Mitochondrial phosphoenolpyruvate carboxykinase from the chicken. Comparison of the cDNA and protein sequences with the cytosolic isozyme

Abstract

The amino acid sequence of the mitochondrial form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK-M) from the chicken was deduced from the 3571 nucleotide sequence of three overlapping cDNA clones. The derived protein sequence, which includes 607 amino acids of the mature enzyme and a leader sequence, was aligned with nine tryptic peptides of PEPCK-M and the primary sequence of the cytosolic form of PEPCK from chicken. Secondary structure predictions for the two PEPCK isozymes indicated similar packing elements of conserved, hydrophobic beta strands in the central core of the primary sequence. This core protein, which contained three GTP-binding consensus elements, was 80% identical in the two chicken isozymes, although the overall level of identity was only 63% for amino acids and 60% for nucleotides. The untranslated regions of the two cDNAs were dissimilar, although both mRNAs have potential for significant secondary structure. The PEPCK-M mRNA contained several G-C-rich regions which demonstrated free energies of formation in dyad symmetry programs up to -70 kcal/mol. The 1.6-kilobase (kb) 3'-untranslated region contained several repeat elements including one of 11 base pairs, which was present 30 times; but, a signal sequence for polyadenylation was not present. Each of the three PEPCK-M cDNA clones recognized two mRNAs of 4.2 and 3.4 kb in the livers and kidneys of starved or normally fed chickens. However, the level of these two related PEPCK-M mRNAs changed in response to cAMP treatment, with the larger mRNA predominant at 20 and 160 min and the 3.4-kb mRNA present at intermediate times. In contrast, the level of the 2.8-kb PEPCK-C mRNA increased dramatically upon addition of the cyclic nucleotide, particularly in the liver where it was not detected without cAMP induction. Thus, PEPCK-M and PEPCK-C, clearly represented the products of two distinct genes, which were distinguished by altered protein sequences and non-cross-hybridizing, differentially regulated mRNAs.