Structurally different rat liver medium-chain acyl-CoA dehydrogenase directed by complementary DNA's differing in their 5'-region

Abstract

Different forms of rat liver medium-chain acyl CoA dehydrogenase (MCAD) (EC 1.3.99.3) were produced in Escherichia coli carrying expression plasmids (pRMCADm-1−9) differing at the 5′-region of the cDNA. The proteins expressed could be readily extracted from the cells. The protein (not, vert, similar44 kDa) directed by pRMCADm-3 showed the highest activity and was readily purified to homogeneity. The purified enzyme contained non-covalently bound FAD and was similar to rat liver mitochondrial enzyme in all respects examined. The purified protein (not, vert, similar45 kDa) directed by pRMCADm-1 did not contain FAD and showed no enzymatic activity. Therefore, the leader peptide disturbs the binding of FAD to the apoprotein. The purified protein (not, vert, similar40 kDa) directed by pRMCADm-6 did not contain FAD. Thus, the deletion of the NH2-terminal portion of the apoprotein to some extent results in its inability to combine with FAD.