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Title: cDNA cloning of rat and human medium chain acyl-CoA dehydrogenase (MCAD)

Abstract

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 ratmore » and human mRNAs encoding pMCAD were 2.2 kb and 2.4 kb, respectively, as determined by Northern blotting.« less

Authors:
; ; ;
Publication Date:
Research Org.:
Yale Univ. School of Medicine, New Haven, CT
OSTI Identifier:
5179931
Report Number(s):
CONF-8606151-
Journal ID: CODEN: FEPRA
Resource Type:
Conference
Journal Name:
Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)
Additional Journal Information:
Journal Volume: 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
Subject:
59 BASIC BIOLOGICAL SCIENCES; 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

Citation Formats

Matsubara, Y, Kraus, J P, Rosenberg, L E, and Tanaka, K. cDNA cloning of rat and human medium chain acyl-CoA dehydrogenase (MCAD). United States: N. p., 1986. Web.
Matsubara, Y, Kraus, J P, Rosenberg, L E, & Tanaka, K. cDNA cloning of rat and human medium chain acyl-CoA dehydrogenase (MCAD). United States.
Matsubara, Y, Kraus, J P, Rosenberg, L E, and Tanaka, K. 1986. "cDNA cloning of rat and human medium chain acyl-CoA dehydrogenase (MCAD)". United States.
@article{osti_5179931,
title = {cDNA cloning of rat and human medium chain acyl-CoA dehydrogenase (MCAD)},
author = {Matsubara, Y and Kraus, J P and Rosenberg, L E and Tanaka, K},
abstractNote = {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.},
doi = {},
url = {https://www.osti.gov/biblio/5179931}, journal = {Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)},
number = ,
volume = 45:6,
place = {United States},
year = {Thu May 01 00:00:00 EDT 1986},
month = {Thu May 01 00:00:00 EDT 1986}
}

Conference:
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