Fujii S, Tahara Y, Toyomoto M, Hada S, Nishimura H, Inoue S, Ikeda K, Inagaki Y, Katsumura S, Samejima Y
Department of Biochemistry, Osaka University of Pharmaceutical Sciences, Japan.
Biochem J. 1995 May 15;308 ( Pt 1)(Pt 1):297-304. doi: 10.1042/bj3080297.
Chemical modification and inactivation of bovine pancreatic, porcine pancreatic, Naja naja atra and Pseudechis australis phospholipases A2 (PLA2s), belonging to Group I, and of Trimeresurus flavoviridis, Vipera russelli russelli and Agkistrodon halys blomhoffii PLA2s, belonging to Group II, were investigated by the use of a manoalide (MLD)-analogue, 1-(2,5-dihydro-hydroxy-5-oxo-3-furanyl)-8,12-dimethyl-4-formyl-3,7, 11-tridecatrienol. At appropriate time intervals, residual PLA2 activities towards monodispersed, anionic mixed micellar and non-ionic mixed micellar substrates were measured. We tested the protective effect of micellar n-dodecylphosphocholine (n-C12PC) on enzyme inactivation. Inactivation of pancreatic PLA2s (Group I) was only observed towards anionic mixed micellar substrates. This inactivation was completely prevented by the presence of micellar n-C12PC. From a fragmentation study of modified bovine pancreatic PLA2 using lysyl endopeptidase, we speculated that Lys-56 of this enzyme was modified by MLD-analogue and that this modification was responsible for enzyme inactivation. Inactivation of non-pancreatic PLA2s was observed towards all types of substrate, except that no significant inactivation of N. naja atra PLA2 (Group I) towards monodispersed substrate was noted. Micellar n-C12PC protected N. naja atra PLA2 (Group I) completely from inactivation by MLD-analogue, but had lesser protective effects on P. australis PLA2 (Group I), T. flavoviridis and V. russelli russelli PLA2s (Group II). However, no significant protection of A. halys blomhoffii PLA2s (Group II) activity was observed. These results indicate that the inactivation of pancreatic and N. naja atra PLA2s originates from the modification of Lys residues at the interfacial recognition site, and that inactivation of P. australis, T. flavoviridis and V. russelli PLA2s arises from the modification of Lys residues at the catalytic site, interfacial recognition site and regions outside both sites. The inactivation of A. halys blomhoffii PLA2 was assumed to be due to the modification of Lys residues outside the two sites described above.
利用一种 manoalide（MLD）类似物 1-(2,5-二氢-羟基-5-氧代-3-呋喃基)-8,12-二甲基-4-甲酰基-3,7,11-十三碳三烯醇，研究了属于第 I 组的牛胰、猪胰、中华眼镜蛇和澳洲拟眼镜蛇磷脂酶 A2（PLA2s）以及属于第 II 组的竹叶青、圆斑蝰和日本蝮蛇 PLA2s 的化学修饰和失活情况。在适当的时间间隔，测量了残余 PLA2 对单分散、阴离子混合胶束和非离子混合胶束底物的活性。我们测试了胶束正十二烷基磷酸胆碱（n-C12PC）对酶失活的保护作用。仅观察到胰 PLA2s（第 I 组）对阴离子混合胶束底物的失活。胶束 n-C12PC 的存在完全阻止了这种失活。通过使用赖氨酰内肽酶对修饰的牛胰 PLA2 进行片段化研究，我们推测该酶的 Lys-56 被 MLD 类似物修饰，并且这种修饰导致了酶的失活。观察到非胰 PLA2s 对所有类型的底物均有失活，只是未注意到中华眼镜蛇 PLA2（第 I 组）对单分散底物有明显失活。胶束 n-C12PC 完全保护中华眼镜蛇 PLA2（第 I 组）不被 MLD 类似物失活，但对澳洲拟眼镜蛇 PLA2（第 I 组）、竹叶青和圆斑蝰 PLA2s（第 II 组）的保护作用较小。然而，未观察到对日本蝮蛇 PLA2s（第 II 组）活性有明显保护作用。这些结果表明，胰和中华眼镜蛇 PLA2s 的失活源于界面识别位点 Lys 残基的修饰，而澳洲拟眼镜蛇、竹叶青和圆斑蝰 PLA2s 的失活源于催化位点、界面识别位点以及这两个位点之外区域的 Lys 残基的修饰。日本蝮蛇 PLA2 的失活被认为是由于上述两个位点之外的 Lys 残基的修饰。
