溶血血小板活化因子乙酰基转移酶(LPAFAT)活性检测方法的比较  

Evaluation of three methods for Lyso-PAF acetyl transferase(LPAFAT)activity assay and their appliance in assessing compounds’inhibitory activities

作　　者：胡傢椿 董继斌[1] 楼滨[1] 李英霞[2] 蒋宪成[1] 丁庭波 HU Jia-chun;DONG Ji-bin;LOU Bin;LI Ying-xia;JIANG Xian-cheng;DING Ting-bo(Department of Pharmacology and Biochemistry,School of Pharmacy,Fudan University,Shanghai 201203,China;Department of Medicinal Chemistry,School of Pharmacy,Fudan University,Shanghai 201203,China;Experimental Teaching Center of Pharmaceutical Sciences,School of Pharmacy,Fudan University,Shanghai 201203,China)

机构地区：[1]复旦大学药学院药理与生化教研室,上海201203 [2]复旦大学合成药物化学教研室,上海201203 [3]复旦大学药学实验教学中心,上海201203

出　　处：《复旦学报（医学版）》2020年第4期488-495,共8页Fudan University Journal of Medical Sciences

基　　金：国家自然科学基金(31371190,81172975)。

摘　　要：目的通过3种方法检测溶血血小板活化因子(platelet activating factor,PAF)乙酰基转移酶(Lyso-PAF acetyl transferase,LPAFAT)的活性并测定抑制剂对酶活性的抑制作用,比较这些方法的优劣。方法分别采用LC/MS/MS检测产物PAF,巯基反应探针结合分光光度法检测产物CoA-SH,TLC检测荧光标记产物法鉴定阳性和假阳性两种不同化合物对LPAFAT酶活性的抑制作用。结果LC/MS/MS方法通过检测天然产物PAF的含量测定酶活性,抑制剂筛选结果准确可靠。巯基反应探针结合分光光度法虽然可以监测产物CoA-SH的含量,但反应体系中产生了大量干扰性的CoA-SH,因此并不能真实反映LPAFAT酶活性,亦不适于抑制剂活性筛选。TLC检测荧光标记产物法因底物NBD-Lyso PC结构已被修饰,不能代表天然底物的真实反应情况,可能存在错筛和漏筛。结论LC/MS/MS检测产物PAF测定LPAFAT活性以及筛选抑制剂,方法灵敏,数据结果可靠,但成本较高。监测产物CoA-SH的含量不适合于测定LPAFAT酶活性。TLC检测荧光标记产物,操作简便,灵敏度高,但筛选抑制剂时存在假阳性的可能,只适用于抑制剂活性的初筛。Objective To evaluate the advantages and deficiencies of methods for measuring Lysoplatelet activating factor(PAF)acetyl transferase(LPAFAT)activity and their appliance in assessing compounds’inhibitory activities.Methods We used three typical methods to measure the product PAF by LC/MS/MS.We measured the product Co A-SH by thiol-reactive probes combined with spectrophotography,and measured the fluorescence labeled product by thin layer chromatography to determine the LPAFAT activity and the inhibitory effect of two compounds,a positive inhibitor and a false positive inhibitor.Results LPAFAT activity and the compounds’inhibitory effect could be determined accurately and sensitively by LC/MS/MS method.Although the Co A-SH could be measured sensitively by thiol-reactive probes combined with spectrophotography,this method could not be used to determine the exact LPAFAT activity because a large amount of unspecific Co A-SH was generated by reactions such as protein acetylation,which made it unsuitable either for screening inhibitors.LPAFAT activity could be determined by measuring the fluorescence labeled PAF analogue after TLC separation.However,since the structure of substrate NBD-lyso PC had been modified and could not represent the real reaction of natural substrates,activities of inhibitors or activators could not be correctly assessed when this method was applied in compound screening.Conclusion LC/MS/MS is sensitive,accurate and reliable for the determination of LPAFAT activity and inhibitors screening,in spite of its high cost consumption.LPAFAT activity cannot be determined accurately by measuring the amount of product Co A-SH.Determination of LPAFAT activity by measuring the fluorescence labeled product after TLC separation is sensitive and simple,which make it suitable for compounds screening,though some compounds may not be assessed accurately,such as the false positive inhibitor in this article.

关 键 词：血小板活化因子(PAF) Lyso-PAF乙酰基转移酶(LPAFAT) 抑制活性测定 LC/MS/MS 

分 类 号：R963[医药卫生—微生物与生化药学]