Chiu A Y, Hunkapiller M W, Heller E, Stuart D K, Hood L E, Strumwasser F
Proc Natl Acad Sci U S A. 1979 Dec;76(12):6656-60. doi: 10.1073/pnas.76.12.6656.
Egg-laying hormone (ELH), a neuropeptide synthesized by the bag cell neurons, induces egg laying and its correlated behavior in Aplysia californica. In the present study, ELH has been purified to homogeneity and its primary structure has been determined. We find this molecule to have 36 amino acid residues with a M(r) of 4385 and a calculated isoelectric point of 9.7. Direct microsequence analysis revealed a single amino acid sequence that is in agreement with the amino acid composition determined after acid hydrolysis of ELH: H-Ile-Ser-Ile-Asn-Gln-Asp-Leu-Lys-Ala-Ile-Thr-Asp-Met-Leu-Leu-Thr-Glu-Gln- Ile-Arg-Glu-Arg-Gln-Arg-Tyr-Leu-Ala-Asp-Leu-Arg-Gln-Arg-Leu-Leu-Glu-Lys-OH. Enzyme data indicate that the COOH-terminal lysine may be modified but its exact nature remains to be determined. There is no similarity between the amino acid sequence of ELH and that of presently known vertebrate neuropeptides. The two-step purification procedure, starting with a homogenate of bag cell clusters, consisted of cation exchange chromatography on SP C25 (Sephadex) followed by gel filtration on Bio-Gel P-6. Our purification results in a 100-fold enrichment of ELH from bag cell homogenates and a 36% recovery of purified radiolabeled marker ELH. Analysis of purified ELH radiolabeled with [(35)S]methionine or [(3)H]leucine on isoelectric focusing gels and on 8 M urea/sodium dodecyl sulfate gels showed only a single peak containing 90% of the radiolabel. Radiolabeled ELH migrated with a pI of 9.0-9.2 and an apparent M(r) of 3500-5700. ELH retained egg-laying bioactivity when eluted from this segment of the gel. We find that 2.5 nmol of pure ELH consistently induces egg laying at 20 degrees C.
产卵激素（ELH）是一种由包细胞神经元合成的神经肽，可诱导加州海兔产卵及其相关行为。在本研究中，ELH已被纯化至同质，其一级结构已被确定。我们发现该分子含有36个氨基酸残基，分子量为4385，计算得出的等电点为9.7。直接微量序列分析揭示了一个单一的氨基酸序列，该序列与ELH酸水解后确定的氨基酸组成一致：H-Ile-Ser-Ile-Asn-Gln-Asp-Leu-Lys-Ala-Ile-Thr-Asp-Met-Leu-Leu-Thr-Glu-Gln-Ile-Arg-Glu-Arg-Gln-Arg-Tyr-Leu-Ala-Asp-Leu-Arg-Gln-Arg-Leu-Leu-Glu-Lys-OH。酶学数据表明，COOH末端的赖氨酸可能被修饰，但其确切性质仍有待确定。ELH的氨基酸序列与目前已知的脊椎动物神经肽的氨基酸序列没有相似性。从包细胞簇匀浆开始的两步纯化程序，包括在SP C25（葡聚糖）上进行阳离子交换色谱，然后在Bio-Gel P-6上进行凝胶过滤。我们的纯化使包细胞匀浆中的ELH富集了100倍，纯化的放射性标记标志物ELH的回收率为36%。在等电聚焦凝胶和8M尿素/十二烷基硫酸钠凝胶上对用[(35)S]甲硫氨酸或[(3)H]亮氨酸放射性标记的纯化ELH进行分析，结果显示只有一个含有90%放射性标记的单一峰。放射性标记的ELH迁移时的pI为9.0-9.2，表观分子量为3500-5700。当从凝胶的这一部分洗脱时，ELH保留了产卵生物活性。我们发现，2.5 nmol的纯ELH在20摄氏度时始终能诱导产卵。
