Direct imaging by cryo-TEM shows membrane break-up by phospholipase A2 enzymatic activity

被引:44
作者
Callisen, TH
Talmon, Y
机构
[1] Tech Univ Denmark, Dept Chem, DK-2800 Lyngby, Denmark
[2] Technion Israel Inst Technol, Dept Chem Engn, IL-32000 Haifa, Israel
关键词
D O I
10.1021/bi980255d
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Phospholipid hydrolysis to free fatty acid and l-lyso-phospholipid by water-soluble phospholipase A(2) (PLA(2)) at the surface of lipid membranes exhibits a poorly understood transition from a low-activity lag phase to a burst regime of rapid hydrolysis. Understanding this kinetic phenomenon may increase our insight into the function of PLA2 under physiological conditions as well as into general interfacial catalysis. In the present study we apply for the first time cryo-transmission electron microscopy (cryo-TEM) and high-performance liquid chromatography (HPLC) to characterize the PLA2 hydrolysis of phospholipid vesicles with respect to changes in lipid composition and morphology. Our direct experimental results show that the initial reaction conditions are strongly perturbed during the course of hydrolysis, Most strikingly, cryo-TEM reveals that starting in the lag phase, vesicles become perforated and degrade into open vesicles, bilayer fragments, and micelles, This structural instability extends throughout the system in the activity burst regime. In agreement with earlier reported correlations between initial phospholipase activity and substrate morphology, our results suggest that the lag-burst phenomenon reflects a cascade process. The PLA(2)-induced changes in lipid composition transform the morphology which in turn results in an acceleration of the rate of hydrolysis because of a strong coupling between the PLA(2) activity and the morphology of the lipid suspension.
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收藏
页码:10987 / 10993
页数:7
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