Catalytic mechanism and function of phosphatidate-specific phospholipases A2

从大鼠睾丸匀浆上清液中纯化出磷脂酶A2(PLA2)。纯化的63 kDa酶不需要Ca~(2+)~(2+)和Gt~(2+)离子，具有磷脂酸偏好PLA2和单酰基甘油脂肪酶(MGL)活性，对不饱和酰基链有一定的专一性，但对溶血磷脂酶和二酰甘油脂肪酶和三酰甘油脂肪酶活性没有影响。阴离子洗涤剂增强了这些活性。丝氨酸修饰的不可逆抑制剂(对氨基)-苯甲基磺酰氟和甲基丙烯酰基氟磷酸盐对这两种活性的抑制程度相似，表明在PLA2和MGL的催化中只有一个活性部位参与。我们用毛细管反相高效液相色谱/电喷雾电离(ESI)离子陷阱质谱仪对大约10个PA-PLA2的胰酶多肽进行了测序，发现它属于一个新的PLA2酶家族。PLA2活性的最适pH(约5.5)与MGL活性的最适pH(约8.0)不同，但这两种活性的最适pH范围都在pH 5.5-7.5的生理范围内。在pH 5.5时，该酶还能水解单(单甘油)磷酸(LBPA)，LBPA迄今被认为是一种抗PLA2的磷脂，也是晚期的内吞体标记物。从氯喹处理的大鼠肝溶酶体粗提物中制备LBPA富集组分，用过量的胰腺PLA2处理，然后用来测定LBPA的水解性。采用微孔正相高效液相色谱/电喷雾质谱鉴定LBPA及其反应产物。这些酶学性质表明，在中等酸性的环境中，该酶可以在内切体/溶酶体系统中代谢磷脂酸、溶二磷脂酸和单酰甘油。

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.