OUTER MEMBRANE PROTEINS OF ESCHERICHIA-COLI .1. EFFECT OF PREPARATIVE CONDITIONS ON MIGRATION OF PROTEIN IN POLYACRYLAMIDE GELS
OUTER MEMBRANE PROTEINS OF ESCHERICHIA-COLI .1. EFFECT OF PREPARATIVE CONDITIONS ON MIGRATION OF PROTEIN IN POLYACRYLAMIDE GELS
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DOI：
10.1016/0003-9861(73)90673-5
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发表时间：
1973-01-01
影响因子：
3.9
通讯作者：
SCHNAITMAN, CA
中科院分区：
文献类型：
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作者：
SCHNAITMAN, CA
通过将膜溶解在有机溶剂中，然后透析到十二烷基硫酸钠 (SDS) 溶液中，或通过将膜直接溶解在 SDS 溶液中，然后透析到 SDS-尿素溶液中，并在 100 °C 下短暂加热，制备用于聚丙烯酰胺凝胶电泳的大肠杆菌外膜蛋白，从而在含有 SDS 的凝胶上产生简单的多肽谱。该多肽模式的特征在于单一主要蛋白质条带迁移，表观分子量约为42,000道尔顿，约占凝胶上总蛋白质的70%。然而，如果将外膜蛋白溶解在不含尿素的 SDS 溶液中并在 70 °C 下加热，则在凝胶的三个区域中观察到主要条带：靠近凝胶顶部的宽条带或一组条带，其表观分子量远大于 42,000 道尔顿（峰 A），第二条带与煮沸样品中的 42,000 道尔顿条带具有相同的迁移率（峰 B），以及第三条迁移较快的条带，其表观分子量远大于 42,000 道尔顿（峰 A）。分子量小于 42,000 道尔顿（峰 C）。从 A 或 C 中洗脱蛋白质，然后在 100 °C 下加热，将该蛋白质转化为随峰 B 迁移的形式。如果将外膜蛋白在 37 °C 下溶解在 SDS 溶液中，无需进一步加热，然后涂在凝胶上，则峰 B 完全消失，A 和 C 增加。通过尿素处理，这些可以部分转化为峰 B。在 SDS 存在下，通过 Sephadex 色谱法分离峰 A 和 C 中的蛋白质，并在煮沸之前和之后测量该蛋白质的特性粘度。 A峰蛋白质的特性粘度在煮沸前后均为35cc/g，而C峰蛋白质的特性粘度在煮沸前为28cc/g，煮沸后为35cc/g。这些结果最好的解释是假设峰 A 中的蛋白质代表 42,000 道尔顿物质的聚集体，通过煮沸或溶剂处理解离，而峰 C 中的蛋白质代表 42,000 道尔顿蛋白质的单体形式，该蛋白质未与 SDS 完全反应，并且仅在煮沸或溶剂处理时才转化为蛋白质-SDS 复合物的“刚性杆”构象特征。
Outer membrane protein ofEscherichia coliprepared for polyacrylamide gel electrophoresis by solubilization of the membrane in an organic solvent followed by dialysis into sodium dodecyl sulfate (SDS) solution or by solublization of the membrane directly in SDS solution followed by dialysis into a SDS-urea solution and brief heating at 100 °C resulted in a simple polypeptide profile on SDS-containing gels. This polypeptide pattern was characterized by a single major protein band migrating with an apparent molecular weight of about 42,000 daltons which accounted for about 70% of the total protein on the gel. However, if the outer membrane protein is dissolved in SDS solution without urea and heated at 70 °C, major bands are observed in three regions of the gel: A broad band or group of bands near the top of the gel with an apparent molecular weight of much greater than 42,000 daltona (peak A), a second band with the same mobility as the 42,000-dalton band in boiled samples (peak B), and a third, faster-migrating band with an apparent molecular weight of less than 42,000 daltons (peak C).Elution of protein from A or C followed by heating at 100 °C converts this protein to a form migrating with peak B. If the outer-membrane protein is dissolved in SDS solution at 37 °C with no further heating and applied to gels, peak B dissappears completely and A and C increase. These can be partially converted to peak B by urea treatment. Protein from peaks A and C was isolated by chromatography on Sephadex in the presence of SDS, and the intrinsic viscosity of this protein was measured before and after boiling. The intrinsic viscosity of protein from peak A was 35 cc/g both before and after boiling, while the intrinsic viscosity of protein from peak C was 28 cc/g before boiling and 35 cc/g after boiling. These results are best explained by assuming that the protein in peak A represents aggregates of a 42,000-dalton species which are dissociated by boiling or solvent treatment and that the protein in peak C represents a monomeric form of the 42,000-dalton protein which is not fully reacted with SDS and which is converted to the “rigid rod” conformation characteristic of protein-SDS complexes only upon boiling or solvent treatment.