Zhu H, Shi J, Cregg J M, Woldegiorgis G
Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland 97291-1000, USA.
Biochem Biophys Res Commun. 1997 Oct 20;239(2):498-502. doi: 10.1006/bbrc.1997.7494.
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.
人心脏肌肉肉碱棕榈酰转移酶I（M-CPTI）基因在含有约24个表达载体拷贝的甲基营养型酵母毕赤酵母菌株中高水平表达。M-CPTI的水平比我们小组之前报道的单拷贝菌株（《生物化学与生物物理学档案》，即将发表）高出十多倍，足以对该膜蛋白进行重组研究，这是该酶纯化和结构分析的关键步骤。在5% Triton X-100中溶解含有M-CPTI的酵母线粒体，会使M-CPTI活性丧失。然后在磷脂存在下通过去除去污剂来重组被去污剂灭活的M-CPTI。重组后的蛋白脂质体表现出的M-CPTI活性为2.4 nmol棕榈酰肉碱形成/mg蛋白质/分钟，恢复到起始线粒体制剂中存在活性的23%。重组后重新激活的M-CPTI对丙二酰辅酶A的敏感性为88%。这是首次证明从任何生物体的溶解材料中直接重新激活丙二酰辅酶A敏感的M-CPTI活性。此前，M-CPTI被认为会被去污剂不可逆地灭活。
