USE OF A SELECTIVE INHIBITOR OF LIVER CARNITINE PALMITOYLTRANSFERASE-I (CPT-I) ALLOWS QUANTIFICATION IF 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

It has recently been established that rat heart mitochondria contain t wo 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-chloroph enoxy)hexyl] oxirane a carboxylic acid (etomoxir) (DNP-Et) was found t o 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 fo rmed 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 K-m for carnitine from similar to 200 to similar to 500 mu M and the I-50 val ue for malonyl-CoA (the concentration needed to suppress enzyme activi ty by 50%) from similar to 0.18 to similar to 0.06 mu M, i.e. the hear t system now behaved exactly the same as that from skeletal muscle. Ta king the K-m for carnitine of L-CPT I and M-CPT I to be 30 and 500 mu M, respectively, it could be calculated that the former contributes si milar to 2% to the total CPT I in heart. When the 82-kDa CPT I isoform s of heart and skeletal muscle were labeled with [H-3]etomoxir and the n exposed to trypsin, the fragmentation patterns obtained were identic al 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 c lass, 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 fro m 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 d ata reaffirm that, in contrast to CPT II, CPT I exists in at least two isoforms and that both are present in rat heart.