Characterization of acidic Ca2+-independent phospholipase A2 of bovine lung
Characterization of acidic Ca2+-independent phospholipase A2 of bovine lung
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DOI：
10.1016/s0305-0491(98)10046-9
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发表时间：
1998-06-01
影响因子：
2.2
通讯作者：
Fisher, AB
中科院分区：
文献类型：
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作者：
Akiba, S;Dodia, C;Fisher, AB
酸性钙非依赖性磷脂酶A（2）（aiPLA（2））以前已从大鼠肺中分离出来，并已描述了人cDNA。本研究将该方法应用于牛肺天然蛋白的大规模分离。基于保守的大鼠/人序列产生针对15个氨基酸合成肽的多克隆抗体。该抗体识别从牛肺匀浆中获得的可溶性级分中估计分子量约为29 kDa的单一蛋白质条带。在连续柱层析中，在含有aiPLA（2）活性的级分中检测到与aiPLA（2）抗肽抗体反应的29 kDa蛋白质。部分纯化的酶在pH 4下显示出在Ca 2+非依赖性悬垂PLA（2）活性方面比匀浆增加176倍。磷脂酰胆碱底物的活性最大，磷脂酰乙醇胺和阴离子磷脂的活性显著降低。该酶在磷脂酰胆碱中没有酰基偏好，对氧化底物也没有偏好，但对1-O-烷基磷脂酰胆碱的活性较低。aiPLA（2）活性可被过渡态磷脂类似物（MJ 33，1-十六烷基-3-三氟乙基甘油基-sn-2-磷酸甲醇）、丝氨酸蛋白酶抑制剂和抗肽抗体抑制，但对花生四烯酰基三氟甲基酮、溴烯醇内酯、对溴苯甲酰甲基溴和ATP不敏感。对29 kDa蛋白的N-末端氨基酸序列的分析表明其与人26 kDa aiPLA具有高度同源性（2）。用糖苷内切酶F处理后，蛋白质的分子量没有显著变化。大鼠肺亚细胞组分的Western印迹显示aiPLA（2）与板层体、溶酶体和胞质组分呈免疫反应性。这些结果表明，从牛肺中分离出大鼠和人酶的29 kDa酸性Ca 2+非依赖性磷脂酶A（2）同系物，并为肺表面活性剂磷脂酰胆碱代谢的特异性提供了证据。(C)1998年爱思唯尔科学公司All rights reserved.
An acidic Ca2+-independent phospholipase A(2) (aiPLA(2)) has been isolated previously from rat lung and a human cDNA has been described. This study applied the method to larger scale isolation of the native protein from the bovine lung. A polyclonal antibody was generated to a 15 amino acid synthetic peptide based on a conserved rat/human sequence. This antibody recognized a single protein band with an estimated molecular mass of approximate to 29 kDa in a soluble fraction obtained from bovine lung homogenate. A 29 kDa protein that reacted with the aiPLA(2) antipeptide antibody was detected in fractions containing aiPLA(2) activity on sequential column chromatographies. The partially purified enzyme showed 176-fold increase over the homogenate in Ca2+-independent pendent PLA(2) activity at pH 4. Activity was maximal with phosphatidylcholine substrate and was significantly less with phosphatidylethanolamine and anionic phospholipids. The enzyme had no acyl group preference in phosphatidylcholine and showed no preference for oxidized substrate, but activity was less with 1-O-alkyl phosphatidylcholine. aiPLA(2) activity was inhibited by a transition state phospholipid analog (MJ33, 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol), serine protease inhibitors, and the anti-peptide antibody but was insensitive to arachidonoyl trifluoromethyl ketone, bromoenol lactone, p-bromophenacyl bromide, and ATP. Analysis of N-terminal amino acid sequence for the 29 kDa protein demonstrated its high homology to human 26 kDa aiPLA(2). These was no significant change in molecular mass of the protein following treatment with endoglycosidase F. Western blot of subcellular fractions from rat lung indicated aiPLA(2) immunoreactivity with lamellar body, lysosomal, and cytosolic fractions. These results indicate isolation from bovine lung of a 29 kDa acidic Ca2+-independent phospholipase A(2) homologue of the rat and human enzyme and provide evidence for specificity in the metabolism of lung surfactant phosphatidylcholine. (C) 1998 Elsevier Science Inc. All rights reserved.