Primary and secondary structural determinants in the receptor binding sequence beta-(38-57) from human luteinizing hormone.
Primary and secondary structural determinants in the receptor binding sequence beta-(38-57) from human luteinizing hormone.
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人黄体生成激素受体结合序列β-(38-57) 的一级和二级结构决定因素。
DOI：
10.1021/bi00425a010
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发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
Ryan,RJ
中科院分区：
文献类型：
--
作者：
Keutmann,HT;Charlesworth,MC;Kitzmann,K;Mason,KA;Johnson,L;Ryan,RJ
材料和方法肽合成。制备的hLH/3-(38-57)的肽和类似物如图1所示。还合成了N端片段38-48及其Ala-38类似物和41-45。 hCG/3-(38-57)和C端片段45-57如先前那样制备(Keutmann等人，1987)。肽通过固相程序（Barany & Merrifield，1979；Stewart & Young，1984）在Applied Biosystems Model430A合成仪（Applied Biosystems，Inc.，Foster City，CA）上以甲基二苯甲基胺树脂作为固体支持物合成。肽通常以 0.5 mmol 的初始偶联残基开始；肽-树脂在合成过程中根据需要进行分割，以提供在给定位置具有替代残基的产品（例如，43A 和 43D、47V 和 47E；图 1）。对于全长38-57类似物，对残基57-44编程单对称酸酐偶联，对残基43-38编程双偶联。定量茚三酮测试用于监测整个偶联效率。即/T-BOC 保护的氨基酸购自Applied Biosystems 和Bachem Fine Chemicals (Torrance, CA)。侧链保护基团包括4-甲基苄基(Cys)、O-苄基(Asp、Glu、Thr)、甲苯磺酰基(Arg)和O-(氯苄基)(Tyr)。用含有10%茴香醚和5%二甲硫醚的无水HF从固体支持物上裂解完成的肽，在Kel-F蒸馏装置（肽研究所，大阪，日本）中在0℃下反应1小时。试剂蒸发后，用乙醚处理树脂以除去残留的清除剂，将肽提取到10％乙酸中，用水稀释至体积150-200mL，并冻干。将肽重新溶解在0.01N乙酸（100μg/mL）中，用氢氧化铵将pH调节至pH 6.5，并将溶液在20℃下搅拌4-6小时以闭合二硫键。通过在 Sephadex G-25 (Pharmacia) 上凝胶过滤纯化肽，用 1.0 M 乙酸洗脱，然后在（羧甲基）纤维素 (Whatman CM-52) 上进行离子交换色谱纯化，用乙酸铵线性梯度（pH 4.5，从 0.1 到 2.0 M）洗脱。具有阴离子取代（Glu、Asp）或缺乏精氨酸的衍生物。 (例如，43A、43D；图1)，使用0.1-1.0M乙酸铵的较浅梯度。对于这些制备，添加了反相 HPLC 的最后一步，使用 Bio-Rad RP-300 Hi-Pore C18 半制备柱，用 0.1% 三氟乙酸中的乙腈（通常为 10%-60%）梯度洗脱。酸水解后（6 Mhc1，24 小时，110°C，真空），通过 Beckman 6300 型仪器上的氨基酸分析证实肽的同质性，并通过
Materials and MethodsPeptide Synthesis. The peptides and analogues of hLH/3-(38-57) prepared are shown in Figure 1. Also synthesized were the N-terminal fragments 38-48, and its Ala-38 analogue, and 41-45. hCG/3-(38-57) and the C-terminal fragment 45-57 were prepared as previously (Keutmann et al., 1987). Peptides were synthesized by the solid-phase procedure (Barany & Merrifield, 1979; Stewart & Young, 1984) on the Applied Biosystems Model430A synthesizer (Applied Biosystems, Inc., Foster City, CA) with methylbenzhydrylamine resin as solid support. Peptides typically commenced with 0.5 mmol of initial coupled residue; the peptide-resin was divided as needed during synthesis to provide products with alternative residues at a given position (eg, 43A and 43D, 47V and 47E; Figure 1). For full-length 38-57 analogues, single symmetric anhydride couplings were pro-grammed for residues 57-44, and double couplings for residues 43-38. Quantitative ninhydrin tests were used for monitoring coupling efficiency throughout. ie/T-BOC-protected amino acids were purchased from Applied Biosystems and from Bachem Fine Chemicals (Torrance, CA). Side-chain-protecting groups included 4-methylbenzyl (Cys), O-benzyl (Asp, Glu, Thr), tosyl (Arg), and O-(chlorobenzyl)(Tyr). Completed peptides were cleaved from the solid support with anhydrous HF containing 10% anisóle and 5% dimethyl sul-fide, reacted 1 h at 0 C in a Kel-F distillation apparatus (Peptide Institute, Osaka, Japan). After evaporation of reagents, the resin was treated with diethyl ether to remove residual scavenger, and the peptide was extracted into 10% acetic acid, diluted with water to a volume of 150-200 mL, and lyophilized. The peptide was redissolved in 0.01 N acetic acid (100¿ ig/mL), the pH was adjusted to pH 6.5 with am-monium hydroxide, and the solution was stirred 4-6 h at 20C to close the disulfide bond. The peptides were purified by gel filtration on Sephadex G-25 (Pharmacia) eluted with 1.0 M acetic acid, followed by ion-exchange chromatography on (carboxymethyl) cellulose (Whatman CM-52) eluted with a linear gradient of ammo-nium acetate, pH 4.5, from 0.1 to 2.0 M. Forderivatives with anionic substitutions (Glu, Asp) or those lacking arginine (eg, 43A, 43D; Figure 1), a shallower gradient of 0.1-1.0 M am-monium acetate was used. For these preparations a final step of reversed-phase HPLC was added, using a Bio-Rad RP-300 Hi-Pore C18 semipreparative column eluted with a gradient of acetonitrile (typically 10%-60%) in 0.1% trifluoroacetic acid. Homogeneity of the peptides was confirmed by amino acid analysis on the Beckman Model 6300 instrument after acid hydrolysis (6 Mhc1, 24 h, 110 C, in vacuo) and by
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影响因子：
2.9
作者：
G. Tregear;J. van Rietschoten;R. Sauer;H. Niall;H. Keutmann;J. Potts
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DOI：
--
发表时间：
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期刊：
影响因子：
--
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通讯作者：
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影响因子：
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作者：
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影响因子：
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作者：
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