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NEW APPROACH TO PURIFICATION OF MEMBRANE-BOUND ENZYME - APPLIED TO PAF SYNTHETIC ENZYME PURIFICATION

膜结合酶纯化新方法——应用于 PAF 合成酶纯化

基本信息

批准号：
12672120
负责人：
SETAKA Morio
金额：
$ 2.11万
依托单位：
TEIKYO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

Biosynthesis of PAF is known to occur by de novo or remodeling pathways. The final step of de novo synthesis of PAF is catalyzed by the CDP-choline: 1-O-alkyl-2-acetyl-sn-glycerol Cholinephosphotransferase (AAG-CPT), which transfers the phosphocholine base group from CDP-choline to 1-0-alkyl-2-acetyl-sn-glycerol. To understand the roles of this enzyme, we attemplted solubilization and purification of the enzyme from porcine spleen microsomes.1. AAG-CPT was solubilized by digitonin into the active form from porcine spleen microsome and partially purified by sequential column chromatographies on Toyopearl HW-65 and DEAE-Toyopearl 650. Solubilized AAG-CPT was deduced to be the molecular complex of 440kDa. The activity of solubilized AAG-CPT was found to be reduced by DEAE column Chromatography.2. The activity of partially purified enzyme was markedly enlarged and stabilized by adding phospholipids such as eggPC, DOPC, DOPE, DOPG and DOPS to the enzyme. However, the addition of DOPA inhibi … More ted the enzyme activity. The increased enzyme activity stimulated by DOPC was completely canceled with DOPA at 1:1 molar ratio to DOPC. The inhibitory effects of DOPA on AAG-CPT activity were also detected with porcine spleen microsome itself, instead of partially purified enzyme. Namely, treatment of microsome with exogenously added phospholipaseD brought about the increase of PA content as well as the reduction of AAG-CPT activity included in microsome.3. The reduction of AAG-CPT activity during DEAE column purification was found to be caused by adsorption of AAG-CPT on the solid surface of column resins, perhaps due to the distortion of the steric structure of AAG-CPT. So we developed the soft surface column chromatography, in which column resin surface was coverd with DEAE-dextran polymers, thus preventing the reduction of AAG-CPT activity.4. We also developed the native digitonin electrophoresis, in which Sephacryl-1000 resins were used instead of polyacrylamide gels, because the molecular weight of AAG-CPT complex, 440kDa was too big to move in gels.5. We synthesized AAG photolabel, which has azocompound at the end of the carbon chain of AAG, in order to identify AAG-CPT out ot many proteins in partially purified preparations. The sample preparation was added with AAG photolabel, then photoirradiated and incubated with ^<14>C-CDP choline, at 37℃. This process is expected to label AAG-CPT specifically with ^<14>C isotope. Infact, preliminary experiments indicated that some protein was labeled with ^<14>C. Further experiments are in progress. Less

已知PAF的生物合成通过从头或重塑途径发生。PAF从头合成的最后一步由CDP-胆碱：1-O-烷基-2-乙酰基-sn-甘油胆碱磷酸转移酶（AAG-CPT）催化，其将磷酸胆碱碱基团从CDP-胆碱转移到1-O-烷基-2-乙酰基-sn-甘油。为了了解这种酶的作用，我们从猪脾微粒体中提取了这种酶的溶解和纯化。用毛地黄皂苷将AAG-CPT从猪脾微粒体中溶解成活性形式，并通过Toyopolysis HW-65和DEAE-Toyopolysis 650连续柱层析进行部分纯化。AAG-CPT的可溶性可能是分子量为440 kDa的复合物。DEAE柱层析发现AAG-CPT增溶后活性降低.加入卵磷脂、DOPC、DOPE、DOPG、DOPS等磷脂后，部分纯化的酶活力明显增强，并有稳定作用。然而，添加多巴酚丁胺 ...更多信息测定酶活性。当DOPC与DOPA的摩尔比为1：1时，DOPC对酶活性的促进作用被完全抵消。用猪脾微粒体代替部分纯化的AAG-CPT酶，检测了DOPA对AAG-CPT酶活性的抑制作用。即外源性磷脂酶D处理微粒体后，微粒体中PA含量增加，AAG-CPT活性降低.在DEAE柱纯化过程中AAG-CPT活性的降低被发现是由于AAG-CPT在柱树脂的固体表面上的吸附引起的，可能是由于AAG-CPT的空间结构的扭曲。因此，我们开发了软表面柱层析，在柱树脂表面覆盖了DEAE-葡聚糖聚合物，从而防止了AAG-CPT活性的降低.由于AAG-CPT复合物的分子量为440 kDa，在凝胶中难以移动，我们还建立了毛地黄皂苷的天然电泳方法，用Sephacryl-1000树脂代替聚丙烯酰胺凝胶.我们合成了AAG光标记物，它在AAG的碳链末端含有偶氮化合物，用于从许多部分纯化的蛋白质中鉴定AAG-CPT。向样品制备液中加入AAG光标记物，然后进行光照射，并<14>在37℃下与^ C-CDP胆碱孵育。预期该过程用13 C同位素特异性标记AAG-CPT<14>。事实上，初步实验表明，一些蛋白质是用^ C标记<14>的。进一步的实验正在进行中。少