Purification and properties of a phospholipase A2/lipase preferring phosphatidic acid, bis(monoacylglycerol) phosphate, and monoacylglycerol from rat testis
Purification and properties of a phospholipase A2/lipase preferring phosphatidic acid, bis(monoacylglycerol) phosphate, and monoacylglycerol from rat testis
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DOI：
10.1074/jbc.m202817200
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发表时间：
2002-11-15
影响因子：
4.8
通讯作者：
Tojo, H
中科院分区：
文献类型：
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作者：
Ito, M;Tchoua, U;Tojo, H
从大鼠睾丸匀浆的上清液部分中将磷脂酶 A(2) (PLA(2)) 纯化至同质。纯化的 63-kDa 酶不需要 Ca2+ 离子即可发挥活性，并且表现出偏好磷脂酸的 PLA(2) 和单酰基甘油脂肪酶活性，并对不饱和酰基链具有适度的特异性。阴离子洗涤剂增强了这些活性。丝氨酸修饰不可逆抑制剂（对脒基苯基）甲磺酰氟和甲基花生四烯基氟膦酸酯对两种活性的抑制程度相似，表明 PLA(2) 和脂肪酶活性涉及单个活性位点。纯化酶的NH2末端12个氨基酸的序列与来自大鼠肝脏的羧酸酯酶的序列相同。 PLA(2) 活性的最佳 pH 值（约 5.5）与脂肪酶活性的最佳 pH 值（约 8.0）不同。在 pH 5.5 时，该酶还可水解双（单酰基甘油）磷酸盐或溶血双磷脂酸 (LBPA)，后者迄今为止被称为分泌性 PLA(2) 抗性磷脂和晚期内体标记物。富含 LBPA 的级分是从氯喹治疗的大鼠的肝脏溶酶体级分中制备的，用过量的胰腺 PLA(2) 处理，然后用于测定 LBPA 水解活性。通过微孔正相高效液相色谱/电喷雾电离离子阱质谱鉴定LBPA和反应产物。这些酶特性表明该酶可以代谢细胞酸性区室中的磷脂酸和溶血双磷脂酸。
Phospholipase A(2) (PLA(2)) was purified to homogeneity from the supernatant fraction of rat testis homogenate. The purified 63-kDa enzyme did not require Ca2+ ions for activity and exhibited both phosphatidic acid-preferring PLA(2) and monoacylglycerol lipase activities with a modest specificity toward unsaturated acyl chains. Anionic detergents enhanced these activities. Serine-modifying irreversible inhibitors, (p-amidinophenyl) methanesulfonyl fluoride and methylarachidonyl fluorophosphonate, inhibited both activities to a similar extent, indicating a single active site is involved in PLA(2) and lipase activities. The sequence of NH2-terminal 12 amino acids of purified enzyme was identical to that of a carboxylesterase from rat liver. The optimal pH for PLA(2) activity (around 5.5) differed from that for lipase activity (around 8.0). At pH 5.5 the enzyme also hydrolyzed bis(monoacylglycerol) phosphate, or lysobisphosphatidic acid (LBPA), that has been hitherto known as a secretory PLA(2)-resistant phospholipid and a late endosome marker. LBPA-enriched fractions were prepared from liver lysosome fractions of chloroquine-treated rats, treated with excess of pancreatic PLA(2) and then used for assaying LBPA-hydrolyzing activity. LBPA and the reaction products were identified by microbore normal phase high performance liquid chromatography/electrospray ionization ion-trap mass spectrometry. These enzymatic properties suggest that the enzyme can metabolize phosphatidic and lysobisphosphatidic acids in cellular acidic compartments.