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Electrophoretically slow and fast forms of the alpha 2 macro globulin molecule

Biochemical Journal 181(2): 401-418
Electrophoretically slow and fast forms of the alpha 2 macro globulin molecule
.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.

Accession: 005349221

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