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Glycosylation of Membrane Sterol Associated with Cell Differentiation

与细胞分化相关的膜甾醇的糖基化

基本信息

批准号：
63580114
负责人：
MUROFUSHI Kimiko
金额：
$ 1.15万
依托单位：
Ochanomizu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

Haploid myxoamoebae of Physarum polycephalum conjugate in pairs to form diploid zygotes and differentiate into plasmodia. During the course of differentiation, an expression of a nonpolar glycoconjugate in membrane fraction was detected. This substance was purified and structural studies were performed by GC, GC/MS, FAB/MS, NMR and enzymatic hydrolysis, ant the substance has been identified as poriferasterol monoglucoside (published in J. Biol. Chem. 262, 16719-16723).UDP-glucose:poriferasterol glucosyltransferase which synthesizes poriferasterol monoglucoside was also expressed during a differentiation from myxoamoebae to plasmodia. The enzyme activity was not detected in the amoeboid cells, but after conjugation, the enzyme activity appeared and increased. The enzyme was partially purified and characterized, and its molecular weight was determined to be 72,000, and pH optimum was 7.0. It catalyzed the glucose transfer from UDP-glucose to sitosterol and campesterol as well as to poriferasterol, but stigmasterol which is C-24 epimer of poriferasterol was a poor substrate for this enzyme reaction. Apparent Km values were 1.2 x 10^<-4>M for UDP-glucose and 4.8 x 10^<-6>M for poriferasterol, respectively. (Biochim. Biophys. Acta 992, 412-415).Colonia strain of Physarum polycephalum is a mutant which differentiates from myxoamoebae to plasmodia without nuclear DNA change. The poriferasterol monoglucoside and its synthesizing enzyme were detected in both stages, myxoamoebae and plasmodia, in this mutant strain.Changes in phospholipid composition and phospholipase D activity were also observed during a differentiation from myxoamoebae to diploid plasmodia in both, wild-typed and mutant strains (Biochim. Biophys. Acta,in press). Sterol contents were examined and compared, then the major one was determined to be poriferasterol (Cell Struct. Funct. 12, 519-524).

多头绒泡菌单倍体粘阿米巴成对结合形成二倍体受精卵并分化为疟原虫。在分化过程中，在膜组分中检测到非极性糖结合物的表达。通过GC、GC/MS、FAB/MS、核磁共振和酶解等手段对该物质进行了分离纯化和结构鉴定，确定其为猪瘟甾醇单糖。化学。UDP-葡萄糖：在粘阿米巴向疟原虫分化的过程中，也表达了合成孔非甾醇单糖苷的孔非甾醇葡萄糖转移酶。在阿米巴细胞中未检测到该酶的活性，但接合后，该酶的活性出现并增强。对该酶进行了部分纯化和性质鉴定，测得其相对分子质量为72,000，最适pH为7.0。它能催化葡萄糖从UDP-葡萄糖转移到谷甾醇、菜籽甾醇以及猪粪甾醇，但豆甾醇是猪粪甾醇的C-24同分异构体，不适合该酶反应。表观Km值分别为1.2×10~(-4)M和4.8×10~(-6)M。(Biochim.生物群落。Acta 992,412-415)。多头绒泡菌的克隆株是一株从粘阿米巴分化为原虫的突变体，核DNA没有变化。该突变株在粘阿米巴和原虫两个阶段都检测到了猪瘟甾醇单糖苷及其合成酶，并观察到从粘阿米巴向二倍体原虫分化过程中磷脂组成和磷脂酶D活性的变化。生物群落。ACTA，在印中)。对甾醇含量进行了测定和比较，确定其主要成分为孔雀石醇(Cell Struct.)功能。12,519-524)。