Expression of a cDNA for rat liver carnitine palmitoyltransferase I in yeast establishes that catalytic activity and malonyl-CoA sensitivity reside in a single polypeptide

Nicholas F. Brown, Victoria Esser, Daniel W. Foster, J. Denis McGarry

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Abstract

A cDNA encoding full-length carnitine palmitoyltransferase I (CPT I) from rat liver was expressed in Saccharomyces cerevisiae, a system devoid of endogenous CPT activity. The recombinant enzyme was of the expected size (as deduced from immunoblots), membrane-bound, and detergent-labile. It was also potently inhibited by malonyl-CoA, with an I50 value (concentration causing 50% inhibition) of ~5 μM, similar to that of the native enzyme in rat liver mitochondria. A truncated variant of the enzyme that lacked the amino- terminal 82 residues encompassing the first hydrophobic domain retained catalytic function but was much less sensitive to malonyl-CoA (I50 > 80 μM). Deletion of the cDNA segment encoding amino acids 31-148 (which includes both first and second hydrophobic stretches) resulted in no detectable product. The data establish unequivocally that a single polypeptide possesses both catalytic and malonyl-CoA binding domains, as well as the other properties previously attributed by us to native CPT I in mammalian mitochondria, and should thus put to rest the controversy surrounding this issue (Kerner, J., Zaluzec, E., Gage, D., and Bieber, L. L. (1994) J. Biol. Chem. 269, 8209-8219). In addition, the results strengthen the view that one site of interaction of malonyl-CoA with the rat liver enzyme involves the NH2-terminal region of the molecule.

Original languageEnglish (US)
Pages (from-to)26438-26442
Number of pages5
JournalJournal of Biological Chemistry
Volume269
Issue number42
StatePublished - Oct 21 1994

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Malonyl Coenzyme A
Carnitine O-Palmitoyltransferase
Liver
Yeast
Rats
Catalyst activity
Complementary DNA
Yeasts
Peptides
Mitochondria
Enzymes
Liver Mitochondrion
Detergents
Saccharomyces cerevisiae
Catalytic Domain
Membranes
Amino Acids
Molecules

ASJC Scopus subject areas

  • Biochemistry

Cite this

Expression of a cDNA for rat liver carnitine palmitoyltransferase I in yeast establishes that catalytic activity and malonyl-CoA sensitivity reside in a single polypeptide. / Brown, Nicholas F.; Esser, Victoria; Foster, Daniel W.; McGarry, J. Denis.

In: Journal of Biological Chemistry, Vol. 269, No. 42, 21.10.1994, p. 26438-26442.

Research output: Contribution to journalArticle

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