Analysis of function and strueture of receptor protein for storageprotein SPI in Bomyxmori.

1)利用5龄第9天幼虫的脂肪体制剂，研究了储存蛋白与质膜组分的结合。在pH值为5且有二价阳离子存在的情况下，SP1与质膜的结合效果最佳，其表观Km为3.5 × 10^<-8> m。SP1结合的竞争实验强烈表明SP1与脂肪体质膜的结合具有较高的特异性，SP1和SP2可能被不同的受体识别。从脂肪体质膜组分中对sp1结合蛋白进行增溶和表征，鉴定和纯化sp1结合蛋白。免疫沉淀和配体印迹实验表明，脂肪体膜组分中有一个100 kDa的蛋白是SPl结合蛋白。2)在Ultrogel ACA34柱上进行凝胶过滤，在Ca^<++>和制备物g . 0存在的情况下，在抗SP1抗体- sepharose柱上进行亲和层析，分离SP1结合蛋白。免疫印迹结果显示，a蛋白与mol.wt.。L00k仅在5龄幼虫至吐丝时的脂肪体组织中出现，在蛹发育时消失。电镜分析表明，纯化后的sp1结合蛋白是一种位于质膜网状系统和基底层的膜蛋白。3)利用5日龄5龄雌幼虫脂肪体mRNA构建cDNA表达文库。用针对sp1结合蛋白的亲和纯化抗体筛选文库，获得7个免疫阳性克隆。以标记的cDNA为探针重新筛选cDNA文库。通过筛选5 × 10^5 pfu，分离出3.5 kb的cdna克隆。采用cDNA末端快速扩增法克隆sp1结合蛋白mRNA的5′末端区域。sp1结合蛋白cDNA克隆区有一个编码1025个氨基酸残基的开放阅读框，推导出的肽段分子量为114,000，略高于SDS聚丙烯酰胺凝胶电泳估计的分子量100,000。从mRNA序列推断，sp1结合蛋白具有两种可能的跨膜结构。少

1) The binding of storage protein to the plasma membrane fraction was studied using the fat body preparation from the fifth instar day-9 larvae. SP1 binding to the plasma membrane was optimal at pH 5 in the presence of divalent cations and apparent Km for SP1 was determined to be 3.5 x 10^<-8> M.The competition experiments of SP1 binding strongly suggested that SP1 binds to the plasma membrane of fat body with high specificity, and that SP1 and SP2 might be recognized by each distinct receptor. Solubilization and characterization of the SP1-binding protein were performed from the fat body plasma membrane fraction to identify and purify the SP1-binding protein. Immunoprecipitate and ligand blot assay demonstrated that a l00 kDa protein in the fat body membrane fraction is SPl binding protein.2) SP1-binding proteins was separated by the gel filtration on an Ultrogel ACA34 column, affinity chromatography on an anti-SP1 antibody-Sepharose column in the presence of Ca^<++> and preparative g … More el electrophoresis, and an antibody was raised against the purified SP1 binding protein. Immunoblotting result show that the a protein with mol.wt.l00k revealed only in the fat body tissue from the fifth instar day-3 larvae to silk-spinning and disappears at pupal development. Electron microscopic analyzes suggested that the purified SP1-binding protein is a membrane protein locating at the plasma membrane reticular system and basal lamina.3) A cDNA expression library was constructed from the fat body mRNA of 5-day old fifth instar female larvae. Screening of the library with the affinity-purified antibodies against the SP1-binding protein yielded seven immuno-positive clones. The cDNA library was rescreened using labeled cDNA as a probe. By screening 5 x 10^5 pfu, a 3.5 kb cDNA-clone was isolated.The 5' terminal region of SP1-binding protein mRNA was cloned by the method of rapid amplification of cDNA end. There is an open reading frame coding for 1,025 amino acid residues in the cloned region of SP1-binding protein cDNA and deduced peptide was a molecular weight of 114,000 that is a little higher than molecular weight 100,000 estimated with SDS polyacrylamide gel electrophoresis. Primary structure deduced from mRNA sequence revealed that SP1-binding protein has two putative membrane-spanning structures. Less