Role of cationic residues in cytolytic activity: modification of lysine residues in the cardiotoxin from Naja nigricollis venom and correlation between cytolytic and antiplatelet activity.
Role of cationic residues in cytolytic activity: modification of lysine residues in the cardiotoxin from Naja nigricollis venom and correlation between cytolytic and antiplatelet activity.
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阳离子残基在溶细胞活性中的作用：黑眼镜蛇毒液心脏毒素中赖氨酸残基的修饰以及溶细胞活性和抗血小板活性之间的相关性。
DOI：
10.1021/bi00449a037
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发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Evans,HJ
中科院分区：
文献类型：
--
作者：
Kini,RM;Evans,HJ
材料和方法毒液和试剂。黑颈眼镜蛇毒液、鸡蛋 PC、心磷脂、丹酰氯、O-甲基异脲和羟胺购自密苏里州圣路易斯的 Sigma Chemical Co.。 4-乙烯基吡啶和琥珀酸酐购自威斯康星州密尔沃基的 Aldrich Chemical Co.，氰化钾购自新泽西州菲利普斯堡的 J. T. Baker Chemical Co.。用于红细胞和血小板聚集研究的正常人血液是从健康志愿者新鲜抽取的。所有其他试剂均为分析纯。心脏毒素CTX-1的纯化和均质性。来自 N. nigricollis crawshawii 毒液的主要心脏毒素 CTX-1 通过 Pharmacia Mono S 柱上的快速蛋白液相色谱纯化，然后在 Bio-Gel P-10 柱上进行凝胶过滤（Kini 等，1987）。 CTX-1 在 Mono S 上重新层析 3 次，在凝胶过滤柱上重新层析 2 次，以尽量减少磷脂酶污染。发现心脏毒素在天然凝胶和十二烷基硫酸钠-聚丙烯酰胺凝胶上都是均质的，并且使用不含去垢剂的鸡蛋悬浮液作为底物，通过标准 pH 统计测定无法检测到磷脂裂解活性（磷脂酶污染 < 0.002%）（Nieuwenhuizen 等，1974）。还原和吡啶乙基化。根据Cavins和Friedman(1970)的方法，将心脏毒素还原并吡啶乙基化。用 0-巯基乙醇（20 mol/mol 二硫化物）处理 6 M 盐酸胍/0.13 M Tris（pH 8.0）中的蛋白质 (10 mg/mL)。在室温下温育3小时后，将混合物用每摩尔3-巯基乙醇3摩尔4-乙烯基吡啶处理。进一步孵育 90 分钟后，将混合物在用 9% 甲酸平衡的 Sephadex G-25 柱上脱盐。该吡啶乙基化CTX-1仅用于序列测定。
Materials and MethodsVenom and Reagents. Naja nigricollis crawshawii venom, PC from hen’s egg, cardiolipin, dansyl chloride, O-methyl-isourea, and hydroxylamine were purchased from Sigma Chemical Co., St. Louis, MO. 4-Vinylpyridine and succinic anhydride were obtained fromAldrich Chemical Co., Mil-waukee, WI, and potassium cyanide was from J. T. Baker Chemical Co., Phillipsburg, NJ. Normal human blood for erythrocytes and for platelet aggregation studies was freshly drawn from healthy volunteers. All other reagents were analytical grade.Purification and Homogeneity of Cardiotoxin CTX-1. The major cardiotoxin, CTX-1, from N. nigricollis crawshawii venom was purified byfast proteinliquid chromatography on a Pharmacia Mono S column followed by gel filtration on a Bio-Gel P-10 column (Kini et al., 1987). CTX-1 was re-chromatographed 3 times on Mono S and 2 times on a gel filtration column to minimize phospholipase contamination. The cardiotoxin was found to be homogeneous on both native and sodium dodecyl sulfate-polyacrylamide gels, and phos-pholipid-splitting activity was undetectable (< 0.002% con-tamination with phospholipase) by a standard pH stat assay, using an egg suspension without detergents as substrate (Nieuwenhuizen et al., 1974). Reduction and Pyridylethylation. The cardiotoxin was reduced and pyridylethylated according to the method of Cavins and Friedman (1970). Protein (10 mg/mL) in 6 M guanidine hydrochloride/0.13 M Tris, pH 8.0, was treated with 0-mercaptoethanol (20 mol/mol of disulfide). After incubation at room temperature for 3 h, the mixture was treated with 3 mol of 4-vinylpyridine per mole of/3-mercaptoethanol. After 90-min further incubation, the mixture was desalted on a Sephadex G-25 column equilibrated with 9% formic acid. This pyridylethylated CTX-1 was used only in thesequence de-termination.