Partial purification and characterization of rat-liver messenger RNA coding for phosphoenolpyruvate carboxykinase (GTP).

The mRNA coding for the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) was partially purified from the liver of cyclic-AMP-treated rats by a procedure involving multiple oligo(dT)-cellulose chromatographies and sucrose gradient fractionations. The purification was monitored by translational assay using a wheat germ extract. Relative to RNA bound once to oligo(dT)-cellulose, the final material was enriched 20-fold in template activity for phosphoenolpyruvate carboxykinase synthesis. With this RNA preparation, cell-free enzyme synthesis amounted to 5% of total mRNA-directed protein synthesis. The apparent sedimentation coefficient of phosphoenolpyruvate carboxykinase mRNA in sucrose gradients was between 20 and 22 S, corresponding to an average molecular weight of 0.93 X 10(6). By formamide/polyacrylamide gel electrophoresis the molecular weight of the enzyme mRNA was estimated at between 0.91 X 10(6) and 1.12 X 10(6). From these estimates, it was concluded that considerable non-coding sequence(s) are present in the mRNA. Approximately 20% of the enzyme mRNA in rat liver failed to bind to oligo(dT)-cellulose, presumably because of the absence of a poly(A) segment. The translation of phosphoenolpyruvate carboxykinase mRNA by the wheat germ extract was inhibited in the presence of 7-methylguanosine 5'-phosphate. The enzyme mRNA appears therefore to have a 'cap' at the 5' end.     

Infulence of hormones and medium composition on the degradation of phosphoenolpyruvate carboxykinase (GTP) and total protein in Reuber H35 cells.

Reuber H35 cells were pulse-labeled with radioactive leucine and the influence of hormones, serum, and amino acids on protein degradation was investigated during a subsequent chase period. Radioactive, immunoprecipitable phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) had a half-life of 5 to 6 hours which was not influenced by either N6, O2-dibutyryl adenosine 3':5'-monophosphate, dexamethasone, or insulin. The rate of phosphoenolpyruvate carboxykinase degradation was the same under steady state conditions as during the approach to a new steady state following hormonal induction or deinduction of the enzyme. Therefore, hormonal regulation of enzyme activity in vivo is the result of changes in the rate of enzyme synthesis. The rate of proteolysis for total cell proteins was increased under nutritional step-down conditions produced by the removal of serum or amino acids, or both, from the medium. This effect was completely prevented by insulin. Cycloheximide and puromycin, but not actinomycin D or cordycepin, inhibited protein degradation under step-down conditions but did not further decrease the basal rate of proteolysis measured in the presence of either insulin or serum plus amino acids. There was a good correlation between changes in proteolysis produced by serum and amino acids and changes in the degradation rate of phosphoenolpyruvate carboxykinase. Also, inhibition of proteolysis with cycloheximide and puromycin was accompanied by a decrease in the degradation rate for enzyme antigen. It is suggested that nutritional step-down leads either to the synthesis or activation of a proteolytic system.     

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.     

The phosphoryl-transfer mechanism of Escherichia coli phosphoenolpyruvate carboxykinase from the use of AlF(3).

The mechanism of reversible transfer of the gamma-phosphate group of ATP by Escherichia coli phosphoenolpyruvate carboxykinase (PCK) on to its substrate is of great interest. It is known that metallofluorides are accurate analogs of the transition state in the context of kinase mechanisms. Therefore, two complexes of PCK, one with AlF(3), Mg(2+) and ADP (complex I), the other with AlF(3), Mg(2+), ADP and pyruvate (complex II) were crystallized. The X-ray crystal structures of these two complexes were determined at 2.0 A resolution. The Al atom has trigonal bipyramidal geometry that mimics the transition state of phosphoryl transfer. The Al atom is at a distance of 2.8 A and 2.9 A from an oxygen atom of the beta-phosphoryl group of ADP in complex I and II, respectively. A water molecule in complex I and an oxygen atom of the pyruvate in complex II are located along the axis of the trigonal bipyramid on the side opposite to the beta-phosphoryl oxygen with respect to the equatorial plane, suggesting that the complexes are close mimics of the transition state. Along with the presence of positively charged species around the AlF(3) moiety, these results indicate that phosphoryl transfer occurs via a direct displacement mechanism with associative qualities.     

Induction by cAMP of the mRNA encoding the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the chicken. Identification and characterization of a cDNA clone for the enzyme

Previous work from our laboratory (Hod, Y., Utter, M. F., and Hanson, R. W. (1982) J. Biol. Chem. 257, 13787-13794) has demonstrated that chicken kidney contains both mitochondrial and cytosolic forms of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) and that the two forms are distinct proteins. Using poly(A+) RNA from chicken kidney, a double-stranded cDNA library was constructed. DNA clones containing sequences complementary to the mRNA for the cytosolic form of phosphoenolpyruvate carboxykinase were initially identified by colony hybridization with 32P-labeled cDNA transcribed from an RNA fraction enriched for the enzyme mRNA. The identity of plasmids containing phosphoenolpyruvate carboxykinase cDNA was confirmed by hybrid-selected translation. Mature mRNA for cytosolic phosphoenolpyruvate carboxykinase of the chicken is 2.8 kilobases in length, similar to that previously noted for mRNA coding for the same enzyme in the rat. The cDNA for the chicken enzyme hybridizes with several restriction fragments of the corresponding cDNA for the rat cytosolic phosphoenolpyruvate carboxykinase, indicating conservation of nucleotide sequences during evolution. Wide spread conservation of sequence homology is also demonstrated by the hybridization of the cDNA for the rat phosphoenolpyruvate carboxykinase with a 2.8-kilobase RNA from the livers of a variety of vertebrates including amphibian, avian, and primate species. Specific mRNA coding for the cytosolic form of phosphoenolpyruvate carboxykinase was present in chicken kidney but absent from the liver, even in animals starved for 48 h. However, the administration of cAMP to normal fed chickens caused a rapid induction of phosphoenolpyruvate carboxykinase mRNA. These findings suggest that the gene for the cytosolic enzyme in chicken liver can be expressed if the proper hormonal stimuli are present.