Isoform-specific membrane insertion of secretory phospholipase A2 and functional implications.
Isoform-specific membrane insertion of secretory phospholipase A2 and functional implications.
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分泌型磷脂酶 A2 的异构体特异性膜插入及其功能意义。
DOI：
10.1021/bi060898q
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发表时间：
2006
期刊：
影响因子：
2.9
通讯作者：
Tatulian,SurenA
中科院分区：
文献类型：
--
作者：
Pande,AbhayH;Qin,Shan;Nemec,KathleenN;He,Xiaomei;Tatulian,SurenA
尽管越来越多的证据表明，界面酶的膜结合模式（包括膜插入深度）对其功能至关重要，但磷脂酶A2（PLA2）的膜插入尚未得到系统的研究。在这里，我们分析了人类IB群PLA2（hIBPLA2）的膜插入，并将其与结构同源的人类IIA群PLA2（V3W- hiiapla2）的V3W突变体以及结构不同的III群蜂毒PLA2（bvPLA2）进行了比较。膜上阴离子电荷的增加导致hIBPLA2的trp3荧光蓝移，丙烯酰胺猝灭作用减弱，酶活性增加，反映了PLA2的膜结合增强。溴化脂质的荧光猝灭表明trp3在流体POPC/POPG膜中有明显的渗透，但在固体DPPC/DPPG膜中很少插入。膜流动性的增加也支持hibpla2的活性，这表明hibpla2的膜插入是由膜流动性控制的，是酶充分活性的必要条件。水溶猝灭剂和膜溶猝灭剂对V3W-hIIAPLA2和bvPLA2的色氨酸荧光猝灭表明，V3W-hIIAPLA2的trp3有大量的膜插入，与hIBPLA2相似，而bvPLA2的色氨酸没有插入。我们的研究结果提供了证据(a)结构相似的IB和IIA组PLA2s，但结构不同的III组PLA2，显著渗透到膜中；(b)膜插入受膜流动性控制，并促进IB和IIA PLA2s的激活；(c)在脂质水解过程中，结构不同的pla2亚型可能采用不同的底物加入/产物释放策略。
Despite increasing evidence that the membrane-binding mode of interfacial enzymes including the depth of membrane insertion is crucial for their function, the membrane insertion of phospholipase A2(PLA2) enzymes has not been studied systematically. Here, we analyze the membrane insertion of human group IB PLA2(hIBPLA2) and compare it with that of a structurally homologous V3W mutant of human group IIA PLA2(V3W-hIIAPLA2) and with a structurally divergent group III bee venom PLA2(bvPLA2). Increasing the anionic charge of membranes results in a blue shift of the fluorescence of Trp3of hIBPLA2, a decrease in quenching by acrylamide, and an increase in enzyme activity, reflecting an enhancement in the membrane binding of PLA2. Fluorescence quenching by brominated lipids indicates significant penetration of Trp3into fluid POPC/POPG membranes but little insertion into the solid DPPC/DPPG membranes. Increased membrane fluidity also supports hIBPLA2activity, suggesting that membrane insertion of hIBPLA2is controlled by membrane fluidity and is necessary for the full activity of the enzyme. Trp fluorescence quenching of the V3W-hIIAPLA2and bvPLA2by water- and membrane-soluble quenchers indicates substantial membrane insertion of Trp3of V3W-hIIAPLA2, similar to that found for hIBPLA2, and no insertion of tryptophans of bvPLA2. Our results provide evidence that (a) structurally similar group IB and IIA PLA2s, but not structurally diverse group III PLA2, significantly penetrate into membranes; (b) membrane insertion is controlled by membrane fluidity and facilitates activation of IB and IIA PLA2s; and (c) structurally distinct PLA2isoforms may employ different tactics of substrate accession/product release during lipid hydrolysis.