Locust ion transport peptide (ITP): primary structure, cDNA and expression in a baculovirus system.
Locust ion transport peptide (ITP): primary structure, cDNA and expression in a baculovirus system.
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发表时间：
1996-05
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通讯作者：
J. Meredith;M. Ring;A. Macins;J. Marschall;N. Cheng;D. Theilmann;H. Brock;John E. Phillips
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作者：
J. Meredith;M. Ring;A. Macins;J. Marschall;N. Cheng;D. Theilmann;H. Brock;John E. Phillips
从蝗虫神经心体（CC）中纯化的离子转运肽（ITP）先前已被证明可以刺激Schistocerca gregaria回肠的盐和水重吸收并抑制酸分泌。我们使用纯化ITP的部分氨基酸序列来推导简并引物。这些用于使用逆转录和聚合酶链式反应（RtPCR）从脑RNA扩增cDNA。使用锚定PCR延伸该序列以产生部分的517bp cDNA克隆。该cDNA编码一个假定的ITP激素原，其可以在两个二元氨基酸位点处被切割以产生72个残基的活性酰胺化肽。推导的氨基酸序列与纯化ITP的氨基酸序列和分子量（8564Da）完全一致。相对于甲壳类高血糖激素（CHH）家族，ITP中的所有6个半胱氨酸残基和许多其他氨基酸残基都是保守的，这表明ITP是一种同源物。然而，CHH，蟹眼柄和CC提取物从远亲昆虫没有行动，而CC提取物从密切相关的昆虫是活性的蝗虫ITP测定，表明生物测定是选择性的。用含有我们的部分cDNA的杆状病毒转染的昆虫Sf9细胞分泌了蝗虫回肠运输的有效刺激物，证实了由我们的ITP克隆编码的肽具有生物活性。ITP的mRNA仅限于大脑和CC。有趣的是，在ITP生物测定中无活性的其他组织中观察到相关mRNA。
Ion transport peptide (ITP) purified from locust nervous corpus cardiacum (CC) has previously been shown to stimulate salt and water reabsorption and inhibit acid secretion in the ileum of Schistocerca gregaria. We used the partial amino acid sequence of purified ITP to derive degenerate primers. These were used to amplify a cDNA from brain RNA using reverse transcription and the polymerase chain reaction (RtPCR). This sequence was extended using anchored PCR to yield a partial, 517bp cDNA clone. This cDNA encodes a putative ITP prohormone which could be cleaved at two dibasic amino acid sites to yield a 72 residue active amidated peptide. The deduced amino acid sequence from the cDNA agrees completely with the amino acid sequence and molecular mass (8564Da) derived from analysis of purified ITP. Relative to a family of crustacean hyperglycaemic hormones (CHH), all six cysteine residues and many other amino acid residues are conserved in ITP, establishing that ITP is a homologue. However, CHH, crab eyestalk and CC extracts from distantly related insects have no action, whereas CC extracts from closely related insects are active on the locust ITP assay, showing that the bioassay is selective. Insect Sf9 cells transfected with a baculovirus containing our partial cDNA secreted a potent stimulant of locust ileal transport, confirming that the peptide encoded by our ITP clone has biological activity. The mRNA for ITP is restricted to the brain and CC. Interestingly, a related mRNA is observed in other tissues which are not active on the ITP bioassay.