Klarskov K, Højrup P, Andersen S O, Roepstorff P
Department of Molecular Biology, University of Odense, Denmark.
Biochem J. 1989 Sep 15;262(3):923-30. doi: 10.1042/bj2620923.
The complete amino acid sequence of a structural protein, protein 8, isolated from the pharate cuticle of the locust Locusta migratoria was determined. Protein 8 contains 148 amino acid residues and has an Mr of 15,224. By the extensive use of information obtained by plasma-desorption mass spectrometry (p.d.m.s.) it was possible to reduce the need for conventional sequence determination and to improve the reliability of the results. On the basis of the determined Mr of the intact protein all the peptides that constitute the complete sequence could be isolated from a time-course enzymic digestion. The isolated peptides were sequenced by using a combination of Edman degradation and carboxypeptidase digestion monitored by p.d.m.s. The alignment of the peptides was established from the time-course digestion and further verified by a second enzymic digestion. The primary structure of the protein consists of two hydrophilic and two hydrophobic regions. The hydrophobic regions are enriched in alanine, valine and proline and dominated by a repetitive sequence Ala-Ala-Pro-(Ala/Val). The sequence strengthens the view that the cuticle proteins belong to a unique family of structural proteins.
已确定从飞蝗（Locusta migratoria）的蛹皮中分离出的一种结构蛋白——蛋白8的完整氨基酸序列。蛋白8含有148个氨基酸残基，分子量为15224。通过广泛利用等离子体解吸质谱（p.d.m.s.）获得的信息，减少了对传统序列测定的需求并提高了结果的可靠性。基于完整蛋白确定的分子量，所有构成完整序列的肽段都可从酶解时间进程中分离出来。使用埃德曼降解法和羧肽酶消化法相结合并通过p.d.m.s.监测对分离出的肽段进行测序。肽段的比对根据酶解时间进程确定，并通过第二次酶解进一步验证。该蛋白的一级结构由两个亲水区域和两个疏水区域组成。疏水区域富含丙氨酸、缬氨酸和脯氨酸，以重复序列Ala-Ala-Pro-(Ala/Val)为主。该序列强化了角质层蛋白属于一个独特结构蛋白家族的观点。
