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

1)用5龄9龄幼虫的脂肪体制剂研究了贮藏蛋白与质膜部分的结合。在二价阳离子存在下，SP1与质膜结合的最佳pH为5，表观Km值为3.5×10~(-8)M。SP1与细胞膜的竞争结合实验表明，SP1与脂肪体质膜的结合具有很高的特异性，SP1和SP2可能被各自不同的受体识别。对脂肪体质膜组分中的SP1结合蛋白进行增溶和鉴定，以鉴定和纯化SP1结合蛋白。免疫沉淀和配基印迹分析表明，脂肪体膜组分中约100 kDa的蛋白质是Sp1结合蛋白。2)经UltroGel ACA34凝胶过滤、抗Sp1抗体-Sepharose柱亲和层析和制备g…分离得到Sp1结合蛋白。进一步电泳法检测，纯化的SP1结合蛋白产生抗体。免疫印迹结果表明，分子量为100k的α蛋白仅在5龄3龄至吐丝期的脂肪体组织中出现，并在蛹发育时消失。电子显微镜分析表明，纯化的SP1结合蛋白是一种位于质膜网状结构和基底膜的膜蛋白。3)以5日龄五龄雌幼虫脂肪体mRNA为模板，构建了一个表达文库。用亲和纯化的抗SP1结合蛋白抗体对文库进行筛选，得到7个免疫阳性克隆。以标记的c DNA为探针，对文库进行再筛选。通过对5×10~(-5)pfu的筛选，获得了一个3.5kb的克隆，并通过快速扩增cDNA端的方法克隆了SP1结合蛋白基因的5‘端区。在SP1结合蛋白基因的克隆区域有一个编码1,025个氨基酸残基的开放阅读框，推导出的多肽的相对分子质量为11.4万，略高于SDS-聚丙烯酰胺凝胶电泳法估计的相对分子质量100,000。从mRNA序列推断的一级结构表明，SP1结合蛋白具有两种可能的跨膜结构。较少

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