Phosphoenolpyruvate carboxykinase in plants exhibiting crassulacean Acid metabolism

Phosphoenolpyruvate carboxykinase has been found in significant activities in a number of plants exhibiting Crassulacean acid metabolism. Thirty-five species were surveyed for phosphoenolpyruvate carboxykinase, phosphoenolpyruvate carboxylase, ribulose diphosphate carboxylase, malic enzyme, and malate dehydrogenase (NAD). Plants which showed high activities of malic enzyme contained no detectable phosphoenolpyruvate carboxykinase, while plants with high activities of the latter enzyme contained little malic enzyme. It is proposed that phosphoenolpyruvate carboxykinase acts as a decarboxylase during the light period, furnishing CO₂ for the pentose cycle and phosphoenolpyruvate for gluconeogenesis.     

Phosphoenolpyruvate carboxykinase is induced in growth-arrested hepatoma cells

Phosphoenolpyruvate carboxykinase (PEPCK) mRNA is elevated in H4IIEC3 rat hepatoma cells cultured at high density, suggesting that PEPCK expression and growth arrest may be coordinately regulated. Induction of growth arrest either by contact inhibition (high culture density) or by serum deprivation correlated with significant increases in PEPCK protein and its mRNA. The observation that PEPCK mRNA was induced by contact inhibition in the presence of serum indicates that the effect of high density is independent of insulin or any other serum component. The magnitudes of the changes in PEPCK expression during growth arrest were greatly enhanced in KRC-7 cells, an H4IIEC3 subclone that is much more sensitive to growth arrest than its parental cell line. Restimulation of proliferation in growth-arrested KRC-7 cells, either by addition of serum or insulin to serum-deprived cells or by replating contact-inhibited cells at low density, caused a rapid decrease in PEPCK expression. However, PEPCK mRNA is not always reduced in proliferating cells since treatment of serum-starved cells with epidermal growth factor stimulated entry into the cell cycle but did not affect PEPCK mRNA levels. Finally, dexamethasone induction of PEPCK mRNA was blunted in cells cultured at high density but was unaffected by the presence or absence of serum. Collectively, these data suggest the possibility of cross-talk between the control of PEPCK expression and growth arrest in KRC-7 cells.     

Phosphoenolpyruvate carboxykinase is a nuclear enzyme in rats and chickens

The presence of phosphoenolpyruvate carboxykinase (PEPCK) in the nuclei of chicken liver cells was confirmed using two experimental designs. PEPCK was found to be enriched in the nuclear fraction of rat liver, a species whose hepatic PEPCK is reported to be predominantly cytosolic. We suggest that PEPCK plays a role in nuclear synthesis of N-acetyl-neuraminic acid.     

Phosphoenolpyruvate carboxykinase overexpression selectively attenuates insulin signaling and hepatic insulin sensitivity in transgenic mice

The ability of insulin to suppress gluconeogenesis in type II diabetes mellitus is impaired; however, the cellular mechanisms for this insulin resistance remain poorly understood. To address this question, we generated transgenic (TG) mice overexpressing the phosphoenolpyruvate carboxykinase (PEPCK) gene under control of its own promoter. TG mice had increased basal hepatic glucose production (HGP), but normal levels of plasma free fatty acids (FFAs) and whole-body glucose disposal during a hyperinsulinemic-euglycemic clamp compared with wild-type controls. The steady-state levels of PEPCK and glucose-6-phosphatase mRNAs were elevated in livers of TG mice and were resistant to down-regulation by insulin. Conversely, GLUT2 and glucokinase mRNA levels were appropriately regulated by insulin, suggesting that insulin resistance is selective to gluconeogenic gene expression. Insulin-stimulated phosphorylation of the insulin receptor, insulin receptor substrate (IRS)-1, and associated phosphatidylinositol 3-kinase were normal in TG mice, whereas IRS-2 protein and phosphorylation were down-regulated compared with control mice. These results establish that a modest (2-fold) increase in PEPCK gene expression in vivo is sufficient to increase HGP without affecting FFA concentrations. Furthermore, these results demonstrate that PEPCK overexpression results in a metabolic pattern that increases glucose-6-phosphatase mRNA and results in a selective decrease in IRS-2 protein, decreased phosphatidylinositol 3-kinase activity, and reduced ability of insulin to suppress gluconeogenic gene expression. However, acute suppression of HGP and glycolytic gene expression remained intact, suggesting that FFA and/or IRS-1 signaling, in addition to reduced IRS-2, plays an important role in downstream insulin signal transduction pathways involved in control of gluconeogenesis and progression to type II diabetes mellitus.     

Phosphoenolpyruvate carboxykinase is an acid-induced, chromosomally encoded virulence factor in Agrobacterium tumefaciens

The pckA gene, encoding phosphoenolpyruvate carboxykinase, catalyzes the reversible decarboxylation and phosphorylation of oxaloacetate to form phosphoenolpyruvate. Located on the circular chromosome of Agrobacterium, this locus is adjacent to the loci chvG and chvI, encoding a two-component regulatory system that has been shown to be important in virulence. Using a reporter gene fusion, studies showed that the pckA gene is induced by acidic pH but not by acetosyringone. This acid induction is regulated by the chvG-chvI regulatory system, which controls acid-inducible genes. A pckA mutant had no demonstrable PckA enzyme activity and grew on AB minimal medium with glucose but did not grow on the same medium with succinate as the sole carbon source and was more inhibited in its growth than the wild-type strain by an acidic environment. A pckA mutant was highly attenuated in tumor-inducing ability on tobacco leaf disks and was severely attenuated in vir gene expression. Although vir gene induction was completely restored when a constitutive virG gene was introduced into the mutant strain, virulence was only partially restored. These results suggest that avirulence may be due to a combination of the inhibition of this mutant in the acidic plant wound environment and the poor induction of the vir genes.     

Phosphoenolpyruvate carboxykinase plays a role in interactions of carbon and nitrogen metabolism during grape seed development

Phosphoenolpyruvate carboxykinase (PEPCK) was shown to be present in a range of developing seeds by measurement of its activity and by immunoblotting. Its function was investigated during grape (Vitis vinifera L.) seed development. The maximum abundance of PEPCK coincided with the deposition of storage reserves. At this stage of development, immunohistochemistry showed that PEPCK was very abundant in a layer of cells located at the boundary of developing storage tissues and in the chalaza (close to the termination of the vascular supply to the seed) and was also present in the palisade layer of the seed coat (the inner layer of the outer integument). Earlier in development PEPCK was also present in the developing palisade layer and in the inner region of the nucellus which surrounds the developing endosperm. At later stages of development, PEPCK was located in the outer region of the endosperm. However, PEPCK was present in the phloem of the seed at all stages of development. Feeding of asparagine to developing grape seeds led to a strong induction of PEPCK. We suggest that, in developing grape seeds, both the chalaza and palisade tissue may distribute imported assimilates from the vasculature to the developing storage tissues and that PEPCK may play a role in the metabolism of nitrogenous assimilates during their delivery from the vasculature to the storage tissues. Received: 22 April 1999 / Accepted: 8 July 1999     

Phosphoenolpyruvate carboxykinase and gluconeogenesis in cotyledons of Cucurbita pepo

1. The aim of this work was to investigate the role of phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating) EC 4.1.1.49) in the conversion of fat to sugar by the cotyledons of seedlings of Cucurbita pepo. 2. The enzyme was partially purified from the cotyledons of 5-day-old seedlings. The Michaelis constants for oxaloacetate and ATP were 56 and 119 micron, respectively. The decarboxylation reaction was optimum at pH 7.4. A range of intermediary metabolites did not affect the activity of the enzyme, but 3-mercaptopicolinic acid at micron concentrations was an effective inhibitor. 3. Centrifugation of extracts of 5-day-old cotyledons sedimented appreciable proportions of the ribuloseibisphosphate carboxylase, isocitrate lyase and fumarate hydratase present but very little of the phosphoenolpyruvate carboxykinase. 4. Measurements of phosphoenolpyruvate carboxykinase of cotyledons during germination showed that the maximum catalytic activity exceeded, and changed coincidently with, the rate of gluconeogenesis. 5. 3-Mercaptopicolinic acid inhibited gluconeogenesis from [1-14C]- and [2-14C]acetate supplied to excised cotyledons. The detailed distribution of 14C indicated inhibition of the conversion of oxaloacetate to phosphoenolpyruvate. 6. It is concluded that in marrow cotyledons phosphoenolpyruvate carboxykinase is in the soluble phase of the cytoplasm and catalyses a component reaction of gluconeogenesis.     

Phosphoenolpyruvate carboxykinase in developing pea seeds is associated with tissues involved in solute transport and is nitrogen-responsive

The aim of this work was to investigate the occurrence of phosphoenolpyruvate carboxykinase (PEPCK) in developing pea (Pisum sativum) seeds in relation to their nitrogen supply. PEPCK was present throughout development, with the peak of PEPCK protein and activity in the seed coat and cotyledons preceding protein accumulation in the cotyledons. It showed a different developmental pattern from enzymes involved in amino acid metabolism (phosphoenolpyruvate carboxylase, glutamine synthetase and glutamate dehydrogenase). Immunolocalization showed that PEPCK was present in parts of the developing seed that are involved in the transport and metabolism of assimilates. Early in development, it was associated with the inner integument of the ovule, the endospermic cytoplasm and the outer cells of the embryo. In the middle of development, around the peak of activity, PEPCK was abundant at the outer surface of the developing cotyledons, in the embryonic axis and in the vasculature of the seed coat. Later in development, PEPCK was associated with the embryonic leaf primordia and meristem and cortex of the radicle. PEPCK protein was strongly induced in vitro in the seed coat by nitrate, ammonium and asparagine, in the cotyledons by asparagine and in planta by the supply of nitrogen, which led to an increase in asparagine secretion by empty seed coats. It is suggested that PEPCK is involved in the metabolism of nitrogenous solutes in developing pea seeds.     

Phosphoenolpyruvate carboxykinase in cucumber plants is increased both by ammonium and by acidification,and is present in the phloem

In cucumber (Cucumis sativus L.), phosphoenolpyruvate carboxykinase (PEPCK) was shown by activity measurements and immunoblots to be present in leaves, stems, roots, flowers, fruit and seed. However, immunolocalisation showed that it was present only in certain cell types. PEPCK was present in the companion cells of the adaxial phloem of minor veins, the adaxial and abaxial phloem of larger veins, the internal and external phloem of vascular bundles in petioles and stems, the phloem in roots and the extra-fascicular phloem in leaves, cotyledons, petioles and stems. Immunohistochemical evidence suggests that both the extra-fascicular phloem and the adaxial phloem are involved in the transport of amino acids. In roots and stems, the abundance of PEPCK was greatly increased by watering plants with a solution of ammonium chloride at low, but not at high pH. PEPCK also increased in leaves, but not roots or stems, of seedlings grown in an atmosphere containing 5% CO₂, and in roots and stems of seedlings watered with butyric acid. All these treatments are known to lower the pH of plant cells. Amino acid metabolism in the phloem may produce an excess of carbon skeletons, pH perturbations and an imbalance in the production/utilisation of NADH. This raises the possibility that PEPCK may function in the conversion of these carbon skeletons to PEP, which, depending on the energy requirements of the phloem, is subsequently utilised by either gluconeogenesis or the Krebs cycle, which both consume protons.Abbreviations Asp Aspartate - Asn Asparagine - Glu Glutamate - Gln Glutamine - NADP-ME NADP-malic enzyme - OAA Oxaloacetate - PEP Phosphoenolpyruvate - PEPC Phosphoenolpyruvate carboxylase - PEPCK Phosphoenolpyruvate carboxykinase     

Cholesterol is necessary for triacylglycerol-phospholipid emulsions to mimic the metabolism of lipoproteins

Emulsions with lipid compositions similar to the triacylglycerol-rich lipoproteins were metabolized similarly to natural chylomicrons or very-low-density lipoproteins when injected intravenously in rats. Radioactive labels tracing the emulsion triacylglycerols and cholesteryl esters were both removed rapidly from the blood stream, but the removal rate of triacylglycerols was faster than that of cholesteryl ester. Most of the removed cholesteryl ester label was found in the liver, but only a small fraction of the triacylglycerol label was found in this organ, consistent with hepatic uptake of the remnants of the injected emulsion. Emulsions otherwise identical but excluding unesterified cholesterol were metabolized differently. The plasma removal of triacylglycerols remained fast, but the cholesteryl esters were removed very slowly. Heparin stimulated lipolysis, but failed to increase the rate of removal of cholesteryl esters from emulsions lacking cholesterol. Evidently, emulsions lacking cholesterol were acted on by the enzyme lipoprotein lipase, but the resultant triacylglycerol-depleted remnant particle remained in the plasma instead of being rapidly taken up by the liver. Therefore, the presence of emulsion cholesterol is a critical determinant of early metabolic events, and the findings point to a similar role for cholesterol in the natural triacylglycerol-rich lipoproteins.     

Phosphoenolpyruvate carboxykinase ferroactivator 1. Mechanism of action and identity with glutathione peroxidase

A cytosolic protein factor (ferroactivator) facilitates the activation of phosphoenolpyruvate carboxykinase by ferrous ions (Bentle, L. A., and Lardy, H. A. (1977) J. Biol. Chem. 252, 1431-1440). We have extended our studies on the interaction of Fe2+ with this enzyme to establish the conditions under which it is an activator or an inhibitor. Preincubation of phosphoenolpyruvate carboxykinase with Fe2+ and dithiothreitol resulted in irreversible loss of enzyme activity within minutes of Fe2+ addition. This was attributed to an active oxygen species produced by aerobic oxidation of the divalent metal ion in the presence of dithiothreitol as suggested by lack of inhibition in preincubation experiments with Fe2+ under mildly acidic pH; ferroactivation by many H2O2 scavenging enzymes; and lack of inhibition on preincubation under anaerobic conditions. We conclude that Fe2+ per se can activate phosphoenolpyruvate carboxykinase and that ferroactivator protein helps to overcome the deleterious effects of aerobic oxidation. Mechanistic details of ferroactivation and a comparison of the known properties of ferroactivator indicated the similarity of this protein with rat liver glutathione peroxidase. The identity of ferroactivator as glutathione peroxidase was confirmed by the demonstration of catalytic activity, selenium content, and immunological cross-reactivity.     

Phosphoenolpyruvate carboxykinase of kidney. Subcellular distribution and response to acid–base changes

1. A method for the assay of phosphoenolpyruvate carboxykinase is presented, based on the enzymic determination of the phosphoenolpyruvate produced by the enzyme reaction. 2. The subcellular distribution of phosphoenolpyruvate carboxykinase in the kidney of several animal species resembled the distribution in the liver. 3. The rise in enzyme activity in the kidney cortex of rats made acidotic by feeding with ammonium chloride was not prevented by administration of ethionine or actinomycin. 4. The possibility is suggested that in the kidney acidosis causes activation of an inactive form of the enzyme already present.     

Mitochondrial creatine kinase is critically necessary for normal myocardial high-energy phosphate metabolism

The individual functional significance of the various creatine kinase (CK) isoenzymes for myocardial energy homeostasis is poorly understood. Whereas transgenic hearts lacking the M subunit of CK (M-CK) show unaltered cardiac energetics and left ventricular (LV) performance, deletion of M-CK in combination with loss of sarcomeric mitochondrial CK (ScCKmit) leads to significant alterations in myocardial high-energy phosphate metabolites. To address the question as to whether this alteration is due to a decrease in total CK activity below a critical threshold or due to the specific loss of ScCKmit, we studied isolated perfused hearts with selective loss of ScCKmit (ScCKmit(-/-), remaining total CK activity approximately 70%) using (31)P NMR spectroscopy at two different workloads. LV performance in ScCKmit(-/-) hearts (n = 11) was similar compared with wild-type hearts (n = 9). Phosphocreatine/ATP, however, was significantly reduced in ScCKmit(-/-) compared with wild-type hearts (1.02 +/- 0.05 vs. 1.54 +/- 0.07, P < 0.05). In parallel, free [ADP] was higher (144 +/- 11 vs. 67 +/- 7 microM, P < 0.01) and free energy release for ATP hydrolysis (DeltaG(ATP)) was lower (-55.8 +/- 0.5 vs. -58.5 +/- 0.5 kJ/mol, P < 0.01) in ScCKmit(-/-) compared with wild-type hearts. These results demonstrate that M- and B-CK containing isoenzymes are unable to fully substitute for the loss of ScCKmit. We conclude that ScCKmit, in contrast to M-CK, is critically necessary to maintain normal high-energy phosphate metabolite levels in the heart.     

Phosphoenolpyruvate carboxykinase assayed at physiological concentrations of metal ions has a high affinity for CO2.

The effect of Mn2+/Mg2+ concentration on the activity of intact, homogeneous phosphoenolpyruvate carboxykinase (PEPCK) from leaves of the C4 grass, Guinea grass (Panicum maximum), have been investigated. Assay conditions were optimized so that PEPCK activity could be measured at concentrations of Mn2+/Mg2+ similar to those found in the cytosol (low micromolar Mn2+ and millimolar Mg2+). PEPCK activity was totally dependent on Mn2+ and was activated at low micromolar concentrations of Mn2+ by millimolar concentrations of Mg2+. Therefore, at physiological concentrations of Mn2+, PEPCK has a requirement for Mg2+. Assay at physiological concentrations of Mn2+/Mg2+ led to a marked decrease in its affinity for ATP and a 13-fold increase in its affinity for CO2. The Km (CO2) was further decreased by assay at physiological ATP to ADP ratios, reaching values as low as 20 microM CO2, comparable with the Km (CO2) of ribulose 1,5-bisphosphate carboxylase-oxygenase. This means that PEPCK will catalyze a reversible reaction and that it could operate as a carboxylase in vivo, a feature that could be particularly important in algal CO2-concentrating systems.     

Phosphoenolpyruvate carboxykinase activity patterns in the liver acinus of diabetic and diabetic and estrogen treated rats

The acinar activity pattern of phosphoenolpyruvate carboxykinase (PEPCK) was investigated in livers of streptozotocin diabetic male and female rats and in addition in livers of diabetic males, which had undergone estrogen treatment. In all diabetic animals blood glucose levels were supranormal and liver PEPCK activity was increased. This increase in activity was greatest in estrogen treated diabetic males and lowest in diabetic females. Plasma insulin levels were reduced after the application of streptozotocin to otherwise normal male and female rats. Yet, in males treated in addition with estrogens the plasma insulin levels reached the normal range again. The PEPCK activity showed a heterotopic distribution along the acinus. The periportal to perivenous gradient was steeper in males compared to females in the untreated as well as in the diabetic state. The application of estrogens to males resulted in a further steepening of the gradient.