Title

Adipose pyruvate carboxylase: Molecular cloning ofcDNA and regulation in 3T3-L1 cells

Date of Award

1994

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Fazal Ahmad, Committee Chair

Abstract

The complete primary structure of mouse adipocyte pyruvate carboxylase (PC) has been deduced by sequencing overlapping cDNA clones isolated by screening an adipocyte cDNA library constructed in the $\lambda$ Zap vector. The PC cDNA contains 4,067 nucleotides, including a 3,534 nucleotide coding sequence and noncoding regions of 100 and 433 nucleotides at the 5$\sp\prime$ and 3$\sp\prime$ ends, respectively. The PC protomer contains 1,178 amino acids (calculated molecular weight of apo-PC = 129,784). The lysine which is biotinated is located 35 residues from the COOH-terminus and is in the consensus sequence, Ala-Met-Lys-Met. Except the activator (acetyl CoA) binding site, location of all of the functional domains along the PC protomer polypeptide chain has been identified and are in the order: mitochondrial targeting signal, biotin carboxylase, carboxyltransferase and the biotin carboxyl carrier protein.The regulation of PC gene in 3T3-L1 cells has also been investigated. The specific activity of PC, in parallel with its protein content, as estimated by Western blotting, increases about 25 fold when 3T3-L1 fibroblasts differentiate to adipocytes. This increase in PC protein is accompanied by a 9 to 10 fold increase in the steady-state levels of PC mRNA as estimated by Northern blotting using a cDNA probe coding for a segment of adipocyte PC. Exposure of adipocytes to cAMP for 24 h decreases cellular protein by about 25% with marked decreases in PC activity and in PC mRNA content. After 24 h treatment with cAMP, both the PC mRNA and catalytic activity decreased to 12% and 2% of control values, respectively. After 48 h exposure to cAMP, PC activity and mRNA decreased to barely detectable levels. Insulin added simultaneously with cAMP partially depressed the cAMP-evoked changes affecting the activity and mRNA content of adipocyte PC. In adipocytes fed medium containing cAMP + insulin, PC activity and its mRNA content declined more slowly during the first 24 h of exposure but after 48 h of treatment their levels dropped to values nearing those of adipocytes treated with cAMP alone. These data suggest that cAMP regulates adipocyte PC by altering the cellular content of its mRNA.Further studies conducted on the mechanism of control of adipocyte PC by cAMP showed that cAMP-treated adipocytes contained PC protein at levels close to those of control adipocytes. This finding indicates that PC may become catalytically inactive in cAMP-treated cells. Additional work is needed to unravel the mechanism by which active PC is converted to the inactive form in adipocytes exposed to cAMP.

Keywords

Biology, Molecular; Biology, Cell

Link to Full Text

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