Purification of phosphoenolpyruvate carboxykinase (GTP) by affinity chromatography on agarose-hydrazide-GTP

The cytosolic form of phosphoenolpyruvate carboxykinase (GTP; EC 4.1.1.32) from rat liver was purified by a procedure involving affinity chromatography on agarose-hydrazide-GTP. Phosphoenolpyruvate carboxykinase is retained quantitatively by the affinity medium in the presence of manganese and can be specifically eluted by a pulse of GTP. On the contrary, no binding to agarose-hydrazide-GTP occurs in the absence of manganese. This suggests that the affinity of the enzyme for GTP is enhanced by prior interaction with manganese. A combination of several conventional purification steps followed by affinity chromatography provides pure phosphoenolpyruvate carboxykinase in good yields. The final specific activity is 19 U/mg protein. The enzyme migrates as a single polypeptide of molecular weight 70,600 during electrophoresis on sodium dodecyl sulfate polyacrylamide gels.     

Induction of mRNA for phosphoenolpyruvate carboxykinase (GTP) by dexamethasone in cultured rat hepatocytes requires on-going protein synthesis.

Dexamethasone is necessary and sufficient to induce mRNA for phosphoenolpyruvate carboxykinase (GTP) (PEPCK) by 19-fold in rat hepatocytes cultured in serum-free medium. However, the time required for maximum induction is 16 h. The slow induction suggested that glucocorticoids regulate the expression of an intermediate gene product(s) which is required for glucocorticoid stimulation of PEPCK-gene expression. Consistent with this notion was the finding that cycloheximide completely blocked the response to dexamethasone. In contrast, cycloheximide did not block the response to a cyclic AMP analogue.     

trans activation of rat phosphoenolpyruvate carboxykinase (GTP) gene expression by micro-coinjection of rat liver mRNA in Xenopus laevis oocytes.

To study the liver-specific trans activation of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene, the PEPCK promoter was linked to a reporter gene and was microinjected into Xenopus laevis oocytes alone or in conjunction with rat liver poly(A)+ RNA. The rat liver mRNA markedly enhanced the expression of the PEPCK-chimeric construct. This effect appeared to be sequence specific, as it was dependent on the presence of the intact promoter. Moreover, the RNA effect was limited to mRNA preparations from PEPCK-expressing tissues only. Finally, microinjection of size-fractionated liver mRNA revealed that the trans-acting factor(s) is encoded by RNA of 1,600 to 2,000 nucleotides, providing a direct bioassay for the gene(s) involved in this tissue-specific trans-activation process.     

Cysteine 288: an essential hyperreactive thiol of cytosolic phosphoenolpyruvate carboxykinase (GTP)

Phosphoenolpyruvate carboxykinase from the cytosol of rat liver has 13 cysteines, at least one of which is known to be very reactive and essential for catalytic activity (Carlson, G. M., Colombo, G., and Lardy, H. A. (1978) Biochemistry 17, 5329-5338). In order to identify the essential cysteine, this enzyme was modified with the fluorescent sulfhydryl reagent N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide. Incubation of phosphoenolpyruvate carboxykinase with a 10% molar excess of this maleimide at 0 degrees C results in the rapid and nearly complete loss of catalytic activity. Under these conditions, 1 mol of the maleimide is incorporated per mol inactivated enzyme. The substrate GDP provides almost complete protection against inactivation and modification, while phosphoenolpyruvate protects against the rate, but not the extent, of modification. The pH dependence of the rate of enzyme inactivation suggests that the modified residue has a pK alpha of approximately 7.0. Purification and sequencing of the labeled peptide identifies the hyperreactive essential cysteine as Cys-288. This cysteine lies between two putative phosphoryl-binding domains and within a hydrophobic sequence.     

Regulation of phosphoenolpyruvate carboxykinase (GTP) in adipose tissue in vivo by glucocorticoids and insulin.

1. The regulation of the synthesis of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) in epididymal adipose tissue, liver and kidney in vivo was studied immunochemically. 2. Phosphoenolpyruvate carboxykinase (GTP) synthesis in adipose tissue is increased by starvation, diabetes and noradrenaline, and decreased by re-feeding and insulin. These changes were also seen in adrenalectomized rats and are qualitatively similar to those observed for the liver enzyme. This indicates the involvement of cyclic AMP as an inducer and insulin as a de-inducer in the regulation of phosphoenolpyruvate carboxykinase (GTP) in both tissues. (Induction and de-induction are defined as selective increase and decrease respectively in the rate of enzyme synthesis, regardless of the mechanism involved.)3. Adrenalectomy had little effect on phosphoenolpyruvate carboxykinase (GTP) synthesis in liver and kidney, but increased the synthesis rate of the adipose-tissue enzyme. Starvation and adrenalectomy had additive effects in increasing the synthesis rate of adipose-tissue phosphoenolpyruvate carboxykinase (GTP). In adrenalectomized diabetic rats glucocorticoids increased phosphoenolpyruvate carboxykinase (GTP) synthesis in liver and kidney while decreasing enzyme synthesis in adipose tissue. De-induction of adipose tissue phosphoenolpyruvate carboxykinase (GTP) is therefore regulated independently by glucocorticoids and insulin. 4. Although liver, kidney and adipose-tissue phosphoenolpyruvate carboxykinases (GTP) are seemingly identical, there is an apparent tissue-specific differentiation in regulatory systems for the enzyme.     

Photochemical cross-linking of guanosine 5'-triphosphate to phosphoenolpyruvate carboxykinase (GTP).

Mammalian phosphoenolpyruvate carboxykinase (PEPCK) specifically requires a guanosine or inosine nucleotide as a substrate; however, the structural basis for this nucleotide specificity is not yet known. Because affinity labels derived from guanosine have not yielded a stable, modified peptide in quantities sufficient for sequence analysis, we have investigated the utility of direct photochemical cross-linking of GTP to PEPCK in order to identify the nucleotide binding site. UV irradiation at a distance of 2 cm by a Mineralight lamp (330 microW/cm2) results in the attachment of [alpha-32P]GTP to PEPCK via a stable, covalent linkage in a reaction that is dependent upon GTP concentration and duration of irradiation. After 10 min of irradiation, more than 0.2 mol of [alpha-32P] GTP is incorporated per mole of PEPCK; under these conditions the GTP concentration required for half-maximal labeling is 69 microM. The substrates phosphoenolpyruvate, ITP, and GDP provide protection against photolabeling, as do Mn2+ and Mg2+. One major and one minor radioactive peptide derived from proteolytic digests of photolabeled PEPCK have been isolated and identified. The major modified peptide has been provisionally assigned to an acidic region near the C-terminus, and the minor peptide has been identified as Ser462-Lys471.     

Effects of glucagon, dexamethasone and triiodothyronine on phosphoenolpyruvate carboxykinase (GTP) synthesis and mRNA level in rat liver cells

Acute hormonal effects on the synthesis rate of the cytosolic form of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (GTP), were investigated using rat hepatocytes maintained in short-term suspension culture. Cells were pulse-labeled with [3H]leucine or [35S]methionine and the rate of synthesis of phosphoenolpyruvate carboxykinase was estimated after immunoprecipitation of cell extracts with specific antibodies or following high-resolution two-dimensional gel electrophoresis of cell proteins. Total RNA was also extracted from cultured cells and subsequently translated in a wheat germ cell-free protein-synthesis system, in order to quantify the level of functional mRNA coding for phosphoenolpyruvate carboxykinase. Glucagon, the single most effective inducer, causes a 15--20-fold increase in the level of specific mRNA in 2 h, accompanied by a similar increase in enzyme synthesis rate. The extent of induction is further amplified about threefold when dexamethasone is added to the culture medium. The synergistic action of dexamethasone does not require pre-exposure of the cells to the glucocorticoid, but on the contrary occurs without lag upon simultaneous addition of glucagon and dexamethasone. The induction of phosphoenolpyruvate carboxykinase mRNA by glucagon is markedly depressed in hepatocytes inhibited for protein synthesis by cycloheximide. Cycloheximide-inhibited cells, however, display a considerable induction of the message after joint stimulation with dexamethasone and glucagon. Thus, the synergistic action of dexamethasone does not require concomitant protein synthesis. These data provide indirect evidence for a primary effect of the glucocorticoids on the expression of the phosphoenolpyruvate carboxykinase gene. Besides glucagon and dexamethasone, the thyroid hormones are shown to influence the rate of phosphoenolpyruvate carboxykinase synthesis in isolated liver cells. The stimulatory effect of 3,5,3'-triiodothyronine (T3) is best demonstrated as a twofold increase in relative rate of enzyme synthesis in cells supplied with T3 plus glucagon, as compared to cells challenged with glucagon alone. The effect of T3 relies on a pretranslational mechanism, as shown by a commensurate increase in functional mRNA coding for phosphoenolpyruvate carboxykinase. Dose-response experiments with T3 as well as dexamethasone demonstrate effects at very low hormone levels, consistent with a role for these hormones as physiological modulators of phosphoenolpyruvate carboxykinase expression.     

The gene encoding the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the chicken

The gene for cytosolic phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) from the chicken was isolated from a recombinant library containing the chicken genome in phage lambda Charon 4A. The isolated clone, lambda PCK1cc, contains the complete gene for the enzyme as well as both 5' and 3' flanking sequences. The gene is approximately 8 kilobases in length divided into 8 exons, as demonstrated by restriction endonuclease mapping and DNA-RNA heteroduplex analysis. Southern blotting of chicken chromosomal DNA digested with various restriction enzymes shows a pattern predicted from the restriction map of lambda PCK1cc. The phosphoenolpyruvate carboxykinase gene is present as a single copy in the haploid chicken genome. The 5' region of the gene was defined by S1 nuclease mapping and by sequencing. Two mRNA species with discrete 5' ends were observed using S1 nuclease mapping. The ratio between the amounts of these multiple forms of mRNA is the same in chicken kidney and liver and is not affected by induction of the enzyme mRNA by cAMP. Examination of sequence homologies with the gene for rat cytosolic phosphoenolpyruvate carboxykinase indicates a putative control region contained in flanking sequences at the 5' end of the gene.     

Mechanism of 3-mercaptopicolinic acid inhibition of hepatic phosphoenolpyruvate carboxykinase (GTP).

The hypoglycemic agent 3-mercaptopicolinic acid inhibits gluconeogenesis from lactate by isolated, perfused livers from fasted rats and guinea pigs. A 3-mercaptopicolinate concentration of 50 muM caused a sharp decrease in glucose synthesis, with virtually complete inhibition at 100 muM. This inhibitory effect was reversed completely when 3-mercaptopicolinate was removed and the rate of glucose synthesis returned to normal values within 2 min. Oxygen consumption was not altered, even at the highest concentration of inhibitor. Gluconeogenesis from glycerol by guinea pig liver was blocked completely by 100 muM 3-mercaptopicolinate but was inhibited only partially in rat liver. After removal of the inhibitor glucose synthesis returned to levels higher than noted before the addition of this compound. The formation of P-enolpyruvate bu isolated guinea pig liver mitochondria metabolizing alpha-ketoglutarate (State 3) was inhibited markedly by 3-mercaptopicolinate, but malate conversion to P-enolpyruvate was considerably less sensitive. Kinetic studies with purified P-enolpyruvate carboxykinase from rat liver cytosol indicate that 3-mercaptopicolinate is a noncompetitive inhibitor with respect to both oxalacetate and MnGTP2-, and that simulataeous saturation with both substrates does not diminish this inhibition. The inhibitory effects of 3-mercaptopicolinate occur primarily by decreasing the rate of product formation while having relatively minor effects on the apparent Michaelis constants for substrates. Inhibition constants for slope and intercept effects ranged from 3 to 9 muM 3-mercaptopicolinate, and the inhibition patterns were dependent on the concentration of free Mn2+ present. Comparison of the inhibition constants with the observed inhibition of gluconeogenesis in livers perfused with 3-mercaptopicolinate supports the contention that P-enolpyruvate carboxykinase is the site of action of this inhibitor. The possibility that 3-mercaptopicolinate inhibition occurs by binding either free or bound manganese was eliminated by determination of the dissociation constant of 0.51 mM for the manganese-3-mercaptopicolinate complex. In addition, no tightly bound, slowly exchanging metal was bound to purified enzyme protein. These results suggest that 3-mercaptopicolinate inhibits by the removal of a tightly bound, rapidly exchanging metal ion other than Mn2+.     

3-Aminobenzamide inhibits poly(ADP ribose) synthetase activity and induces phosphoenolpyruvate carboxykinase (GTP) in H4IIE hepatoma cells

The purpose of this study was to determine whether changes in ADP-ribosylation affect expression of the gene encoding the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) in H4IIE hepatoma cells. Treatment with 3-aminobenzamide, a specific inhibitor of poly(ADP ribose) synthetase, caused an 89% decrease of ADP-ribosylation in isolated nuclei, and resulted in a two- to threefold induction of immunoassayable PEPCK in cultured cells. In contrast, the structurally related compound p-aminobenzoic acid had no significant effect on either process. In vivo labeling of proteins with [35S]methionine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, showed that the induction of immunoreactive PEPCK by 3-aminobenzamide was due to a selective increase in the synthesis of the protein. The specific induction of PEPCK synthesis by 3-aminobenzamide was accounted for by a twofold increase of mRNAPEPCK which reached its maximal value 4 h after the addition of 3-aminobenzamide and returned to the basal level by 10 h. A possible role of ADP-ribosylation in cAMP or glucocorticoid induction of PEPCK was investigated in experiments in which H4IIE cells were treated with combinations of 3-aminobenzamide and either dexamethasone or a cAMP analog. In each case the effects on PEPCK induction were additive, indicating that glucocorticoids and cAMP induce PEPCK by pathways different from that used by 3-aminobenzamide.