cDNA cloning of rat and human medium chain acyl-CoA dehydrogenase (MCAD)
MCAD is one of three mitochondrial flavoenzymes which catalyze the first step in the ..beta..-oxidation of straight chain fatty acids. It is a tetramer with a subunit Mr of 45 kDa. MCAD is synthesized in the cytosol as a 49 kDa precursor polypeptide (pMCAD), imported into mitochondria, and cleaved to the mature form. Genetic deficiency of MCAD causes recurrent episodes of hypoglycemic coma accompanied by medium chain dicarboxylic aciduria. Employing a novel approach, the authors now report isolation of partial rat and human cDNA clones encoding pMCAD. mRNA encoding pMCAD was purified to near homogeneity by polysome immunoadsorption using polyclonal monospecific antibody. Single-stranded (/sup 32/P)labeled cDNA probe was synthesized using the enriched mRNA as template, and was used to screen directly 16,000 colonies from a total rat liver cDNA library constructed in pBR322. One clone (600 bp) was detected by in situ hybridization. Hybrid-selected translation with this cDNA yielded a 49 kDa polypeptide indistinguishable in size from rat pMCAD and immunoprecipitable with anti-MCAD antibody. Using the rat cDNA as probe, 43,000 colonies from a human liver cDNA library were screened. Four identical positive clones (400 bp) were isolated and positively identified by hybrid-selected translation and immunoprecipitation. The sizes of rat and human mRNAs encoding pMCAD were 2.2 kb and 2.4 kb, respectively, as determined by Northern blotting.
- Research Organization:
- Yale Univ. School of Medicine, New Haven, CT
- OSTI ID:
- 5179931
- Report Number(s):
- CONF-8606151-
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 45:6; Conference: 76. annual meeting of the Federation of American Society for Experimental Biology, Washington, DC, USA, 8 Jun 1986
- Country of Publication:
- United States
- Language:
- English
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DEUTERIUM
ISOTOPE EFFECTS
HYDROXYLASES
BIOCHEMICAL REACTION KINETICS
TRITIUM COMPOUNDS
BENZOIC ACID
NADH2
PSEUDOMONAS
BACTERIA
CARBOXYLIC ACIDS
COENZYMES
ENZYMES
HYDROGEN ISOTOPES
ISOTOPES
KINETICS
LABELLED COMPOUNDS
LIGHT NUCLEI
MICROORGANISMS
MONOCARBOXYLIC ACIDS
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
OXIDOREDUCTASES
REACTION KINETICS
STABLE ISOTOPES
550201* - Biochemistry- Tracer Techniques