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首页> 外文期刊>Biochemistry >Interfacial catalysis by human 85 kDa cytosolic phospholipase A2 on anionic vesicles in the scooting mode.
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Interfacial catalysis by human 85 kDa cytosolic phospholipase A2 on anionic vesicles in the scooting mode.

机译：人85 kda胞质磷脂脂磷脂酶A2在扫描模式中阴离子囊泡的界面催化。

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摘要

Analysis of phospholipases A2 on model phospholipid bilayers in which enzyme is essentially irreversibly bound at the lipid-water interface, termed "scooting mode", is a useful tool for studying the kinetic properties of interfacial enzymes. It is shown that human cytosolic 85 kDa phospholipase A2 (cPLA2) hydrolyzes sn-2-arachidonyl-containing phospholipids or the gamma-linolenoyl ester of 7-hydroxycoumarin (GLU) dispersed in vesicles of 1,2-dioleoyl-sn-glycero-3-phosphomethanol (L-DOPM) in the scooting mode. Trapping of cPLA2 on L-DOPM vesicles is rapid and independent of product formation. Slowing of cPLA2-catalyzed hydrolysis of substrates present in phosphatidylmethanol and phosphatidylcholine vesicles is primarily due to apparent inactivation rather than to substrate depletion. cPLA2 phosphorylated on serine 505 by mitogen-activated protein kinase displays a 30% increase in the rate of sn-2-arachidonylphosphatidylcholine hydrolysis in the scooting mode compared to that of the nonphosphorylated enzyme. Kinetic parameters of cPLA2 acting on a variety of different phosphatidylmethanol vesicles were evaluated, and the results are discussed in terms of active site affinities for substrates and of lateral organization of substrates in the bilayer. A key result is that the sigmoidal kinetics reported previously using 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) vesicles are most prominent near the phase transition temperature of DMPM. No sigmoidal kinetics was observed using L-DOPM vesicles. The results of kinetic experiments and the behavior of a fluorescent substrate analog are consistent with nonideal mixing of substrate in DMPM vesicles, but not in L-DOPM vesicles, suggesting that apparent saturation and sigmoidal kinetics are more a result of nonideal mixing of substrate in DMPM vesicles than of active site binding of substrate. The fluorescence assay described using L-DOPM/GLU vesicles is useful for evaluating the interfacial behavior of cPLA2, including its substrate preferences and the effect of active site-directed inhibitors.
机译：磷脂酶A2对模型磷脂双层的磷脂酶A2，其中酶在脂质 - 水界面上基本上不可逆转地结合，称为“Scooting模式”，是研究界面酶的动力学性质的有用工具。结果表明，人细胞溶溶胶85kDa磷脂酶A2（CPLA2）水解含Sn-2-花生泛酰基的磷脂或7-羟基苏格林（Glu）的γ-亚麻酰基酯，分散在1,2-Dioleyl-Sn-Glycero-3的囊泡中在Scooting模式下 - 膦醇（L-DOPM）。 L-DOPM囊泡上的CPLA2捕获是快速的，与产品形成无关。在磷脂酰甲醇和磷脂酰胆碱囊泡中存在的底物的CPLA2催化水解速度主要是由于表观灭活而不是底物耗尽。通过促丝磷酸化蛋白激酶磷酸丝氨酸505磷酸化的CPLA2显示，与非磷酸化酶的酶相比，SN-2-植物酰基膦酰胺水解的速率增加30％。评估作用于各种不同磷脂酰甲醇囊泡的CPLA2的动力学参数，并就底物的活性位点亲和力讨论了结果，双层中的衬里衬底的侧向组织。一个关键的结果是先前使用1,2-Dimyristoyl-Sn-甘油-3-磷甲醇（DMPM）囊泡的六样动力学在DMPM的相转变温度附近最突出。使用L-DOPM囊泡观察到不观察到乙型骨。动力学实验的结果和荧光衬底模拟的行为与DMPM囊泡中的底物的非膜混合一致，但不是在L-DOPM囊泡中，表明表观饱和度和乙状动力学在DMPM中的底物的非膜混合的结果是更加饱和度囊泡比底物的活性位点结合。使用L-DOPM / GLU囊泡描述的荧光测定可用于评估CPLA2的界面行为，包括其基材偏好和活性位点定向抑制剂的效果。

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