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 approximately 24 copies of the expression vector. Levels of M-CPTI were more than ten-fold higher than previously reported 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 analysis of the enzyme. Solubilization of yeast mitochondria containing M-CPTI in 5% Triton X-100 abolished M-CPTI activity. The detergent-inactivated 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 mitochondrial preparation. The malonyl-CoA sensitivity of the reconstituted reactivated M-CPTI was 88%. This is the first demonstration of direct reactivation of malonyl-CoA-sensitive M-CPTI activity from solubilized materials from any organism. Previously, M-CPTI was presumed to be irreversibly inactivated by detergents.
Zhu
H
H
Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland 97291-1000, USA.
Shi
J
J
Cregg
J M
JM
Woldegiorgis
G
G
D000429:Alcohol Oxidoreductases / Q000235:genetics; D002334:Carnitine O-Palmitoyltransferase / Q000096:biosynthesis* / Q000235:genetics*; D018628:Gene Dosage; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000096:biosynthesis; D006801:Humans; D008928:Mitochondria / Q000201:enzymology / Q000235:genetics; D018482:Muscle, Skeletal / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology*; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D011401:Promoter Regions, Genetic
D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D002334:Carnitine O-Palmitoyltransferase