Kim A J, Cha G H, Kim K, Gilbert L I, Lee C C
Department of Molecular Biology, College of Natural Sciences, Seoul National University, Korea.
Proc Natl Acad Sci U S A. 1997 Feb 18;94(4):1130-5. doi: 10.1073/pnas.94.4.1130.
The prothoracicotropic hormone (PTTH) of Drosophila melanogaster is a modulator of ecdysteroid (molting hormone) synthesis and was isolated and characterized from extracts of whole larvae (approximately 4 x 10(5) larvae). The purification protocol included delipidation, salt-extraction, heat treatment, conventional column chromatography, and HPLC, and yielded about 50 microg of pure hormone. Biological activity was followed using a ring gland in vitro assay in which ecdysteroidogenesis by control ring glands as measured by radioimmunoassay was compared with ring gland incubations containing active fractions. The molecular weight of the purified PTTH was 45 kDa and N-terminal amino acid sequence analysis indicated that those analyzed sequences displayed no significant homology with known peptides or peptide hormones, including PTTH from the silkmoth, Bombyx mori. Western blot analysis indicated that the native form of Drosophila PTTH was a single 66-kDa polypeptide with N-linked carbohydrate chains and intrachain disulfide bonds. The purified 45-kDa peptide is the deglycosylated form, a result of glycosidase activity present during preparation of the PTTH extract. The deglycosylated form shows heterogeneity, presumably as a result of varying degrees of deglycosylation at the N terminus.
黑腹果蝇的促前胸腺激素（PTTH）是蜕皮甾体（蜕皮激素）合成的调节剂，它是从整个幼虫（约4×10⁵只幼虫）的提取物中分离并鉴定出来的。纯化方案包括脱脂、盐提取、热处理、常规柱色谱和高效液相色谱，得到了约50微克的纯激素。通过体外环腺测定法跟踪生物活性，其中通过放射免疫测定法测量对照环腺的蜕皮甾体生成，并与含有活性组分的环腺孵育进行比较。纯化的PTTH的分子量为45 kDa，N端氨基酸序列分析表明，所分析的序列与已知的肽或肽激素没有显著同源性，包括家蚕的PTTH。蛋白质免疫印迹分析表明，果蝇PTTH的天然形式是一种单一的66 kDa多肽，带有N-连接的糖链和链内二硫键。纯化的45 kDa肽是去糖基化形式，这是PTTH提取物制备过程中存在的糖苷酶活性的结果。去糖基化形式表现出异质性，可能是由于N端去糖基化程度不同所致。
