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Catalytic mechanism and function of phosphatidate-specific phospholipases A2

磷脂酸特异性磷脂酶A2的催化机制和功能

基本信息

批准号：
11670120
负责人：
TOJO Hiromasa
金额：
$ 2.5万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

A phospholipase A2 (PLA2) was purified to homogeneity from the supernatant fraction of rat testis homogenate. The purified 63-kDa enzyme did not require Ca^<2+> ions for activity, and exhibited both phosphatidic acid-preferring PLA2 and monoacylglycerol lipase (MGL) activities with a modest specificity toward unsaturated acyl chains, but not lysophospholipase, and di- and triacylglycerol lipase activities. Anionic detergents enhanced these activities. Serine-modifying irreversible inhibitors, (p-amidino)-phenylmethanesulfonyl fluoride and methylarachidonyl fluorophosphonate inhibited both activities to a similar extent, indicating a single active site is involved in PLA2 and MGL catalysis. We sequenced about 10 tryptic peptides of PA-PLA2 by capillary reverse phase HPLC/electrospray ionization (ESI) ion trap mass spectrometry, and found that it belonged to a new family of PLA2 enzymes. The optimal pH for PLA_2 activity (around 5.5) differed from that for MGL activity (around 8.0), but both activities broadly extended over a physiologically relevant pH range from pH 5.5 to 7.5. At pH 5.5 the enzyme also hydrolyzed bis (monoacylglycerol) phosphate (lysobisphosphatidic acid, LBPA) that has been hitherto known as a PLA2-resistant phospholipid and a late endosome marker. LBPA-enriched fractions were prepared from crude liver lysosome fractions of chloroquine-treated rats, treated with excess of pancreatic PLA2, and then used for assaying LBPA-hydrolyzing activity. LBPA and the reaction products were identified by microbore normal-phase HPLC/ESI ion-trap mass spectrometry. These enzymatic properties suggest that the enzyme can metabolize phosphatidic acid, lysobisphosphatidic acid, and monoacylglycerol in the endosome/lysosome system with mildly acidic milieu.

从大鼠睾丸匀浆上清中纯化出磷脂酶A2 （PLA2）。纯化后的63-kDa酶不需要Ca^<2+>离子来维持活性，并且具有磷脂酸偏好PLA2和单酰基甘油脂肪酶（MGL）活性，对不饱和酰基链具有适度的特异性，但不具有溶血磷脂酶、二酰基甘油脂肪酶和三酰基甘油脂肪酶的活性。阴离子洗涤剂增强了这些活性。丝氨酸修饰不可逆抑制剂（对氨基）-苯基甲基磺酰氟和甲基四烯酰基氟膦酸盐对这两种活性的抑制程度相似，表明PLA2和MGL的催化作用涉及一个单一的活性位点。采用毛细管反相高效液相色谱/电喷雾离子阱质谱法对PA-PLA2的10个色氨酸肽进行了测序，发现其属于一个新的PLA2酶家族。PLA_2活性的最佳pH值（约5.5）与MGL活性的最佳pH值（约8.0）不同，但两者的活性都在生理相关的pH值范围内（pH 5.5 ~ 7.5）广泛扩展。在pH 5.5时，该酶还水解单酰基甘油磷酸（溶双磷脂酸，LBPA），迄今为止，它一直被认为是一种抗pla2的磷脂和一个晚期内体标记物。以氯喹处理大鼠肝溶酶体粗馏分制备富含lbpa的馏分，并用过量的胰腺PLA2处理，然后测定lbpa水解活性。采用微孔正相高效液相色谱/ESI离子阱质谱法对LBPA及其反应产物进行鉴定。这些酶的性质表明该酶可以在温和酸性环境的内核体/溶酶体系统中代谢磷脂酸、溶二磷脂酸和单酰基甘油。