Lichtenbergova L, Yoon E T, Cho W
Department of Chemistry (M/C 111), University of Illinois at Chicago 60607-7061, USA.
Biochemistry. 1998 Oct 6;37(40):14128-36. doi: 10.1021/bi980888s.
To determine the mechanism of calcium-dependent membrane binding of cytosolic phospholipase A2 (cPLA2), we measured the interactions of cPLA2 with phospholipid monolayers and polymerizable mixed liposomes containing various phospholipids. In the presence of calcium, cPLA2 showed much higher penetrating power than secretory human pancreatic PLA2 toward anionic and electrically neutral phospholipid monolayers. cPLA2 also showed ca. 30-fold higher binding affinity for nonpolymerized 2, 3-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-1-phosphoglycerol (D-BLPG) liposomes than for polymerized ones where the membrane penetration of protein is significantly restricted. Consistent with this difference in membrane binding affinity, cPLA2 showed 20-fold higher activity toward fluorogenic substrates, 1-O-(1-pyrenedecyl)-2-arachidonoyl-sn-glycero-3-phosphocholine, inserted in nonpolymerized D-BLPG liposomes than the same substrate in polymerized D-BLPG liposomes. Furthermore, cPLA2 showed much higher sn-2 acyl group specificity (arachidonate specificity) and headgroup specificity in nonpolymerized D-BLPG liposomes than in polymerized D-BLPG liposomes. Finally, diacylglycerols, such as 1, 2-dioleoyl-sn-glycerol, selectively enhanced the membrane penetration, hydrophobic membrane binding, and interfacial enzyme activity of cPLA2. Taken together, these results indicate the following: (1) calcium not only brings cPLA2 to the membrane surface but also induces its membrane penetration. (2) This unique calcium-dependent membrane penetration of cPLA2 is necessary for its interfacial binding and substrate specificity. (3) Diacylglycerols might work as a cellular activator of cPLA2 by enhancing its membrane penetration and hydrophobic membrane binding.
为了确定胞质型磷脂酶A2（cPLA2）钙依赖性膜结合的机制，我们测量了cPLA2与磷脂单层以及含有各种磷脂的可聚合混合脂质体之间的相互作用。在有钙存在的情况下，cPLA2对阴离子和电中性磷脂单层的穿透能力比分泌型人胰腺磷脂酶A2高得多。cPLA2对未聚合的2,3-双[12-（硫辛酸氧基）十二烷酰基]-sn-甘油-1-磷酸甘油（D-BLPG）脂质体的结合亲和力也比对蛋白质膜穿透受到显著限制的聚合脂质体高约30倍。与膜结合亲和力的这种差异一致，cPLA2对插入未聚合D-BLPG脂质体中的荧光底物1-O-（1-芘癸基）-2-花生四烯酰基-sn-甘油-3-磷酸胆碱的活性比对聚合D-BLPG脂质体中的相同底物高20倍。此外，cPLA2在未聚合的D-BLPG脂质体中比在聚合的D-BLPG脂质体中表现出更高的sn-2酰基特异性（花生四烯酸特异性）和头部基团特异性。最后，二酰基甘油，如1,2-二油酰基-sn-甘油，选择性地增强了cPLA2的膜穿透、疏水膜结合和界面酶活性。综上所述，这些结果表明：（1）钙不仅使cPLA2到达膜表面，还诱导其膜穿透。（2）cPLA2这种独特的钙依赖性膜穿透对于其界面结合和底物特异性是必要的。（3）二酰基甘油可能通过增强cPLA2的膜穿透和疏水膜结合而作为其细胞激活剂。
