The electrophoretically ‘slow’ and ‘fast’ forms of the α2-macroglobulin molecule

Abstract

alpha 2-Macroglobulin (alpha 2M) was isolated from human plasma by a four-step procedure: poly(ethylene glyco) 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). The electrophoretic mobility of the F-alpha 2M resulting from reaction with NH4+ salts was identical with that of proteinase complexes. We attribute the change in electrophoretic mobility of the alpha 2M to a conformation change, but there was no evidence of a change in pI or Strokes radius. Electrophoresis of S-alpha 2M in the presence of sodium dodecylsulphate gave results consistent with the view that the alpha 2M molecule is a tetramer of identical subunits, assembled as a non-covalent pair of disulphide-linked dimers. Some of the subunits seemed to be 'nicked' into two-thires-length and one-third-length chains, however. This was not apparent with F-alpha 2M produced by ammonium salts. F-alpha 2M produced by trypsin showed two 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 the 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 sodium dodecyl sulphate. A much greater degree of cross-linking was observed with the F-alpha 2M, indicating that the subunits interact most closely in this form of the molecule. Exposure of S-alpha 2M to 3 M-urea or pH3 resulted in dissociation to the disulphide-bonded half-molecules; these did not show the 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 per subunit. Inact 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 the thiol groups of the quarter-subunits of S-alpha 2M were blocked by carboxymethylation, oxidative reassociation did not occur. Nevertheless treatment of these subunits with methylammonium salts or a proteinase caused the reassembly of half-molecules and intact (F-) tetramers. It is emphasized that F-alpha 2M does not have the properties of a denatured form of the protein...

Parameters influencing the attachment of hepatitis A virus to a variety of continuous cell lines

“…When the proteins were fully denatured and reduced prior to electrophoresis, the reaction with the immobilized proteins was still observed, but at a reduced level. However the electrophoretic migration of the reactive species under these conditions corresponded to an Mr of approximately 170000, consistent with the size of the ot2M subunit and with a smaller Mr protein which may represent a protease-'nicked' form of ~t2 M (Barrett et al, 1979). Thus it appears that the reaction of HAV with ~2M or FCS is not dependent on the quaternary structure of the protein.…”

“…When the samples were boiled in SDS and 2-mercaptoethanol prior to electrophoresis, virus still bound to the immobilized proteins. Under these conditions the major reacting species in the FCS lane had an Mr of approximately 170K, which corresponds to the reported size of the ct2M monomer (Barrett et al, 1979). A similar protein could be seen in the fraction 1 sample but virus bound more extensively to a band migrating at approximately 110K, the reported Mr of a proteolytic fragment derived from at2M (Barrett et al, 1979).…”

“…Under these conditions the major reacting species in the FCS lane had an Mr of approximately 170K, which corresponds to the reported size of the ct2M monomer (Barrett et al, 1979). A similar protein could be seen in the fraction 1 sample but virus bound more extensively to a band migrating at approximately 110K, the reported Mr of a proteolytic fragment derived from at2M (Barrett et al, 1979). The band in purified 0¢2M binding virus most strongly also had an Mr of l l0K, suggesting that this material had been extensively cleaved.…”

Direct kinetics of bait region cleavage of α‐2‐macroglobulin by a rapid quenching method

“…accumulated knowledge on the trap mechanism, we know that (1) one molecule of tetrameric human c~2M can trap up to two molecules of small proteinases such as chymotrypsin or trypsin, (2) when two mol of chymotrypsin are trapped per mol of c~2M under molar abundance of the former, all four bait regions of tetrameric ~2M are cleaved. The structural changes of CZzM and its homologues that takes place after bait region cleavage can be detected by a variety of physical and biochemical methods including electron microscopy [5,6], X-ray solution scattering [7], circular dichroism [8], electrophoresis [9], gel chromatography [10], fluorescence spectroscopy and sedimentation velocity [11]. We have little information, however, on the kinetic aspects of the reaction such as (1) how fast the bait regions are cleaved after the encounter of ~2 M with proteinases, (2) how many bait regions must be cleaved before the proteinases are trapped, (3) how closely connected in time are the bait region cleavage and the structural change?…”

Sequence determination of the thiolester site of the fourth component of human complement.

“…Thus, the autolytic cleavage is dependent on the integrity of the internal thiolester. Nucleophile inactivation, autolytic cleavage on denaturation, and the ability to form covalent bonds (with proteases) have also been described for a2-macroglobulin (a2M) (25)(26)(27)(28)(29)(30)(31). We and others have shown that nucleophile inactivation again correlates with the appearance of a thiol group (10,(32)(33)(34).…”