Code Christian, Domanov Yegor, Jutila Arimatti, Kinnunen Paavo K J
Helsinki Biophysics and Biomembrane Group, Medical Biochemistry, Institute of Biomedicine, University of Helsinki, Finland.
Biophys J. 2008 Jul;95(1):215-24. doi: 10.1529/biophysj.108.128710. Epub 2008 Mar 13.
The lag-burst behavior in the action of phospholipase A(2) (PLA(2)) on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was investigated at temperatures slightly offset from the main phase transition temperature T(m) of this lipid, thus slowing down the kinetics of the activation process. Distinct stages leading to maximal activity were resolved using a combination of fluorescence parameters, including Förster resonance energy transfer between donor- and acceptor-labeled enzyme, fluorescence anisotropy, and lifetime, as well as thioflavin T fluorescence enhancement. We showed that the interfacial activation of PLA(2), evident after the preceding lag phase, coincides with the formation of oligomers staining with thioflavin T and subsequently with Congo red. Based on previous studies and our findings here, we propose a novel mechanism for the control of PLA(2), involving amyloid protofibrils with highly augmented enzymatic activity. Subsequently, these protofibrils form "mature" fibrils, devoid of activity. Accordingly, the process of amyloid formation is used as an on-off switch to obtain a transient burst in enzymatic catalysis.
在略偏离1,2 - 二棕榈酰 - sn - 甘油 - 3 - 磷酸胆碱主相变温度T(m)的温度下，研究了磷脂酶A(2)（PLA(2)）作用于该脂质时的滞后 - 爆发行为，从而减缓了活化过程的动力学。通过结合荧光参数，包括供体和受体标记酶之间的Förster共振能量转移、荧光各向异性和寿命，以及硫黄素T荧光增强，解析了导致最大活性的不同阶段。我们表明，在先前的滞后阶段之后明显的PLA(2)界面活化，与用硫黄素T染色随后用刚果红染色的寡聚体形成相吻合。基于先前的研究和我们在此的发现，我们提出了一种控制PLA(2)的新机制，涉及具有高度增强酶活性的淀粉样原纤维。随后，这些原纤维形成“成熟”纤维，失去活性。因此，淀粉样蛋白形成过程被用作一个开关，以在酶催化中获得短暂的爆发。
