Anaphylatoxin from the fifth component of porcine complement. Purification and partial chemical characterization.
Anaphylatoxin from the fifth component of porcine complement. Purification and partial chemical characterization.
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来自猪补体第五种成分的过敏毒素。
DOI：
10.1016/s0021-9258(18)50370-7
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发表时间：
1979
期刊：
影响因子：
--
通讯作者：
T. Hugli
中科院分区：
文献类型：
--
作者：
C. Gerard;T. Hugli
描述了一种用于猪C5 a纯化的新方法，其从1升补体活化血清产生毫克量的过敏毒素。分离的策略采用酸沉淀的活化血清，凝胶过滤的酸溶性馏分，和SP-Sephadex色谱法，使用梯度洗脱。使用QAE-Sephadex色谱法实现最终纯化。所得产物在电泳后在十二烷基硫酸钠或pH 4.5聚丙烯酰胺凝胶上迁移为单一条带，并且在pH 8.5的醋酸纤维素条上微区带电泳后呈现均一性。用Sephadex G-50凝胶过滤法测定其表观分子量为8500。与人C5 a不同，猪多肽在聚丙烯酰胺凝胶中用高碘酸-希夫试剂染色时没有显示碳水化合物的证据。自动化的NH z-末端序列分析提供了猪C5 a的以下部分结构：NHz-M！+ Leu-Gln-Lys-Lys-Ile-Glu-Glu-Glu-J&-Ala-Lys-。根据羧肽酶B和Y对多肽的连续降解，提出了羧基末端结构-Gln-Leu-Gly-Arg-COOH。12个NH 4 OH残基中的10个在人和猪C5 a中是相同的，并且对于在COOH末端分配的4个残基观察到完全同源性。纯化的猪过敏毒素在5 × 10- 1 M的浓度下诱导平滑肌（回肠）收缩，并且对人多形核白细胞具有趋化性，ED 50值为3至4 × 10 - 1 M（人C5 a的ED 6为1至3 × 10 - 1 M）。
A novel procedure for porcine C5a purification is described which yields milligram quantities of anaphylatoxin from 1 liter of complement-activated serum. The strategy for isolation employs acid precipitation of the activated serum, gel filtration of the acid-soluble fraction, and SP-Sephadex chromatography using gradient elution. Final purification is achieved using QAE-Sephadex chromatography. The product obtained migrates as a single band on sodium dodecyl sulfate or on pH 4.5 polyacrylamide gels after electrophoresis, and appears homogeneous after microzone electrophoresis on cellulose acetate strips at pH 8.5. The apparent molecular weight is 8500 as determined by gel filtration on Sephadex G-50. Unlike human C5a, the porcine polypeptide shows no evidence of carbohydrate when stained by periodic acid-Schiff reagent in polyacrylamide gels. Automated NHz-terminal sequence analysis provided the following partial structure for porcineC5a: NHz-M!+ Leu-Gln-Lys-Lys-Ile-Glu-Glu-Glu-J&-Ala-Lys-. A COOH-terminal structure-Gin-Leu-Gly-Arg-COOH is proposed based upon sequential degradation of the polypeptide using carboxypeptidases B and Y. Ten of the twelve NH&erminal residues are identical in human and porcine C5a and complete homology is observed for the 4 residues assigned at the COOH terminus. The purified porcine anaphylatoxin induces smooth muscle (ileal) contraction at a concentration of 5 X 10-l’M, and is chemotactic for human polymorphonuclear leukocytes with an EDSo value of 3 to 4 X lo-’M (ED6,, for human C5a is 1 to 3 X lo-’M).