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.

Highlights

  • One of the PEPCK-M peptides, which aligned near the carboxyl terminus of PEPCK-C, was chosen to develop an oligonucleotide probe for PEPCK-M

  • Isolation and sequencing of the cDNA presented in this paper allows the first presentation of the amino acid sequence of the mature form of the mitochondrial isozyme of PEPCK from the chicken

  • The distinctive nucleotide sequence gives rise to the corresponding unique protein sequence from which PEPCKM-specific tryptic peptides were obtained. These results demonstrate that PEPCK-M and PEPCK-C are encoded by separate genes

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Summary

COMPARISON OF THE cDNA AND PROTEIN SEQUENCES WITH THE CYTOSOLIC ISOZYME*

Isolation of full length cDNA (Hod et al, 1984a) and genomic (Hod et al, 1984b) clones for PEPCK-C allowed determination of the amino acid sequence (Cook et al, 1986) of one of the two chicken isozymes. The present study reports the nucleotide sequence of the mRNA and deduced protein sequence for the second isozyme from the chicken, PEPCK-M, and compares them to the corresponding sequences for PEPCK-C. Both the mRNA and amino acid sequences are analyzed for possible consequences due to secondary structure. The level of PEPCK-M mRNA in response to CAMP treatment was determined

PROCEDURES
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DISCUSSION
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