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.
Highlights
From the Department of Vnternal Medicine and §Biochemistry and the Gifford Laboratories of Diabetes Research, University of Texus Southwestern Medica1 Center, Southwestern Medical School, Dallas, Texas 75235
It has recently been established that rat heartmito- Carnitine palmitoyltransferase I (CPT I)' catalyzes thefirst chondria contain two isoforms of carnitine palmitoyl- step specific to themitochondrial oxidation of long chain fatty transferase I (CPT I), the minor 88-kDa variant being acids and exertsmajor control over the process by virtue of its identical to liver CPT I (L-CPTI) and the dominant 82- unique inhibitabilityby malonyl-CoA [1].Unlike CPTI1, CPT I appears to exist in at (M-CPT I)
In the caseof etomoxir itself, terference from the liver isoform. To validate this approach it production of the CoA ester is readily catalyzed by mitochon- was necessary t o establish that under the preincubation condria from liver, heart, and skeletalmuscle, provided that ATP ditions chosen dinitrophenol analog of etomoxir (DNP-Et) would interact exclusively and and CoASH are available for the activatingenzyme pres- completely with all of the liver-type CPT I (L-CPT I) present in heartmitochonent on the outer membrane
Summary
EVIDENCE THAT THE DOMINANT CARDIAC CPT I ISOFORM IS IDENTICAL TO THE SKELETAL MUSCLE ENZYME* Wasstrikingly different.Whereas 40 1.1~DNP-Et-CoA was needed to achieve 85% suppression of muscle CPT I activity, >90% of the liver enzyme wasinhibited at an esterconcentration of only 50 m.
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