ELECTROPHORETICALLY SLOW AND FAST FORMS OF THE ALPHA-2-MACROGLOBULIN MOLECULE
ELECTROPHORETICALLY SLOW AND FAST FORMS OF THE ALPHA-2-MACROGLOBULIN MOLECULE
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DOI：
10.1042/bj1810401
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发表时间：
1979-01-01
影响因子：
4.1
通讯作者：
SAYERS, CA
中科院分区：
文献类型：
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作者：
BARRETT, AJ;BROWN, MA;SAYERS, CA
通过4步程序从人血浆中分离α 2-巨球蛋白（α 2 M）：聚（乙二醇）分级分离、凝胶色谱、优球蛋白沉淀和免疫吸附。通过电泳或免疫沉淀法在最终制剂中未检测到污染物。该蛋白质在凝胶电泳中以单一慢带运行，并命名为S-α 2 M。S-α 2 M结合约2 mol胰蛋白酶/mol。用蛋白酶或铵盐处理S-α 2 M产生在电泳中更移动的且缺乏蛋白酶结合活性的分子形式（F-α 2 M）。与NH 4+盐反应得到的F-α 2 M的电泳迁移率与蛋白酶复合物的电泳迁移率相同。α 2 M的电泳迁移率的变化归因于构象变化，但没有pI或Stokes半径变化的证据。S-α 2 M在十二烷基硫酸钠[SDS]存在下的电泳给出的结果与α 2 M分子是相同亚基的四聚体的观点一致，组装成二硫键连接的二聚体的非共价对。有些亚基似乎被切割成2/3长度和1/3长度的链。这对于由铵盐产生的F-α 2 M并不明显。由胰蛋白酶产生的F-α 2 M显示2条新带，这归因于亚基多肽链在中部附近的裂解。F-α 2 M的免疫测定给出的火箭比用S-α 2 M的那些低12-29%。通过在SDS存在下电泳之前用戊二醛处理每种形式来研究S-α 2 M和F-α 2 M中亚基之间相互作用的性质。用F-α 2 M观察到更大程度的交联，表明亚基在这种形式的分子中相互作用最密切。将S-α 2 M暴露于3 M-尿素或pH 3导致解离成二硫键合的半分子;这些未显示完整α 2 M的蛋白酶结合活性特征。F-α 2 M比S-α 2 M更不容易解离。通过温和还原，S-α 2 M容易解离成四分之一亚基，形成3-4个新的巯基/亚基。然后通过亚基的再氧化可以高产率再生完整的反应性α 2 M。通过与蛋白酶或铵盐反应形成的F-α 2 M在相同条件下不解离，尽管链间二硫键被还原。如果S-α 2 M的四分之一亚基的巯基被羧甲基化阻断，则不发生氧化再结合。用甲基铵盐或蛋白酶处理这些亚基引起半分子和完整的（F-）四聚体的重新组装。F-α 2 M不具有变性形式的蛋白质的性质。对S型和F型之间性质差异的一种解释是S型是平面四聚体，而F型是假四面体。结果被认为与α 2 M与蛋白酶反应的陷阱假说有关。
.alpha.2-Macroglobulin (.alpha.2M) was isolated from human plasma by a 4-step procedure: poly(ethylene glycol) fractionation, gel chromatography, euglobulin precipitation and immunoadsorption. No contaminants were detected in the final preparations by electrophoresis or immunoprecipitation. The protein ran as a single slow band in gel electrophoresis and was designated S-.alpha.2M. S-.alpha.2M bound about 2 mol of trypsin/mol. Treatment of S-.alpha.2M with a proteinase or ammonium salts produced a form of the molecule more mobile in electrophoresis and lacking proteinase-binding activity (F-.alpha.2M). Electrophoretic mobility of the F-.alpha.2M resulting from reaction with NH4+ salts was identical with that of proteinase complexes. Change in electrophoretic mobility of the .alpha.2M was attributed to a conformational change, but there was no evidence of a change in pI or Stokes radius. Electrophoresis of S-.alpha.2M in the presence of sodium dodecyl sulfate [SDS] gave results consistent with the view that the .alpha.2M molecule is a tetramer of identical subunits, assembled as a non-covalent pair of disulfide-linked dimers. Some subunits seemed to be nicked into 2/3 length and 1/3 length chains. This was not apparent with F-.alpha.2M produced by ammonium salts. F-.alpha.2M produced by trypsin showed 2 new bands attributable to cleavage of the subunit polypeptide chain near the middle. Immunoassays of F-.alpha.2M gave rockets 12-29% lower than those with S-.alpha.2M. The nature of interactions between subunits in S-.alpha.2M and F-.alpha.2M was investigated by treating each form with glutaraldehyde before electrophoresis in the presence of SDS. A much greater degree of cross-linking was observed with the F-.alpha.2M, indicating that subunits interact most closely in this form of the molecule. Exposure of S-.alpha.2M to 3 M-urea or pH 3 resulted in dissociation to disulfide-bonded half-molecules; these did not show proteinase-binding activity characteristic of the intact .alpha.2M. F-.alpha.2M was less easily dissociated than was S-.alpha.2M. S-.alpha.2M was readily dissociated to the quarter-subunits by mild reduction, with the formation of 3-4 new thiol groups/subunit. Intact reactive .alpha.2M could then be regenerated in high yield by reoxidation of the subunits. F-.alpha.2M formed by reaction with a proteinase or ammonium salts was not dissociated under the same conditions, although the interchain disulphide bonds were reduced. If thiol groups of the quarter-subunits of S-.alpha.2M were blocked by carboxymethylation, oxidative reassociation did not occur. Treatment of these subunits with methylammonium salts or a proteinase caused the reassembly of half-molecules and intact (F-) tetramers. F-.alpha.2M does not have the properties of a denatured form of the protein. One interpretation of the difference in properties between the S- and F-forms is that the S-form is a planar tetramer, whereas the F-form is pseudotetrahedral. Results are considered in relation to the trap hypothesis for the reaction of .alpha.2M with proteinases.