Abstract
To begin to explore the basis for the tissue-specific expression of mitochondrial carnitine palmitoyltransferase I (CPT I), we focused on three rat tissues (liver, heart, and skeletal muscle) in which the enzyme was known to display very different properties. In Northern blot analysis, a cDNA probe corresponding to liver CPT I readily hybridized to a 4.5-kilobase species of mRNA in liver and heart, but not in skeletal muscle. Using the same probe to screen a neonatal rat heart cDNA library, a full-length clone, surprisingly having 100% sequence identity to the liver CPT I cDNA, was isolated. The paradox was resolved by two additional experiments. First, in Western blots of mitochondrial membranes, an antibody raised against liver CPT I recognized the 88-kDa protein in heart, as well as in liver, but not in skeletal muscle. Second, high specific activity [3H]deschloroetomoxir (a covalent ligand for CPT I) reacted with a single form of CPT I in liver (approximately 88 kDa) and skeletal muscle (approximately 82 kDa), while proteins of both sizes were labeled in the cardiac myocyte. Tritiated ligand binding to the two heart proteins was blocked by excess unlabeled malonyl-CoA. It is concluded that liver and skeletal muscle each contains a single and distinct isoform of CPT I with monomeric size of approximately 88 and 82 kDa, respectively. The heart contains a CPT I protein of approximately 82 kDa in size (probably identical to the skeletal muscle protein) but, importantly, also expresses the liver-type enzyme. The results likely explain why previous studies of heart CPT I yielded an apparent Km for carnitine and I50 value for malonyl-CoA inhibition that were intermediate between those of the liver and skeletal muscle enzymes.
Highlights
Palmitoyltransferase I the mitochondrial oxidatioonf long chain fatty aciadnsd exerts major control over the process by virtue of its potent inhibit
To begin to explore the basis for the tissue-specific expression of mitochondrial carnitine palmitoyltransferase I (CPT I), we focused on three rat tissues in which the enzyme was known to display very different properties
This too was induced in liversof DEHP-treated and muscle mitochondria that malonyl-CoA and agents of the rats.The intriguingfinding was thaat signal of similar oxirane carboxylic acid class compete for a comsize was readily detected in heart(lane 3 ), b unt ot in skeletal mon binding site on CPT I [13].It lent confidence to the muscle
Summary
Ability by malonyl-CoA [1].Previous studies in t h e rat established that the CPT I associated with liver mitochondria exhibits very different kinetic propertiesand sensitivity to malonyl-. 12 mM MgCI,, 12 mM ATP, 0.7 mM GSH, 100PMCoASH, and 1.2% fatty was seen in the membranfersom heart mitochondria (lane acid-free bovine serum albumin), followed by addition of [3Hldeschlo- 21, but not in those from skeletal muscle (lane 3 ) .By contrast, roetomoxir (r3H]DEt()specific activity of 3.08 mCi/mg; Sandoz Pharma- a strong signal representingCPT I1 (-70 kDa in size) wasseen ceuticals) to a final concentratiofn3.0 p ~A.fter incubation for 30 min in all three lanes when a duplicate blot was probed with an a t room temperature (with mixing every min), the mitochondria were antibody to liver CPTI1 The latter were diced into chunks of no larger than 2 mm', and myocytes were
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