Jenkins Christopher M, Han Xianlin, Yang Jingyue, Mancuso David J, Sims Harold F, Muslin Anthony J, Gross Richard W
Division of Bioorganic Chemistry, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Biochemistry. 2003 Oct 14;42(40):11798-807. doi: 10.1021/bi034611q.
Cytosolic phospholipase A(2)gamma (cPLA(2)gamma) is a calcium-independent, membrane-associated phospholipase A(2) that possesses a C-terminal prenylation motif (-CCLA) whose covalent structure cannot be deduced from the primary sequence alone. Accordingly, we overexpressed human cPLA(2)gamma containing an N-terminal His tag ((His)(6)cPLA(2)gamma) in Sf9 cells and quantitatively solubilized and purified the enzyme by sequential immobilized metal affinity and Mono Q column chromatographies. The final preparation appeared as a single 61 kDa band after SDS-PAGE/silver-staining, possessed high lysophospholipase activity (50 micromol min(-1) mg(-1)), and was inhibited by, but did not hydrolyze, palmitoyl-CoA. Radiolabeling of recombinant human cPLA(2)gamma with [(3)H]-mevalonolactone in the absence of statins and subsequent cleavage of prenyl groups with Raney nickel revealed that the enzyme is only farnesylated and is not geranylgeranylated. Analysis of CNBr-digested cPLA(2)gamma by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI/TOF-TOF) mass spectrometry demonstrated the presence of a farnesyl moiety at Cys-538, cleavage of the Cys(538)-Cys(539) bond, and carboxymethylation of the resultant C-terminal prenylated cysteine. Collectively, these results describe the solubilization and purification of recombinant cPLA(2)gamma to homogeneity and identify cPLA(2)gamma as a farnesylated protein that undergoes at least three sequential posttranslational modifications that likely facilitate its targeting and interactions with its membrane substrates.
胞质型磷脂酶A2γ（cPLA2γ）是一种不依赖钙的、与膜相关的磷脂酶A2，其具有一个C末端异戊二烯化基序（-CCLA），其共价结构不能仅从一级序列推导得出。因此，我们在Sf9细胞中过表达了含有N末端His标签（（His）6cPLA2γ）的人cPLA2γ，并通过连续的固定金属亲和色谱和Mono Q柱色谱对该酶进行定量溶解和纯化。SDS-PAGE/银染后，最终制剂呈现为一条单一的61 kDa条带，具有高溶血磷脂酶活性（50 μmol min-1 mg-1），并且受到棕榈酰辅酶A的抑制，但不被其水解。在不存在他汀类药物的情况下，用[3H]-甲羟戊酸内酯对重组人cPLA2γ进行放射性标记，随后用雷尼镍切割异戊二烯基团，结果表明该酶仅被法尼基化，而未被香叶基香叶基化。通过基质辅助激光解吸/电离飞行时间/飞行时间（MALDI/TOF-TOF）质谱分析CNBr消化的cPLA2γ，结果表明在Cys-538处存在法尼基部分，Cys（538）-Cys（539）键发生断裂，并且所得C末端异戊二烯化半胱氨酸发生羧甲基化。总的来说，这些结果描述了重组cPLA2γ的溶解和纯化至均一性，并确定cPLA2γ是一种法尼基化蛋白，它经历了至少三种连续的翻译后修饰，这些修饰可能有助于其靶向定位及其与膜底物的相互作用。
