title: Use of a selective inhibitor of liver carnitine palmitoyltransferase I (CPT I) allows quantification of its contribution to total CPT I activity in rat heart. Evidence that the dominant cardiac CPT I isoform is identical to the skeletal muscle enzyme.
abstract: It has recently been established that rat heart mitochondria contain two isoforms of carnitine palmitoyltransferase I (CPT I), the minor 88-kDa variant being identical to liver CPT I (L-CPT I) and the dominant 82-kDa form resembling the skeletal muscle enzyme (M-CPT I) (Weis, B. C., Esser, V., Foster, D. W., and McGarry, J. D. (1994) J. Biol. Chem. 269, 18712-18715). To quantify the functional contribution of L-CPT I to overall CPT I activity in heart mitochondria a selective inhibitor of the former was needed. The dinitrophenol analog of 2[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylic acid (etomoxir) (DNP-Et) was found to have this property. When liver and skeletal muscle mitochondria were exposed to DNP-Et in the presence of ATP and CoASH, the DNP-Et-CoA formed completely inhibited liver CPT I while leaving the muscle enzyme unaffected. Similar treatment of heart mitochondria blocked only the L-CPT I component. This had the effect of shifting the apparent Km for carnitine from approximately 200 to approximately 500 microM and the I50 value for malonyl-CoA (the concentration needed to suppress enzyme activity by 50%) from approximately 0.18 to approximately 0.06 microM, i.e. the heart system now behaved exactly the same as that from skeletal muscle. Taking the Km for carnitine of L-CPT I and M-CPT I to be 30 and 500 microM, respectively, it could be calculated that the former contributes approximately 2% to the total CPT I in heart. When the 82-kDa CPT I isoforms of heart and skeletal muscle were labeled with [3H]etomoxir and then exposed to trypsin, the fragmentation patterns obtained were identical and quite distinct from that given by CPT I from liver. We conclude that (i) DNP-Et, unlike other agents of the oxirane carboxylic acid class, has remarkable inhibitory selectivity for L-CPT I over M-CPT I; (ii) the previously puzzling observation that rat heart CPT I displays kinetic characteristics intermediate between those of the enzymes from liver and skeletal muscle is entirely accounted for by the low level expression of L-CPT I in the cardiac myocyte; and (iii) the dominant 82-kDa CPT I isoform in heart is identical to the muscle enzyme. The data reaffirm that, in contrast to CPT II, CPT I exists in at least two isoforms and that both are present in rat heart.
authors.affiliation: Department of Internal Medicine, University of Texas Southwestern Medical Center, Southwestern Medical School, Dallas 75235.
authors.forename: B C
authors.initials: BC
authors.lastname: Weis
authors.forename: A T
authors.initials: AT
authors.lastname: Cowan
authors.forename: N
authors.initials: N
authors.lastname: Brown
authors.forename: D W
authors.initials: DW
authors.lastname: Foster
authors.forename: J D
authors.initials: JD
authors.lastname: McGarry
mesh_terms: D000818:Animals; D002334:Carnitine O-Palmitoyltransferase / Q000037:antagonists & inhibitors*; D004852:Epoxy Compounds / Q000494:pharmacology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors*; D008099:Liver / Q000201:enzymology*; D008297:Male; D008928:Mitochondria / Q000201:enzymology; D018482:Muscle, Skeletal / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014357:Trypsin / Q000494:pharmacology
chemical_list: D004852:Epoxy Compounds; D007527:Isoenzymes; D002334:Carnitine O-Palmitoyltransferase; D014357:Trypsin; C054207:etomoxir