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Molecular cloning of cDNA encoding mitochondrial very-long-chain acyl-CoA dehydrogenase from bovine heart
1Department of Chemistry, Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science & Technology, Portland 97291-1000, USA.
Aim:
To clone the cDNA encoding an isoenzyme of mitochondrial very-long-chain acyl-CoA dehydrogenase (VLCAD) from bovine heart lambda gt11 and lambda gt10 cDNA libraries.
Methods:
The clone was isolated with immunoscreening technique and validated by (1) the microsequences of the N-terminus and three internal proteolytic fragments from the purified enzyme; (2) identification of the acyl-CoA dehydrogenase (AD) signature sequence; and (3) high homology of the deduced peptide sequences, as expected, with those of rat liver mitochondrial VLCAD.
Results:
The cDNA (2203 bp) corresponds to a approximately 2.4-kb mRNA band from the same tissue source revealed by a Northern blotting. The deduced peptide sequence of 655 amino acids (70,537 Da) is composed of a 40-amino acid mitochondrial leader peptide moiety (4,346 Da) and a 615-amino acid peptide as a mature protein (66,191 Da). A comparison of the peptide sequences in the AD family shows the major diversity in their signal sequences, suggesting a structural basis for their different mitochondrial locations. The catalytic sites are all highly conserved among VLCAD. Ser-251 analogous to and Cys-215 diversified to other family members. A pseudo-consensus sequence of leucine zipper was found in the C-terminal region from Leu-568 to Leu-589, implying a mechanism whereby the dimer of this protein is formed by zipping these leucine residues from the alpha-helixes of 2 monomers.
Conclusion:
The isolated cDNA clone encodes an isoenzyme of mitochondrial VLCAD in bovine heart.