Studies on the mechanisms of the anti-inflammatory and anti-viral native substances

天然抗炎抗病毒物质的作用机制研究

基本信息

批准号：
08670532
负责人：
OHTSUKI Kenzo
金额：
$ 1.41万
依托单位：
Kitasato University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

The project study was done to investigate (i) purification of the specific binding proteins (gbPs) of anti-inflammatory egent, glycyrrhizin, from GL-sensitive mammalian cells, (ii) identification of gbPs by determination of their N-terminal partial amino acid sequences, and (iii) kinetics of the GL-induced selective inhibition of their physiological activities in vitro.To purify the gbPs from GL-sensitive mammalian cells or mouse tissues (liver and spleen) , a GL-affinity column was prepared. By using this GL-affinity column, several gbPs were purified to homogenous polypeptides after partial purification of the 1.5 M NaC1 clude extracts by combination with CM-cellulose column cromatography and gel filtration on a Superdex 200pg column (HPLC) . Determination of their N-termina partial amino acid sequences revealed that (i) arachidonate cascade enzymes, such as phospholipase A_2 (PLA_2) , 5'-1ipoxygenase (5'-LOX) and lipocortin I,are identified as gbPs ; (ii) serine protease (p32) , hyaluronidase (p55) comperiment C3 and immunoglobulin (Igg) are identified as gbPs in human serum ; and (iii) 14 kDa secetory PLA_2 (14 kDa sPLA_2 ) and serine protease are identified as gbPs in the synovial fluids of patients with rheumatonoid arthritis. Furthermore, kenitic studies revealed that GL,but not GA,at low levels (1-5muM) inhibits the-physiological activities of these gbPs and also selectively inhibits the CK-II-mediated activation of these gbPs in vitro.

该项目研究的目的是调查 (i) 从 GL 敏感哺乳动物细胞中纯化抗炎剂甘草甜素的特异性结合蛋白 (gbP)，(ii) 通过测定其 N 端部分氨基酸来鉴定 gbP序列，以及 (iii) GL 诱导的体外生理活性选择性抑制的动力学。为了从 GL 敏感的哺乳动物细胞或小鼠组织（肝脏和脾脏）中纯化 gbP，制备GL-亲和柱。通过使用该GL亲和柱，通过与CM-纤维素柱色谱法和Superdex 200pg柱（HPLC）上的凝胶过滤相结合，部分纯化1.5M NaCl包括提取物后，数个gbP被纯化为均质多肽。对其N端部分氨基酸序列的测定表明，（i）花生四烯酸级联酶，如磷脂酶A_2（PLA_2）、5'-1环氧合酶（5'-LOX）和脂皮质素I，被鉴定为gbPs； (ii) 丝氨酸蛋白酶 (p32)、透明质酸酶 (p55) 互补体 C3 和免疫球蛋白 (Igg) 在人血清中被鉴定为 gbP； (iii) 14 kDa 分泌型 PLA_2 (14 kDa sPLA_2 ) 和丝氨酸蛋白酶在类风湿性关节炎患者的滑液中被鉴定为 gbP。此外，动力学研究表明，低水平（1-5μM）的 GL（而非 GA）会抑制这些 gbP 的生理活性，并且还会在体外选择性抑制 CK-II 介导的这些 gbP 的激活。