HUMAN C1BAR INHIBITOR - PRIMARY STRUCTURE, CDNA CLONING, AND CHROMOSOMAL LOCALIZATION
HUMAN C1BAR INHIBITOR - PRIMARY STRUCTURE, CDNA CLONING, AND CHROMOSOMAL LOCALIZATION
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DOI：
10.1021/bi00363a018
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发表时间：
1986-07-29
期刊：
影响因子：
2.9
通讯作者：
MAGNUSSON, S
中科院分区：
文献类型：
--
作者：
BOCK, SC;SKRIVER, K;MAGNUSSON, S
材料和方法蛋白质的纯化。人血浆经聚乙二醇沉淀、DEAE-纤维素和己基琼脂糖层析(Nilsson&Wiman，1982)、0.1M NH4HC03凝胶过滤、Sepharose6B凝胶过滤，从人血浆中分离得到CT抑制物。得到的单链材料对CLS具有完全的活性(Chapuis等人，1977)，并在NaDod-S04-PAGE中迁移了105000 Da的表观分子质量。磷的测定采用改进的Fiske-Subbarow方法(Ames，1966)。用Sephacryl S-300凝胶过滤，在40 mM磷酸二氢钠、0.1M氯化钠、0.1M NaN3、0.1%NaDodS04、pH 7.3的溶液中，从CLS-CT缓蚀剂复合体中分离出活化肽。氨基酸测序。CT抑制剂被化学或酶降解，如图1a所示。用胃酶消化，用99%的甲酸溶解蛋白质，然后稀释到5%，酶/底物比为1/100w/w，在室温下孵育3h，用三氟甲磺酸(Edge等人，1981)或碱性硼氢化钠(Spiro&Bhoyroe，1974)处理富含碳水化合物的多肽。多肽首先用Sephadex G-50F或Sephacryl S-200凝胶过滤，大部分用0.1MNH4HC03，然后用0.01-1.0MNH4HC03的线性梯度在DEAE-Sephacel上进行离子交换层析。在Hewlett-Packard 1084B液色仪上用反相高效液相色谱仪进行最终纯化。在应用生物系统模型470A(使用化学品和制造商提供的02N VAc程序)或Beckman 890C测序仪上对多肽进行测序。氨基酸分析在Beckman 121MB仪器上进行。除了我们实验室常用的系统(Skorstengaard等人，1982,1984)外，Vydac C4柱(以异丙醇和三乙胺为洗脱梯度，pH 5.2，在0.1%CF3COOH中)
Materials and MethodsProtein Purification. CT inhibitor was isolated from human plasma by precipitation with polyethylene glycol), chromatography on DEAE-cellulose and hexyl-Sepharose (Nilsson & Wiman, 1982), and gel filtration in 0.1 M NH4HC03, pH8.3, on Sepharose 6B. The resulting single-chain material was fully active against Cls (Chapuis et al., 1977) and migrated with an apparent molecular mass of 105 000 Da in NaDod-S04-PAGE. Phosphate was determined by a modified Fiske-Subbarow method (Ames, 1966). The activation pep-tide was separated from the Cls-CT inhibitor complex by gel filtration on Sephacryl S-300 in 40 mM sodium phosphate, 0.1 M NaCl, 0.1 M NaN3, and 0.1% NaDodS04, pH 7.3. Amino Acid Sequencing. CT inhibitor was degraded chemically or enzymatically as indicated in Figure la. For digestion with pepsin, the protein was dissolved in 99% formic acid and then diluted to 5%; the enzyme/substrate ratio was1/100 w/w, and the digest was incubated at room temperature for 3 h. Carbohydrate-rich peptides were treated with tri-fluoromethanesulfonic acid (Edge et al., 1981) or alkaline sodium borohydride (Spiro & Bhoyroe, 1974). Peptides were purified by an initial gel filtration on Sephadex G-50F or Sephacryl S-200, mostly in 0.1 M NH4HC03, followedby ion-exchange chromatography on DEAE-Sephacel using a linear gradient of 0.01-1.0 M NH4HC03. Final purification was achieved by reversed-phase HPLC on a Hewlett-Packard 1084B liquid chromatograph. Peptides were sequenced on anApplied Biosystems Model 470A (using the chemicals and the 02n vac program supplied by the manufacturer) or on a Beckman 890C sequenator. Amino acid analysis was per-formed on a Beckman 121MB instrument. In addition to the systems commonly used in our laboratory (Skorstengaard et al., 1982, 1984), columns of Vydac C4 (with elution gradients of 2-propanol and triethylamine, pH 5.2, in 0.1% CF3COOH)