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

• Project/Area Number: 12672120
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Biological pharmacy
• Research Institution: TEIKYO UNIVERSITY
• Principal Investigator: SETAKA Morio TEIKYO UNIVERSITY, FACULTY OF PHARMACEUTICAL SCIENCES, PROFESSOR, 薬学部, 教授 (70012630)
• Co-Investigator(Kenkyū-buntansha): SATOH Noriko TEIKYO UNIVERSITY, FACULTY OF PHARMACEUTICAL SCIENCES, RESEARCH ASSOCIATE, 薬学部, 助手 (80162460) ; KARASAWA Ken TEIKYO UNIVERSITY, FACULTY OF PHARMACEUTICAL SCIENCES, ASSISTANT PROFESSOR, 薬学部, 助教授 (50186029)
• Project Period (FY): 2000 – 2002
• Project Status: Completed (Fiscal Year 2002)
• Budget Amount: ¥3,300,000 (Direct Cost: ¥3,300,000); Fiscal Year 2002: ¥600,000 (Direct Cost: ¥600,000); Fiscal Year 2001: ¥900,000 (Direct Cost: ¥900,000); Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
• Keywords: Platelet Activating Factor / membrane bound enzyme / PAF synthetic enzyme / enzyme purification / 膜結合型酵素 / 酵素精製 / PAF合成酵 / 膜結合酵素の精製

Research Abstract

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 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.

Research Products

• [Publications] N.Satoh, A.Harada, K.Yokoyama, K.Karasawa, K.Inoue, M.Setaka: "Regulation of Activities of Cytidine 5'-Diphosphocholine : 1-O-Alkyl-2-Acetyl-sn-Glycerol Cholinephosphotransferase, an Enzyme Responsible for de novo Synthesis of Platelet-Activating factor, by Membrane Phospholipids"J.Health Science. 49(1). 13-21 (2003)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Noriko Satoh, Ayako Harada, Kazuaki Yokoyama, Ken Karasawa, Keizo Inoue and Morio Setaka: "Regulation of Activities of Cytidine 5'-Dephosphocholine: 1-O-Aikyl-2-Acetyl-sn-Glycerol Cholinephosphotransferase, an Enzyme Responsible for de novo Synthesis of Platelet-Activating Factor, by Membrane Phospholipids"J. of Health Science. 49 (1). 13-21 (2003)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] N.Satoh, A.Harada, K.Yokoyama, K.Karasawa, K.Inoue, M.setaka: "Regulation of Activities of Cytidine 5-Diphosphocholine : 1-O-Alkyl-2-Acetyl-sn-Glycerol Cholinephospohtransferase, an Enzyme Responsible for de novo Synthesis of Platelet-Activating factor, by Membrane Phospholipids"J. Health Science. 49(1). 13-21 (2003)
• [Publications] N.Satoh et al.: "Phospholipids-induced modulation of AAG-CPT activities solubilized form porcine spleen microsomes."Chemistry and Physics of Lipids. 107. 30 (2000)