Purification and characterization of cytosol phosphoenolpyruvate carboxykinase from bullfrog (Rana catesbeiana) liver.

Cytosol PEP carboxykinase has been purified to electrophoretic homogeneity from bullfrog liver homogenate. The enzyme is a single polypeptide chain with a molecular weight of approximately 72,000-75,000. The purified enzyme catalyzed oxaloacetate decarboxylation (nucleoside triphosphate-supported), phosphoenolpyruvate carboxylation, and an exchange reaction between oxaloacetate and [14C]HCO3-in the presence of ITP or CTP. Manganese is absolutely required for the enzyme-catalyzed phosphoenolpyruvate carboxylation, whereas it can be replaced by Mg2+ for the oxaloacetate decarboxylation and the exchange reaction. The optimal pH of each reaction is dependent on the divalent metal ion used. The dependence of the enzyme activity on Mn2+ is markedly different in the phosphoenolpyuvate carboxylation and the oxaloacetate decarboxylation reactions.     

Phosphoenolpyruvate carboxykinase (guanosine 5'-triphosphate) from rat liver cytosol. Divalent cation involvement in the decarboxylation reactions

The presence of a divalent metal ion together with a catalytic amount of inosine 5'-diphosphate (IDP) is essential for the formation of pyruvate from oxalacetate catalyzed by purified rat liver cytosol phosphoenolpyruvate carboxykinase (PEPCK). With decreasing order of effectiveness, this pyruvate-forming activity was supported by micromolar levels of Cd2+, Zn2+, Mn2+, and Co2+. At the same concentrations, Mg2+ or Ca2+ was not effective. Combinations of Cd2+ with either Zn2+, Mn2+ or Co2+ were not additive with respect to the pyruvate-forming activity of PEPCK. Kinetic determination, with Cd2+ as the supporting cation, showed a 1:1 stoichiometry of interaction between each enzyme molecule and the nonconsumable substrate IDP. With 10 muM added Cd2+, the apparent Km for oxalacetate was 41 muM, and the apparent Ka for IDP was 0.25 muM. With Zn2+ or Mn2+, the apparent Ka for IDP was 0.2 or 0.13 muM, respectively. The effect of divalent transition-metal ions on PEPCK-catalyzed formation of phosphoenolpyruvate from oxalacetate was also investigated. Under steady-state conditions, the basal activity with MgITP was effectively enhanced with micromolar levels of Mn2+, Cd2+, or Co2+ included in the assay. The Vm increased 7- and 3.6-fold, and the apparent Km for MgITP changed by about a factor of 2 with the optimal concentrations of Mn2+ and Co2+, respectively. The most striking changes were in the apparent Km values for oxalacetate, which decreased to one-third and one-tenth when either Mn2+ or Co2+ was present in the assay together with Mg2+. The possible physiological importance of this kinetic effect is discussed.     

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.     

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.     

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+.     

The effect of 3-mercaptopicolinic acid on phosphoenolpyruvate carboxykinase (GTP) in the rat and guinea pig.

In vitro, 3-mercaptopicolinic acid inhibited phosphoenolpyruvate carboxykinase activity in supernatant fractions of liver, kidney cortex, and adipose tissue obtained from fasted rats. 3-Mercaptopicolinic acid also inhibited enzymatic activity in the mitochondrial and supernatant fractions of liver obtained from fasted guinea pigs. In the fasted rat, the oral administration of 3-mercaptopicolinic acid increased liver carboxykinase activity even though the blood glucose concentrations decreased. Kidney cortex carboxykinase decreased while adipose tissue enzyme was unchanged. In the fasted guinea pig, the oral administration of 3-mercaptopicolinic acid lowered blood glucose concentrations but had no effect on liver mitochondrial or supernatant carboxykinase activity. The elevation in rat liver enzymatic activity appears to be due to protein synthesis, since the concurrent administration of cycloheximide prevents the increase in enzyme activity. 3-Mercaptopicolinic acid appears to be noncompetitive with respect to Mn²⁺.     

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.     

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.     

Inactivation of phosphoenolypyruvate carboxykinase (GTP) by liver extracts.

1. The inactivation of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) in liver extracts was catalysed by the microsomal fraction, and led to the enzyme becoming bound to the microsomal membranes. 2. Inactivation by microsomal fraction, typsin or heating at 48degreesC was accelerated by L-cystine, D-cystine and oxidized glutathione and decreased by dithiothreitol. 3. MnC1(2) and CoC1(2) protected the enzyme from inactivation by heat or microsomal fraction, but did not affect the inactivation caused by trypsin. 4. Several proteinase inhibitors had no effect on the microsomal inactivation reaction, suggesting that proteolysis was not involved. 5. It is argued that the initial step in the degradation of phosphoenolpyruvate carboxykinase (GTP) is an inactivation reaction, perhaps involving oxidized thiol compounds.