Miyake R, Gross R W
Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Biochim Biophys Acta. 1992 Dec 2;1165(2):167-76. doi: 10.1016/0005-2760(92)90183-v.
Three phospholipase A2 activities from canine vascular smooth muscle were identified and characterized including: (1) a cytosolic calcium-independent phospholipase A2 which is activated by nucleotide di- and triphosphates; (2) a cytosolic calcium-dependent phospholipase A2 which is activated by physiologic increments in calcium ion concentration; and (3) a microsomal calcium-independent phospholipase A2 which was highly selective for plasmenylcholine substrate. Vascular smooth muscle cytosolic calcium-independent phospholipase A2 was activated 338% +/- 11 (X+S.E.; n = 15) by physiologic concentrations of ATP. Similar amounts of activation were also present utilizing other nucleotide di- and triphosphates (e.g., ADP, CTP, GDP and GTP) as well as non-hydrolyzable nucleotide triphosphate analogs (e.g., ATP-gamma-S, AMP-PNP and GTP-gamma-S). Vascular smooth muscle cytosolic calcium-dependent phospholipase A2 was purified 455-fold by sequential DEAE-Sephacel, Phenyl-Sepharose, Mono Q, hydroxyapatite and Superose 12 chromatographies. The partially purified calcium-dependent phospholipase A2 was activated by physiologic increments in calcium ion concentration (e.g., 1 microM) and possessed an apparent native molecular weight of 95 kDa, an acidic isoelectric point (pI = 4.8) and a neutral pH optimum (pH 7.0). Vascular smooth muscle microsomal phospholipase A2 activity was predominantly calcium-independent and was over six-fold selective for hydrolysis of plasmenylcholine substrate. Taken together, these results demonstrate the existence of three separate and distinct phospholipase A2 activities in vascular smooth muscle and identify ATP and calcium ion as independent modulators of discrete phospholipase A2 activities in vascular smooth muscle cells.
已鉴定并表征了犬血管平滑肌中的三种磷脂酶A2活性，包括：（1）一种胞质钙非依赖性磷脂酶A2，其被二磷酸和三磷酸核苷酸激活；（2）一种胞质钙依赖性磷脂酶A2，其被钙离子浓度的生理性增加激活；（3）一种微粒体钙非依赖性磷脂酶A2，其对缩醛磷脂酰胆碱底物具有高度选择性。生理性浓度的ATP可使血管平滑肌胞质钙非依赖性磷脂酶A2激活338%±11（X+S.E.；n = 15）。使用其他二磷酸和三磷酸核苷酸（如ADP、CTP、GDP和GTP）以及不可水解的三磷酸核苷酸类似物（如ATP-γ-S、AMP-PNP和GTP-γ-S）时也存在类似程度的激活。通过连续的DEAE-琼脂糖凝胶、苯基琼脂糖凝胶、Mono Q、羟基磷灰石和Superose 12层析，将血管平滑肌胞质钙依赖性磷脂酶A2纯化了455倍。部分纯化的钙依赖性磷脂酶A2被钙离子浓度的生理性增加（如1 microM）激活，其表观天然分子量为95 kDa，酸性等电点（pI = 4.8），最适pH值为中性（pH 7.0）。血管平滑肌微粒体磷脂酶A2活性主要是钙非依赖性的，对缩醛磷脂酰胆碱底物的水解选择性超过六倍。综上所述，这些结果证明了血管平滑肌中存在三种独立且不同的磷脂酶A2活性，并确定ATP和钙离子是血管平滑肌细胞中离散磷脂酶A2活性的独立调节剂。
