substrate specificities of pyruvate kinase and phospho enolpyruvate carboxykinase is discussed as a possible metabolic control factor.

Influence of Diet on Glucose Tolerance, on the Rate of Glucose Utilization and on Gluconeogenic Enzyme Activities in the Dog

Four diets containing 0 to 62% of energy from carbohydrate and 24% to 48% of energy from protein were fed to young dogs. Two of the diets (diets LPLC and HPLC) were carbohydrate-free and two of the diets (diets HPHC and HPLC) contained 46% to 48% protein. The influence of these diets on several parameters of glucose metabolism was ascertained. Following an intravenous glucose load, plasma glucose levels were higher in dogs fed carbohydrate-free diets (diets LPLC and HPLC) than observed in dogs fed carbohydrate-containing diets (diet LPHC and HPHC). Consumption of high-protein diets (diets HPHC and HPLC) also impaired glucose tolerance. Estimates of glucose utilization were obtained. Dogs fed carbohydrate-containing diets exhibited a higher rate of glucose utilization than did dogs fed the carbohydrate-free diets. Fasting the dogs for 48 hours reduced the glucose replacement rate in dogs fed the carbohydrate-containing diets but did not influence the rate of glucose utilization in dogs fed the carbohydrate-free diets. The activities of phosphoenolpyruvate carboxykinase (PEPCK) and of pyruvate carboxylase (PYCAR) in livers and kidneys of these dogs were influenced by the diets fed. Mitochondrial PEPCK and PYCAR activities in both liver and kidney were increased in dogs fed the carbohydrate-free diets. Consumption of the high-protein diets actually decreased the activities of PEPCK in liver and kidney mitochondria.

The Effect of Premature Delivery on the Development of Gluconeogenic Enzymes in the Guinea Pig

The activities of key gluconeogenic enzymes in the livers of newborn guinea pigs were monitored as a function of time following birth either vaginally at term or prematurely by cesarian section at 62 days of gestation. The activity of hepatic glucose-6-phosphatase rose dramatically from 1.40 +/- 0.26 mumol/min/g at birth to a maximum of 6.8 +/- 0.9 mumol/min/g at 24 hr in prematurely delivered animals although there was little significant change in activity in full term animals. The activity of hepatic fructose-1,6-diphosphatase and mitochondrial phosphoenolpyruvate carboxykinase changed little over the first 3 days of life in either full term or premature animals. Cytosolic phosphoenolpyruvate carboxykinase, on the other hand, had low activity at birth being 0.11 +/- 0.03 mumol/min/g in full term and 0.06 +/- 0.04 mumol in premature animals rising to values of 0.71 +/- 0.06 and 1.12 +/- 0.12 mumol/min/g, respectively, at 24 hr of life. Pyruvate carboxylase activities in the premature animals remained significantly lower than those in full term animals in the first 72 hr of life. Transient hypoglycemia was evident in the prematurely delivered animals, but not in the full term animals, the blood glucose values being 82 +/- 7 mg/100 ml for the full term animals and 20 +/- 8 mg/100 ml for the premature infants at 2 hr of life.

Development of Gluconeogenic Enzymes in the Newborn Guinea Pig

The activities of key gluconeogenic enzymes in the liver of newborn guinea pigs delivered vaginally at term were monitored as a function of time following birth. The activities of glucose-6-phosphatase and fructose-1,6-diphosphatase did not show a significant increase over the first 72 h of life, neither did the activity of mitochondrial phosphoenolpyruvate carboxykinase. The mitochondrial enzyme pyruvate carboxylase and the cytosolic phosphoenolpyruvate carboxykinase (PEPCK) both increased significantly in the first 24 h postpartum. Mitochondrial protein and succinate dehydrogenase activities showed only slight increases in the 72-hour period. Rapid depletion of liver glycogen was evident in these animals following birth, but severe hypoglycaemia was not evident. Mitochondrial and cytosolic PEPCK showed similar kinetic behaviour with respect to their affinities for oxalacetate and divalent metal cation Mn++, though the mitochondrial enzyme would accept Mg++ as the divalent metal in place of Mn++. The role of the compartmented PEPCK activities is discussed.

Rat Fetal Brown Adipose Tissue <i>in vitro</i>: Effects of Hormones and Ambient Temperature

Interscapular brown adipose tissue (BAT) from fetal rats was maintained in Eagle’s minimal essential medium for 6–24 h at 37 or 25 °C. Pyruvate kinase (PK), cytoplasmic and mitochondrial α-glycerophosphate dehydrogenase and phosphoenolpyruvate carboxykinase (PEPK) activities were determined at the start and end of incubation. During prenatal development, the activities of all enzymes, except PK, increased significantly. During incubation at 37 °C, PEPK activity decreased by about 60% in 24 h. The other enzyme activities also fell to a lesser extent. Only the mitochondrial enzyme remained unchanged. Both glucagon and insulin significantly slowed down the fall in PEPK activity. Norepinephrine added every hour for 6 h increased PEPK activity, but only in BAT from fetal rats weighing less than 3.5 g. Incubation at 25 °C largely prevented the decrease in enzyme activities seen at 37 °C. Fetal liver reacted in the opposite way. At 37 °C, PEPK activity increased while at 25 °C it decreased. However, postnatal liver responded to incubation temperature like fetal BAT. It is postulated that the initial level of PEPK (high in fetal BAT and postnatal liver and low in prenatal liver) plays a role in the <i>in vitro</i> response.

Changes in the activities of enzymes of kidney gluconeogenesis during development of the rat

1. 1. Changes in the activities of enzymes of kidney gluconeogenesis have been measured from late foetal to adult life. 2. 2. Fructose 1,6-diphosphatase increases in activity after birth, reaching adult values by about 30 days of age. 3. 3. Extramitochondrial phosphoenolpyruvate carboxykinase activity increases during late gestation, varies little between 2 and 10 days and then rises to a maximum at 30–40 days, thereafter falling to adult values. 4. 4. Mitochondrial and extramitochondrial pyruvate carboxylase show a somewhat similar developmental pattern and at all ages the mitochondrial enzyme has greater activity than the extramitochondrial enzyme. 5. 5. The mitochondrial phosphoenolpyruvate carboxykinase exhibited an irregular developmental pattern and at all ages except late foetal was of much lower activity than the extramitochondrial enzyme.

Development of mitochondrial pyruvate metabolism in rat brain.

AbstractThe activities of a number of mitochondrial enzymes involved in the metabolism of pyruvate during development of the rat brain were investigated. The rates of decarboxylation of [1‐14C]pyruvate to 14CO2 via pyruvate dehydrogenase and the fixation of H14CO3− in the presence of pyruvate via pyruvate carboxylase by brain homogenates were very low in newborn rats. These rates increased markedly by about four‐fold and 15‐fold respectively during 10–35 postnatal days. The rates of the fixation of H14CO3− by cerebral homogenates were supported by the development of the activity of pyruvate carboxylase in rat brain. The activities of citrate synthase, aconitase, NAD‐malate dehydrogenase, aspartate aminotransferase, alanine aminotransferase and phosphoenol‐pyruvate carboxykinase were very low in the particulate fraction of the newborn rat brain. The activities of all these enzymes increased makedly by about three‐ to 10‐fold during 10–35 days after birth. The activity of mitochondrial phosphoenolpyruvate carboxykinase from rat brain was not precipitated by an antibody prepared against rat liver cytosolic phosphoenolpyruvate carboxykinase suggesting that cerebral mitochondrial enzyme is immunologically different from that of the cytosolic form in hepatocytes. The significance of the development of the cerebral mitochondrial metabolism is discussed in relation to biochemical maturation of the brain.

A mitochondrial phosphoenolpyruvate carboxykinase from rat brain

Phosphoenolpyruvate carboxykinase from the rat brain has been purified approximately 6000-fold. This purified enzyme was stable at −20 °C for several months. Phosphoenolpyruvate carboxykinase from the rat brain was found to be mitochondrial, in contrast to that in the rat liver where this enzyme was always present in the soluble form. Other properties of the brain phosphoenolpyruvate carboxykinase were largely similar to those of the liver enzyme. The larger Michaelis constant for phosphoenolpyruvate in the direction of CO 2 fixation suggested that its function was more for the decarboxylation of oxaloacetate and formation of phosphoenolpyruvate. The possible implication of this reaction in both gluconeogenesis and coupling to substrate level phosphorylation of α-ketoglutarate were discussed.

The pathway of gluconeogenesis in the cortex of guinea-pig kidney. Use of aminooxyacetate as a transaminase inhibitor.

Aminooxyacetate is a potent inhibitor of glutamate: oxaloacetate transaminases from both rat and guinea pig kidney cortex. In guinea pig kidney cortex, phosphoenolpyruvate carboxykinase activity was found to be 59% cytoplasmic, 41% mitochondrial. Mitochondrial phosphoenolpyruvate carboxykinase activity is expressed only after sonication indicating that the enzyme is located within the inner mitochondrial membrane. Aminooxyacetate inhibits gluconeogenesis from lactate, but not from pyruvate, in both rat and guinea‐pig kidney cortex. It is concluded that gluconeogenesis in guinea‐pig kidney cortex proceeds by a pathway involving cytoplasmic, rather than mitochondrial phosphoenolpyruvate carboxykinase.

The regulation of glyceride synthesis from pyruvate in isolated fat cells. The effects of palmitate and alteration of dietary status.

The incorporation of 14C‐labelled pyruvate into glyceride fatty acids and glyceride glycerol has been measured in fat cells prepared from epididymal adipose tissues of normal rats, rats starved for 24 h, rats starved for 24 h and refed a carbohydrate diet for 24 h or rats starved for 24 h and refed a fat diet for 24 h. Rates of fatty acid synthesis were decreased in starved and starved, fat refed cells and increased in starved, carbohydrate refed cells. In all four dietary states, addition of palmitate to the incubation medium resulted in increased rates of glyceride glycerol formation and decreased rates of fatty acid synthesis from 14C‐labelled pyruvate. In normal cells these effects of palmitate were maximal below 1 mM palmitate. Dietary‐induced alterations in incorporation of 14C‐labelled pyruvate into glyceride glycerol, maximised by addition of palmitate, correlated well with corresponding alterations in the activity of extramitochondrial phosphoenolpyruvate carboxykinase in the cells, but did not correlate with alterations in the activity of pyruvate carboxylase or mitochondrial phosphoenolpyruvate carboxykinase. Use of marker enzymes allowed an assessment to be made of the distribution of these enzymes between extramitochondrial and mitochondrial spaces.

Kinetic studies with cytosol and mitochondrial phosphoenolpyruvate carboxykinases.

1. Measurements of Michaelis constants for oxaloacetate in the reaction catalysed by liver phosphoenolpyruvate carboxykinase give values much lower than previously reported. With Mg(2+) as bivalent cation, the Michaelis constant was approx. 2.5x10(-5)m whether the enzyme used was the mitochondrial phosphoenolpyruvate carboxykinase purified from sheep liver or chicken liver or the cytosol enzyme purified from rat liver or sheep liver. 2. When Mn(2+) replaced Mg(2+) in the reaction a lower Michaelis constant of 9x10(-6)m was found, but only with the mitochondrial enzymes. 3. With all enzymes malate at high concentration was a competitive inhibitor with respect to oxaloacetate when Mn(2+) was the added cation. With Mg(2+) the inhibition by malate was competitive with the mitochondrial enzymes and non-competitive with the cytosol enzymes.

Purification of Phosphoenolpyruvate Carboxykinase from the Cytosol Fraction of Rat Liver and the Immunochemical Demonstration of Differences between This Enzyme and the Mitochondrial Phosphoenolpyruvate Carboxykinase

Abstract Phosphoenolpyruvate carboxykinase (EC 4.1.1.32) has been isolated and purified from the cytosol fraction of rat liver to give a specific activity of 14 to 16 µmoles of bicarbonate fixed per min per mg of protein. The enzyme is homogeneous on disc electrophoresis and Sephadex G-100 chromatography and has a molecular weight of 74,500 with an isoelectric point of 5.04. Antibodies prepared in rabbits against the pure enzyme show identical titration curves against the cytosol P-enolpyruvate carboxykinase of liver and adipose tissue. A single continuous precipitation line is obtained on Ouchterlony double diffusion precipitation analysis when pure enzyme and the cytosol P-enolpyruvate carboxykinase of liver and adipose tissue are compared. On the other hand, the solubilized mitochondrial P-enolpyruvate carboxykinase of rat liver is not precipitated by the antibody to the cytosol enzyme. We conclude that the mitochrondrial and cytosol P-enolpyruvate carboxykinases are distinct enzymes, notwithstanding their uniform kinetic and physical properties.

Metabolic interactions of glucose, lactate, and beta-hydroxybutyrate in rat brain slices.

Metabolic interactions of glucose, lactate, and beta-hydroxybutyrate in rat brain slices.

Renal metabolic response to acid base changes. I. Enzymatic control of ammoniagenesis in the rat.

Experiments were done on rats to investigate the nature of the renal response to metabolic acidosis and the changes in enzyme activity associated with increased ammoniagenesis. When metabolic acidosis was induced with oral feeding of ammonium chloride for 48 hr, there was an increase of activity of the enzyme phosphoenolpyruvate carboxykinase (PEPCK) in whole kidneys as well as in the kidney cortex. There was no change in PEPCK in liver, and glucose-6-phosphatase showed no change in kidney or liver in response to metabolic acidosis. The increase in PEPCK activity in kidney cortex varied with the degree of acidosis and there was a close correlation between cortical PEPCK activity and urinary ammonia. Kidney cortex mitochondrial PEPCK did not change in response to metabolic acidosis. An increase in PEPCK occurred as early as 6 hr after NH(4)Cl feeding, before there was any increase in kidney glutaminase I activity. Rats fed sodium phosphate, or given triamcinolone intramuscularly, developed a metabolic alkalosis, but there was increased urinary ammonia and an increase in activity of renal cortical PEPCK. Triamcinolone plus ammonium chloride induced a greater increase of PEPCK activity than triamcinolone by itself; on the contrary, the rise of glucose-6-phosphatase induced by triamcinolone was not enhanced by acidosis. Glucose-6-phosphatase from control and acidotic rats had identical kinetic characteristics. The results indicate that increased PEPCK activity is constantly related to increases of urinary ammonia. It is proposed that the increase of PEPCK activity is the key event in the ammoniagenesis and gluconeogenesis which follow on metabolic acidosis.

The Enzymatic Carboxylation of Phosphoenolpyruvate: V. Kinetic and 18O studies on liver mitochondrial phosphoenolpyruvate carboxykinase

The Enzymatic Carboxylation of Phosphoenolpyruvate: V. Kinetic and 18O studies on liver mitochondrial phosphoenolpyruvate carboxykinase

The essential thiol group of sheep kidney mitochondrial phosphoenolpyruvate carboxykinase

1. 1. Evidence has been presented that the integrity of a cysteine-residue in sheep kidney mitochondrial phosphoenolpyruvate carboxykinase (GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32) is essential for full expression of catalytic activity. Modification of the enzyme with sulphydryl group reagents such as N-ethylmaleimide, iodoacetamide, iodosobenzoic acid, 5,5′-dithiobis(2-nitro benzoic acid), p-hydroxymercuribenzoic acid and p-hydroxymercuriphenylsulphonic acid all resulted in a loss of catalytic activity. Confirmation of the identity of the reactive residue as cysteine was achieved by characterising the radioactive derivative of the N- ethyl[ I- 14 C] maleimide- labelled enzyme. 2. 2. N-ethylmaleimide modification of the enzyme was first order with respect to both time and inhibitor concentration. Analysis of the kinetic data showed that an average of one molecule of the inhibitor reacted with the enzyme causing inactivation. The pH dependence of N-ethylmaleimide modification shows an inflection at pH 7.3 corresponding to the p K a of the reactive sulphydryl group. 3. 3. Protection against N-ethylmaleimide modification was afforded by the nucleotide substrate and analogues in the preferred order ITP < IDP > IMP > inosine. The dissociation constant, K D, for the enzyme-IDP complex was 8.04 · 10 −6 M ± 0.26. 4. 4.|Kinetic investigations of the modified enzyme showed that both K m (app) and ν max were decreased when phosphoenolpyruvate and IDP were the variable substrates while with HCO 3 − as the variable substrate mixed inhibition patterns were obtained depending on the degree of modification with decreases in ν max. 5. 5. N-ethylmaleimide modification resulted in a completely inactive product. However, methylation of this sulphydryl residue yields a derivative which shows a small residual activity (about 5–10% of that of the native enzyme). 6. 6. It is concluded that the function of the sulphydryl group of phosphoenolpyruvate carboxykinase is in some way associated with the catalytic process as distinct from binding.

The synthesis of phosphoenolpyruvate in the gluconeogenesis. Enzyme levels of chick embryo livers.

Livers of chick embryos at different stages of incubation have been fractionated into mitochondrial and “soluble” fractions by centrifugation.The pyruvate carboxylase and phosphoenolpyruvate carboxykinase activities, which constitute the major pathway for the synthesis of phosphoenolpyruvate, have been measured in both preparations.The pyruvate carboxylase activity occurs only in the mitochondria and parallels the gluconeogenetic ability.The bulk of phosphoenolpyruvate carboxykinase activity appears in the mitochondria. Only traces can be detected in the “soluble” fraction. The level of the mitochondrial phosphoenolpyruvate carboxykinase activity varies during the incubation period in accordance with its presumed role as a major source of phosphoenolpyruvate in the gluconeogenesis.The hypothesis that the pathway of synthesis of phosphoenolpyruvate in the process of gluconeogenesis is mainly located in the mitochondria of chick embryo liver is proposed.