Inhibitors of mitochondrial carnitine palmitoyltransferase I limit the action of proteases on the enzyme

Isolation and partial amino acid analysis of a truncated form of the rat liver isozyme

Victoria Esser, Masamichi Kuwajima, Charles H. Britton, Kousik Krishnan, Daniel W. Foster, J. Denis McGarry

Research output: Contribution to journalArticle

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Abstract

Our objective was to isolate from rat liver mitochondria the malonyl-CoA-regulated and detergent-labile enzyme, carnitine palmitoyltransferase I (CPT I), whose properties and relationship to CPT II have been the subject of debate. After exposure of mitochondria to the dinitrophenol derivative of etomoxir-CoA (DNP-Et-CoA, a covalent inhibitor of CPT I), followed by detergent solubilization and blue Sepharose chromatography, the DNP-Et-labeled CPT I could be readily visualized on immunoblots using an anti-DNP monoclonal antibody. This material was used to raise a rabbit polyclonal antibody that recognized CPT I regardless of whether it was carrying a covalent ligand. Exposure of membranes from untreated mitochondria to a mixture of trypsin and chymotrypsin caused rapid loss of CPT I activity with a concomitant disappearance of immunodetectable protein. However, inclusion of malonyl-CoA in such incubations afforded major protection of CPT I activity. Under these conditions CPT I simply underwent truncation from ∼90 to ∼82 kDa. This was also true if CPT I had first been labeled with Et-CoA or DNP-Et-CoA prior to protease treatment. Thus, the presence of an inhibitor, whether reversible or irreversible, at the active site of CPT I limited the action of trypsin/chymotrypsin to removal of a small portion of the protein which was probably not necessary for catalytic function. These and other experiments with antibodies and proteases provided additional insight into the membrane topology of CPT I. They also strengthened our conviction that CPT I and CPT II are distinct proteins and that the former exists as tissue-specific isoforms. Finally, the 82-kDa truncated form of rat liver CPT I was isolated and subjected to partial amino acid analysis. Four unambiguous peptide sequences were obtained.

Original languageEnglish (US)
Pages (from-to)5810-5816
Number of pages7
JournalJournal of Biological Chemistry
Volume268
Issue number8
StatePublished - Mar 15 1993

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Carnitine O-Palmitoyltransferase
Liver
Isoenzymes
Rats
Peptide Hydrolases
Amino Acids
Enzymes
Mitochondria
Coenzyme A
Malonyl Coenzyme A
Detergents
Dinitrophenols
Membranes
Proteins
Agarose Chromatography
Antibodies
Liver Mitochondrion
Chromatography

ASJC Scopus subject areas

  • Biochemistry

Cite this

Inhibitors of mitochondrial carnitine palmitoyltransferase I limit the action of proteases on the enzyme : Isolation and partial amino acid analysis of a truncated form of the rat liver isozyme. / Esser, Victoria; Kuwajima, Masamichi; Britton, Charles H.; Krishnan, Kousik; Foster, Daniel W.; McGarry, J. Denis.

In: Journal of Biological Chemistry, Vol. 268, No. 8, 15.03.1993, p. 5810-5816.

Research output: Contribution to journalArticle

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abstract = "Our objective was to isolate from rat liver mitochondria the malonyl-CoA-regulated and detergent-labile enzyme, carnitine palmitoyltransferase I (CPT I), whose properties and relationship to CPT II have been the subject of debate. After exposure of mitochondria to the dinitrophenol derivative of etomoxir-CoA (DNP-Et-CoA, a covalent inhibitor of CPT I), followed by detergent solubilization and blue Sepharose chromatography, the DNP-Et-labeled CPT I could be readily visualized on immunoblots using an anti-DNP monoclonal antibody. This material was used to raise a rabbit polyclonal antibody that recognized CPT I regardless of whether it was carrying a covalent ligand. Exposure of membranes from untreated mitochondria to a mixture of trypsin and chymotrypsin caused rapid loss of CPT I activity with a concomitant disappearance of immunodetectable protein. However, inclusion of malonyl-CoA in such incubations afforded major protection of CPT I activity. Under these conditions CPT I simply underwent truncation from ∼90 to ∼82 kDa. This was also true if CPT I had first been labeled with Et-CoA or DNP-Et-CoA prior to protease treatment. Thus, the presence of an inhibitor, whether reversible or irreversible, at the active site of CPT I limited the action of trypsin/chymotrypsin to removal of a small portion of the protein which was probably not necessary for catalytic function. These and other experiments with antibodies and proteases provided additional insight into the membrane topology of CPT I. They also strengthened our conviction that CPT I and CPT II are distinct proteins and that the former exists as tissue-specific isoforms. Finally, the 82-kDa truncated form of rat liver CPT I was isolated and subjected to partial amino acid analysis. Four unambiguous peptide sequences were obtained.",
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