In vitro synthesis of pig kidney general acyl CoA dehydrogenase

In vitro synthesis of general acyl CoA dehydrogenase [EC 1.3.99.3], one of the mitochondrial flavoenzymes, was carried out to elucidate its biosynthetic mechanism. Poly(A)+ RNA isolated from pig kidney was translated in vitro using wheat germ lysate system and the synthesized enzyme was immunoprecipitated by the antibody against purified pig kidney general acyl CoA dehydrogenase. The apparent molecular weight of the synthesized protein was estimated to be approximately 1,000 daltons larger than that of the mature enzyme, indicating that general acyl CoA dehydrogenase in pig kidney is synthesized as a precursor with a larger molecular weight

Immunochemical and molecular analysis of medium-chain acyl CoA dehydrogenase deficiency

Medium-chain acyl CoA dehydrogenase (MCAD) (acylCoA: (acceptor) 2,3-oxidoreductase, EC 1.3.99.3) deficiency in two patients, MY and AH, was examined by use of an anti-MCAD antibody and the cDNA for the enzyme. No MC AD protein was detected by immunoblot analysis in the fibroblast extract from the first patient MY, while it was present. but not catalytically active in the second patient AH. In order to clarify the molecular mechanism of these deficiencies, a cDNA encoding MCAD was isolated from a human placenta cDNA library. The cDNA contained 1.263 nucleotides of the coding region, 64 nuc1eotides of the 5'-noncoding region, and 686 nucleotides of the 3'noncoding region. The level of mRNA for MC AD in the patients was examined by RNA blot analysis with the cDNA as probe, and the results indicate that the patient MY also had the mRNA and that the level of the mRNA in both patients was almost the same as that of the control subject. Thus it seems that the deficiency in the patients is due to a point mutation(s) and that the position of the mutation(s) in the gene of patient MY is different from that of patient AH

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

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