Broekemeier Kimberly M, Iben James R, LeVan Emily G, Crouser Elliott D, Pfeiffer Douglas R
Department of Chemistry, Ohio Northern University, Ada, Ohio 45018, USA.
Biochemistry. 2002 Jun 18;41(24):7771-80. doi: 10.1021/bi020157z.
Mitochondria contain a type IIA secretory phospholipase A(2) that has been thought to hydrolyze phospholipids following Ca(2+) accumulation and induction of the permeability transition. These enzymes normally require millimolar Ca(2+) for optimal activity; however, no dependence of the mitochondrial activity on Ca(2+) can be demonstrated upon equilibrating the matrix space with extramitochondrial Ca(2+) buffers. Ca(2+)-independent activity is seen following protonophore-mediated uncoupling, when uncoupling arises through alamethicin-mediated pore formation, or upon opening the permeability transition pore. Under the latter conditions, activity continues in the presence of excess EGTA but is somewhat enhanced by exogenous Ca(2+). The Ca(2+)-independent activity is best seen in media of high ionic strength and displays a broad pH optimum located between pH 8 and pH 8.5. It is strongly inhibited by bromoenol lactone but not by arachidonyl trifluoromethyl ketone, dithiothreitol, and other inhibitors of particular phospholipase A(2) classes. Immunoanalysis of mitochondria and mitochondrial subfractions shows that a membrane-bound protein is present that is recognized by antibody against an authentic iPLA(2) that was first found in P388D(1) cells. It is concluded that mitochondria contain a distinct Ca(2+)-independent phospholipase A(2) that is regulated by bioenergetic parameters. It is proposed that this enzyme, rather than the Ca(2+)-dependent type IIA phospholipase A(2), initiates the removal of poorly functioning mitochondria by processes involving autolysis.
线粒体含有一种IIA型分泌型磷脂酶A2，人们认为它在钙离子积累和通透性转换诱导后会水解磷脂。这些酶通常需要毫摩尔浓度的钙离子才能达到最佳活性；然而，当用线粒体外钙离子缓冲液平衡基质空间时，无法证明线粒体活性对钙离子的依赖性。在质子载体介导的解偶联后、通过阿拉霉素介导的孔形成导致解偶联时或打开通透性转换孔时，可观察到不依赖钙离子的活性。在后一种情况下，即使存在过量的乙二醇双四乙酸（EGTA），活性仍会持续，但外源钙离子会使其有所增强。不依赖钙离子的活性在高离子强度的介质中最明显，其最适pH值范围较宽，在pH 8至pH 8.5之间。它受到溴烯醇内酯的强烈抑制，但不受花生四烯酰三氟甲基酮、二硫苏糖醇和其他特定磷脂酶A2类抑制剂的抑制。对线粒体和线粒体亚组分的免疫分析表明，存在一种膜结合蛋白，它能被针对最初在P388D(1)细胞中发现的一种真实的iPLA(2)的抗体所识别。结论是线粒体含有一种独特的不依赖钙离子的磷脂酶A2，其受生物能量参数调节。有人提出，该酶而非依赖钙离子的IIA型磷脂酶A2，通过自溶等过程启动对功能不良线粒体的清除。
