Mensa-Wilmot K, Englund P T
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD.
Mol Biochem Parasitol. 1992 Dec;56(2):311-21. doi: 10.1016/0166-6851(92)90180-r.
Glycosyl phosphatidylinositol-specific phospholipase C (GPI-PLC) from Trypanosoma brucei cleaves the glycosyl phosphatidylinositol (GPI) anchor of the trypanosome variant surface glycoprotein (VSG) and other GPI structures. We have expressed this enzyme in Escherichia coli, using a protocol designed to produce the native enzyme rather than a fusion protein. We have purified large amounts of GPI-PLC from E. coli membranes, using a single step immunoaffinity technique. The expressed enzyme is identical to its trypanosome counterpart in enzymatic specificity, mobility on SDS-PAGE, and isoelectric point. Recombinant GPI-PLC is a membrane enzyme; it associates with E. coli membranes and, like the T. brucei GPI-PLC, partitions into the detergent phase in Triton X-114 phase separation experiments. The Michaelis constants for the two enzymes are similar (400 nM, with VSG as substrate). The turnover number (kcat, 72 min-1) of the recombinant enzyme (expressed from a. T. brucei rhodesiense WRATat 1.1 cDNA) is about one-tenth that of GPI-PLC from T. brucei brucei (ILTat 1.3).
来自布氏锥虫的糖基磷脂酰肌醇特异性磷脂酶C（GPI-PLC）可切割锥虫可变表面糖蛋白（VSG）的糖基磷脂酰肌醇（GPI）锚以及其他GPI结构。我们利用一种旨在产生天然酶而非融合蛋白的方案，在大肠杆菌中表达了这种酶。我们采用单步免疫亲和技术，从大肠杆菌膜中纯化出了大量的GPI-PLC。所表达的酶在酶特异性、SDS-PAGE迁移率和等电点方面与其锥虫对应物相同。重组GPI-PLC是一种膜酶；它与大肠杆菌膜结合，并且在Triton X-114相分离实验中，与布氏锥虫GPI-PLC一样，会分配到去污剂相中。这两种酶的米氏常数相似（以VSG为底物时为400 nM）。重组酶（由布氏罗得西亚锥虫WRATat 1.1 cDNA表达）的转换数（kcat，72 min-1）约为布氏布氏锥虫（ILTat 1.3）GPI-PLC的十分之一。
