Bungart D, Hilbich C, Dircksen H, Keller R
Institut für Zoophysiologie, Rheinischen Friedrich Wilhelms-Universität, Bonn, Germany.
Peptides. 1995;16(1):67-72. doi: 10.1016/0196-9781(94)00145-v.
By use of an enzyme immunoassay that was developed for the determination of orcokinin, a myotropic neuropeptide of the sequence NFDEIDRSGFGFN from the crayfish, Orconectes limosus, immunoreactive material was detected in extracts of thoracic ganglia from the shore crab, Carcinus maenas. Isolation of the immunoreactive material was achieved by the following steps: 1) prepurification by gel filtration, 2) immunoaffinity chromatography on an anti-orcokinin IgG protein-A sepharose column, and 3) reversed-phase HPLC. The HPLC profile after affinity purification revealed three main immunoreactive peptides that were rechromatographed. None of these peptides was identical to orcokinin in terms of retention time. Automated gas-phase sequencing revealed these peptides to be analogues of orcokinin differing in one amino acid residue. They were named [Ser9]-, [Ala13]- and [Val13]orcokinin (NFDEIDRSSFGFN, Mr 1549.3; NFDEIDRSGFGFA, Mr 1475.3; NFDEIDRSGFGFV, Mr 1503.9). Carboxypeptidase A treatment of the peptides indicated a free C-terminus. Complete characterization of the three peptides was achieved from approximately 230 thoracic ganglia of Carcinus maenas.
利用一种为测定螯虾促肌动肽（一种来自螯虾奥氏拟螯虾、序列为NFDEIDRSGFGFN的促肌神经肽）而开发的酶免疫测定法，在岸蟹平背蜞胸神经节提取物中检测到了免疫反应性物质。通过以下步骤实现了免疫反应性物质的分离：1）通过凝胶过滤进行预纯化；2）在抗促肌动肽IgG蛋白A琼脂糖柱上进行免疫亲和色谱；3）反相高效液相色谱。亲和纯化后的高效液相色谱图谱显示有三种主要的免疫反应性肽，对其进行了重新色谱分析。这些肽在保留时间方面均与促肌动肽不同。自动气相测序显示这些肽是促肌动肽的类似物，在一个氨基酸残基上有所不同。它们被命名为[Ser9]-、[Ala13]-和[Val13]促肌动肽（NFDEIDRSSFGFN，Mr 1549.3；NFDEIDRSGFGFA，Mr 1475.3；NFDEIDRSGFGFV，Mr 1503.9）。对这些肽进行羧肽酶A处理表明其C末端是游离的。从大约230个平背蜞胸神经节中完成了这三种肽的完整表征。
