The conformational changes of α2-macroglobulin induced by methylamine or trypsin. Characterization by extrinsic and intrinsic spectroscopic probes

Abstract

The conformational changes around the thioester-bond region of human or bovine alpha 2M (alpha 2-macroglobulin) on reaction with methylamine or trypsin were studied with the probe AEDANS [N-(acetylaminoethyl)-8-naphthylamine-1-sulphonic acid], bound to the liberated thiol groups. The binding affected the fluorescence emission and lifetime of the probe in a manner indicating that the thioester-bond region is partially buried in all forms of the inhibitor. In human alpha 2M these effects were greater for the trypsin-treated than for the methylamine-treated inhibitor, which both have undergone similar, major, conformational changes. This difference may thus be due to a close proximity of the thioester region to the bound proteinase. Reaction of trypsin with thiol-labelled methylamine-treated bovine alpha 2M, which retains a near-native conformation and inhibitory activity, indicated that the major conformational change accompanying the binding of proteinases involves transfer of the thioester-bond region to a more polar environment without increasing the exposure of this region at the surface of the protein. Labelling of the transglutaminase cross-linking site of human alpha 2M with dansylcadaverine [N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulphonamide] suggested that this site is in moderately hydrophobic surroundings. Reaction of the labelled inhibitor with methylamine or trypsin produced fluorescence changes consistent with further burial of the cross-linking site. These changes were more pronounced for trypsin-treated than for methylamine-treated alpha 2M, presumably an effect of the cleavage of the adjacent 'bait' region. Solvent perturbation of the u.v. absorption and iodide quenching of the tryptophan fluorescence of human alpha 2M showed that one or two tryptophan residues in each alpha 2M monomer are buried on reaction with methylamine or trypsin, with no discernible change in the exposure of tyrosine residues. Together, these results indicate an extensive conformational change of alpha 2M on reaction with amines or proteinases and are consistent with several aspects of a recently proposed model of alpha 2M structure [Feldman, Gonias & Pizzo (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5700-5704].

The bromocresol green assay, but not the modified bromocresol purple assay, overestimates the serum albumin concentration in nephrotic syndrome through reaction with α2-macroglobulin

“…These cleavage products induce conformational changes in 2 -macroglobulin, and the enzymatic active site of the proteinases is physically trapped and thus inhibited. 22 Among the five 1 -and 2 -globulins measured in this study, only 2 -macroglobulin increased in parallel with the severity of hypoalbuminemia and proteinuria (Table 2).…”

“…Human 2 -macroglobulin is a multifunctional glycoprotein that forms a homotetramer of $725 kDa. 22 2 -Macroglobulin binds non-specifically to various proteinases, which cleave the susceptible site (thioester) of 2 -macroglobulin. These cleavage products induce conformational changes in 2 -macroglobulin, and the enzymatic active site of the proteinases is physically trapped and thus inhibited.…”

α-Macroglobulins Are Present in Some Gram-negative Bacteria

“…In the absence of proteolytic activation, and consequent conformational disturbance around the thiol ester, cleavage of the thiol ester requires a high concentration of a potent, small, nucleophile such as methylamine or hydroxylamine. Such cleavage results in generation of a free thiol (33), and in many cases a large conformational change in the ␣-macroglobulin (34).…”

Probing the functional role of the N-terminal region of cystatins by equilibrium and kinetic studies of the binding of Gly-11 variants of recombinant human cystatin C to target proteinases

“…Quenching of the tryptophan fluorescence of wild-type cystatin C or G1 1W-cystatin C (2 #tM) by increasing concentrations of iodide (0-0.4 M) was measured essentially as in earlier work [18]. The samples were excited at 280 nm and the emission was measured at wavelengths of 345 nm for wild-type cystatin C and 350 nm for G1 1W-cystatin C. The data were corrected for innerfilter effects and were plotted according to a modified SternVolmer equation [19].…”