Studies on the primary structure of acetyl coenzyme A carboxylase

Dong-Hoon Bai, Purdue University

Abstract

A $\lambda$gt11 cDNA library, constructed using enriched poly (A)+ RNA from lactating rat mammary glands, was initially screened with polyclonal antiserum to acetyl-CoA carboxylase. Three positive clones were identified. Western blot analysis revealed that two clones, $\lambda$DHR3 and $\lambda$KHR18, synthesized 165,000 dalton fusion proteins which were recognized by antibodies to acetyl-CoA carboxylase and $\beta$-galactosidase. Immunoprecipitation of these proteins was competed by affinity column purified acetyl-CoA carboxylase. Furthermore, purified antibody isolated by binding to those specific fusion proteins immunoprecipitated acetyl-CoA carboxylase enzyme activity from a crude liver homogenate. In order to complete the cDNA sequence, a 5$\sp\prime$ cDNA fragment of $\lambda$DHR3 and a 3$\sp\prime$ cDNA fragment of $\lambda$KHR18 were used as initial screening probes to isolate additional overlapping cDNAs. Seven major clones, $\lambda$DHR3, $\lambda$KHR18, $\lambda$DHN106, $\lambda$DHN132, $\lambda$KHN18, $\lambda$KHN146, and $\lambda$DHN59, were isolated, and their restriction maps analyzed. Together they span 9 Kb of the mRNA coding for acetyl-CoA carboxylase. Four peptides from a CNBr digest and one peptide containing biocytin from a S. aureus V8 enzyme digest of rat liver acetyl-CoA carboxylase were isolated by high performance liquid chromatography. Amino acid analyses were performed with a gas phase sequenator. The amino acid sequences of these peptides were compared to the amino acid sequence deduced from the overlapping cDNA clones. All amino acid sequences from the peptide sequence analyses were identical to the deduced sequence. The amino acid sequence of the biotin binding site of rat acetyl-CoA carboxylase was Val-Met-Lys-Met. This tetrapeptide was located in close proximity to the amino terminus.

Degree

Ph.D.

Advisors

Kim, Purdue University.

Subject Area

Food science

COinS