CHARACTERIZATION OF THE MALONYL-COA-SENSITIVE CARNITINE PALMITOYLTRANSFERASE (CPT(O)) OF A RAT-HEART MITOCHONDRIAL PARTICLE - EVIDENCE THATTHE CATALYTIC UNIT IS CPT(I)

A post 30,000 x g particulate fraction was isolated from rat heart. Th is mixed membrane fraction is enriched in a carnitine palmitoyltransfe rase which is sensitive to both malonyl-CoA and etomoxiryl-CoA at conc entrations that inhibit the malonyl-CoA-sensitive carnitine palmitoylt ransferase (CPTo/CPT-I) of intact mitochondria. Tritiated etomoxiryl-C oA labels two proteins with the same molecular weight as the labeled p roteins from rat heart mitochondria. Malonyl-CoA-sensitive carnitine p almitoyltransferase in the particulate fraction is stable to freeze-th awing, and the activity is not latent. These data show that the carnit ine palmitoyltransferase associated with this particle is CPTo/CPT-I. Positive Western blots were obtained, with the particle using anti-CPT i/CPT-II at a molecular weight identical with the CPTi/CPT-II purified from rat heart mitochondria. Catalytic activity was purified to near homogeneity in approximately 40% yield. The purified protein has a mol ecular weight identical with CPTi/CPT-II, it cross-reacts with antibod y against CPTiCPT-II, it is not inhibited by malonyl-CoA or etomoxiryl -CoA, and mass spectral analyses of the tryptic peptides give the same molecular masses as CPTi/CPT-II, and, when mixed with equal amounts o f CPTi/CPT-II, one uniform spot is found by two-dimensional electropho resis. These data indicate that the catalytic subunit of CPTo/CPT-I is the same as CPTi/CPT-II. The average inhibition of the CPT of frozen- thawed particles is 71% by 50 nM etomoxiryl-CoA and 62% by 50 nM malon yl-CoA. The inhibitor sensitivity, but not the catalytic activity, is lost by solubilization in 1% Triton X-114; removal of Triton X-114 usi ng Extracti-Gel D restores etomoxiryl-CoA and malonyl-CoA sensitivity (both 50 nM) of CPT to an average of 77 and 48%, respectively. Consist ent with previous reports, these results show that CPTo/CPT-I is NOT i nactivated by detergents, rather detergents both desensitize it to mal onyl-CoA and alter its V-max. These data show the assumption that CPTo /CPT-I is inactivated by detergents is untenable.