Original Articles

Molecular cloning of cDNA encoding mitochondrial very-long-chain acyl-CoA dehydrogenase from bovine heart

Xiao-Cheng Sun, Xiao-Wei Zhang, Wei Liu, Hong-Fa Zhu


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
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.

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