RECONSTITUTION OF HIGHLY EXPRESSED HUMAN HEART-MUSCLE CARNITINE PALMITOYLTRANSFERASE-I

The human heart muscle carnitine palmitoyltransferase I (M-CPTI) gene was expressed at high levels from a strain of the methylotrophic yeast Pichia pastoris containing similar to 24 copies of the expression vec tor, Levels of M-CPTI were more than ten-fold higher than previously r eported by our group with a single-copy strain (Arch, Biochem, Biophys ., in press) and were sufficient to perform reconstitution studies on the membrane protein, a key step in purification and structural analys is of the enzyme. Solubilization of yeast mitochondria containing M-CP TI in 5% Triton X-100 abolished M-CPTI activity, The detergent-inactiv ated M-CPTI was then reconstituted by removal of the detergent in the presence of phospholipids, The reconstituted proteoliposomes exhibited M-CPTI activity of 2.4 nmol, palmitoylcarnitine formed/mg protein/min , a recovery of 23% of the activity present in the starting mitochondr ial preparation. The malonyl-CoA sensitivity of the reconstituted reac tivated M-CPTI was 88%. This is the first demonstration of direct reac tivation of malonyl-CoA-sensitive M-CPTI activity from solubilized mat erials from any organism, Previously, M-CPTI was presumed to be irreve rsibly inactivated by detergents. (C) 1997 Academic Press.