Hixon M S, Ball A, Gelb M H
Department of Chemistry, University of Washington, Seattle 98195, USA.
Biochemistry. 1998 Jun 9;37(23):8516-26. doi: 10.1021/bi980416d.
Cytosolic group IV phospholipase A2 (cPLA2) plays a role in liberating arachidonic acid from the sn-2 position of mammalian cellular phospholipids. The enzyme consists of a catalytic domain joined to an N-terminal calcium-dependent, membrane binding domain (C2 domain). The interfacial binding properties of the full-length, nonphosphorylated enzyme and its C2 domain to phospholipid vesicles were studied as a function of vesicle phospholipid composition and calcium concentration. The binding of cPLA2 to phosphatidylcholine vesicles is mostly governed by its C2 domain; binding is relatively weak, and calcium enhances binding and interfacial catalysis by about 10-fold. Catalytically productive interfacial binding was measured by monitoring the increase in the rate of cPLA2-catalyzed hydrolysis of a fluorimetric substrate present in vesicles as a function of bulk vesicle concentration. Enzyme-vesicle binding was also measured by fluorescence as was enzyme-calcium binding. Compared to zwitterionic vesicles, cPLA2 binding to anionic phosphatidylmethanol vesicles is of higher affinity and calcium-independent, although calcium is required for the binding of the C2 domain to these anionic vesicles. cPLA2 is fully catalytically active on phosphatidylmethanol vesicles in the absence of calcium. Phosphatidylserine is not a good replacement for phosphatidylmethanol for inducing high-affinity, calcium-independent binding of cPLA2. These results reveal two modes of catalytically productive interfacial binding of cPLA2: calcium-dependent anchoring via the C2 domain and a calcium-independent component involving a phosphatidylmethanol recognition element in the catalytic domain. They also show that membrane binding of cPLA2 is not, in general, predicted by the interfacial binding properties of its C2 domain.
胞质溶胶中的IV型磷脂酶A2（cPLA2）在从哺乳动物细胞磷脂的sn-2位释放花生四烯酸的过程中发挥作用。该酶由一个催化结构域和一个N端钙依赖性膜结合结构域（C2结构域）组成。研究了全长非磷酸化酶及其C2结构域与磷脂囊泡的界面结合特性，作为囊泡磷脂组成和钙浓度的函数。cPLA2与磷脂酰胆碱囊泡的结合主要由其C2结构域控制；结合相对较弱，钙可使结合和界面催化增强约10倍。通过监测囊泡中荧光底物的cPLA2催化水解速率随囊泡总体浓度的增加来测量催化活性界面结合。酶-囊泡结合也通过荧光测量，酶-钙结合也是如此。与两性离子囊泡相比，cPLA2与阴离子磷脂酰甲醇囊泡的结合具有更高的亲和力且不依赖钙，尽管C2结构域与这些阴离子囊泡的结合需要钙。在没有钙的情况下，cPLA2在磷脂酰甲醇囊泡上具有完全的催化活性。磷脂酰丝氨酸不是诱导cPLA2高亲和力、不依赖钙结合的磷脂酰甲醇的良好替代品。这些结果揭示了cPLA2催化活性界面结合的两种模式：通过C2结构域的钙依赖性锚定和涉及催化结构域中磷脂酰甲醇识别元件的不依赖钙的成分。它们还表明，cPLA2的膜结合一般不能由其C2结构域的界面结合特性预测。
