CONTINUOUS, VESICLE-BASED FLUOROMETRIC ASSAYS OF 14- AND 85-KDA PHOSPHOLIPASES A(2)
CONTINUOUS, VESICLE-BASED FLUOROMETRIC ASSAYS OF 14- AND 85-KDA PHOSPHOLIPASES A(2)
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DOI：
10.1006/abio.1995.9967
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发表时间：
1995-11-20
影响因子：
2.9
通讯作者：
GELB, MH
中科院分区：
文献类型：
--
作者：
BAYBURT, T;YU, BZ;GELB, MH
本文介绍了85 kDa哺乳动物胞浆磷脂酶A(2)(CPLA(2))和14 kDa人非胰腺分泌型磷脂酶A(2)(SLA(2))的新底物的合成和分析。合成了在sn-2位含有花生四烯基链的磷脂酰胆碱，以及在sn-1位含有10-丙二烯基或10-丙二酰基的磷脂酰胆碱，并在基于荧光的分析中证明它们是CPLA(2)的底物。尽管当底物分散在Triton X-100混合胶束中时，该方法也适用，但大多数分析都使用底物磷脂的小单层囊泡和大单层囊泡。CPLA(2)的分析可以在一个固定的时间点模式下进行，其中一个产物--含芘的溶血磷脂--被快速高效液相色谱检测。或者，当水相中存在牛血清白蛋白时，分析变得连续；这种蛋白质从囊泡中提取含有芘的溶血磷脂，这导致了单体芘标记的荧光。这些检测方法能够检测到亚纳克量的CPLA(2)。在伽马-亚麻酸和7-羟基香豆素之间形成的酯也是CPLA(2)的底物，当结合到阴离子磷脂的囊泡中时，1，2-二油酰基-sn-甘油-3-磷酸甲基提供了一种分析方法，其中酶不离开囊泡表面(滑移模式)。与所有以前报道的基于囊泡的CPLA(2)分析不同，基于DOPM的分析可以看到一个漫长的线性反应过程。利用底物1-palmitoyl-2-(10-pyrenedecanoyl)-sn-glycero-3-phosphomethanol和脂沉，建立了一种具有亚纳克灵敏度的Spla(2)测定方法。后者由磷脂酰胆碱小泡组成，超过底物小泡，它不结合SLA(2)，但为酶产生的10-吡喃甲酸提供了陷阱。检测到了下沉小泡中的单体芘标记的荧光。基于底物1，2-di(10-pyrenedecanoyl)-sn-glycero-3-phosphocholine存在于非水解性阴离子磷脂1，2-二十四烷基甘油-3-磷酸甲醇的囊泡中，底物为10%，建立了第二种使用单一类型囊泡的SPLA(2)分析方法。在这种荧光底物上，sPLA2的作用导致芘的链分离，从而从单体芘中发出荧光。这些CPLA(2)和SLA，是筛选和分析抑制剂以及研究这些酶的界面动力学的理想方法。(C)1995年学术出版社。
This paper describes the synthesis and analysis of new substrates for the 85-kDa, mammalian, cytosolic phospholipase A(2) (cPLA(2)) and the 14-kDa, human nonpancreatic, secreted phospholipase A(2) (sPLA(2)). Phosphatidylcholines containing an arachidonyl chain at the sn-2 position and either a 10-pyrenedecyl or a 10-pyrenedecanoyl chain at the sn-1 position were synthesized and shown to be substrates for cPLA(2) in a fluorescence-based assay. Most of the assays make use of small and large unilamellar vesicles of substrate phospholipid, although the assay also works when the substrate is dispersed in Triton X-100 mixed-micelles. The cPLA(2) assays can be carried out in a fixed time-point mode in which one of the products, the pyrene-containing lysophospholipid, is detected by rapid HPLC. Alternatively, the assay becomes continuous when bovine serum albumin is present in the aqueous phase; this protein extracts the pyrene-containing lysophospholipid from the vesicle, and this leads to the fluorescence of monomeric pyrene label. These assays are capable of detecting subnanogram amounts of cPLA(2). The ester formed between gamma-linolenic acid and 7-hydroxycoumarin is also a substrate for cPLA(2), and when incorporated into vesicles of the anionic phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphomethano provides an assay in which the enzyme does not leave the vesicle surface (scooting mode). Unlike all of the previously reported, vesicle-based cPLA(2) assays, a prolonged linear reaction progress is seen with the DOPM-based assay. An assay of sPLA(2) with subnanogram sensitivity was developed which makes use of the substrate 1-palmitoyl-2-(10-pyrenedecanoyl)-sn-glycero-3-phosphomethanol and a lipid sink. The latter is composed of phosphatidylcholine vesicles, in excess of substrate vesicles, which do not bind sPLA(2) but provide a trap for enzyme-produced 10-pyrenedecanoic acid. The fluorescence of monomeric pyrene label in sink vesicles is detected. A second sPLA(2) assay using a single type of vesicle was developed based on the substrate 1,2-di(10-pyrenedecanoyl)-sn-glycero-3-phosphocholine present at 10 mol% in vesicles of the nonhydrolyzable anionic phospholipid 1,2-ditetradecyl-sn-glycero-3 -phosphomethanol. The action of sPLA2 on this fluorescent substrate leads to a separation of the pyrene chains resulting in fluorescence emission from monomeric pyrene. These cPLA(2) and sPLA, assays are ideal for inhibitor screening and analysis, and for studying the interfacial kinetics of these enzymes. (C) 1995 Academic Press, Inc.