Kinetics of conformational changes and inactivation of human .alpha.2-macroglobulin on reaction with methylamine

Larsson, L J; Olson, Steven T.; Bjoerk, Ingemar

doi:10.1021/bi00328a002

Cited by 21 publications

(20 citation statements)

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“…* To whom correspondence and reprint requests should be sent. similar to that induced by proteinases (Barrett et al, 1979;Gonias et al, 1982;Bjork & Fish, 1982;Strickland & Bhattacharya, 1984;Larsson et al, 1985;Eccleston & Howard, 1985). In contrast, no major conformational change occurs in bovine a2M, and this protein also retains activity.…”

Section: Introductionmentioning

confidence: 74%

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

Larsson¹,

Lindahl²,

Hallén-Sandgren³

et al. 1987

Biochemical Journal

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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].

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Section: Introductionmentioning

confidence: 74%

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

Larsson¹,

Lindahl²,

Hallén-Sandgren³

et al. 1987

Biochemical Journal

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“…Protein concentrations were obtained by absorption measurements at 280 nm. The specific absorption coefficients and Mr values used in the calculations have been reported previously (Bjork & Fish, 1982;Larsson et al, 1985). All concentrations of proteinases were active-site concentrations.…”

Section: Methodsmentioning

confidence: 99%

“…Small primary amines inactivate human a2M (Steinbuch et al, 1968; Barrett et al, 1979), evidently by a nucleophilic attack on the thioester bonds by the unprotonated amine (Sottrup-Jensen et al, 1980;Howard, 1981;Larsson & Bjbrk, 1984). The thioesterbond cleavage leads to at least two sequential conformational changes of the inhibitor, the final conformational state being very similar to that induced by the reaction of a2M with proteinases (Barrett et al, 1979;Bjork & Fish, 1982;Gonias et al, 1982;Dangott et al, 1983;Strickland & Bhattacharya, 1984;Larsson et al, 1985). The thioester bonds of bovine and rat a2M are also cleaved by amines, but, in contrast with human a2M, no major conformational change ensues, and the inhibitor retains activity (Dangott & Cunningham, 1982;Gonias et al, 1983;Feldman et al, 1984;Bjork et al, 1985).…”

Section: Introductionmentioning

confidence: 98%

Binding of proteinases to human α2-macroglobulin with its thioester bonds cleaved by methylamine in the presence of a thiol-group-cyanylating reagent

Björk¹

1985

Biochemical Journal

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After cleavage of the thioester bonds of human alpha 2-macroglobulin (alpha 2M) by methylamine, the inhibitor undergoes an extensive conformational change and loses its ability to bind proteinases. In contrast, similar cleavage in the presence of dinitrophenyl thiocyanate, a reagent that cyanylates the liberated thiol groups, does not change the mobility of alpha 2M in gel electrophoresis, and the inhibitor also retains activity [Van Leuven, Marynen, Cassiman & Van den Berghe (1982) Biochem. J. 203, 405-411]. Analyses in this work show that also the spectroscopic properties of alpha 2M are essentially unperturbed under these conditions. These observations are consistent with the major change of the conformation of the protein having been arrested by the cyanylation reaction. However, several functional properties of the protein are altered, indicating that a limited conformational change does occur. The apparent stoichiometry of binding of trypsin is thus decreased to about 0.5 mol of enzyme/mol of alpha 2M. Nevertheless trypsin induces a similar conformational change in all molecules of the modified inhibitor as that induced in untreated alpha 2M. This behaviour indicates a similar mode of binding of the enzyme to the modified alpha 2M as to intact alpha 2M, but also a high extent of non-productive activation of binding sites in the modified inhibitor. A further difference to untreated alpha 2M is that most of the bound trypsin molecules react considerably faster with soya-bean trypsin inhibitor. The rate of inhibition of thrombin is also greatly decreased, and the modified inhibitor is more sensitive than untreated alpha 2M to proteolysis at sites outside the 'bait' region. The properties of the cyanylated human alpha 2M are thus similar to those of bovine alpha 2M in which the thioester bonds have been cleaved by methylamine in the absence of the cyanylating reagent [Björk, Lindblom & Lindahl (1985) Biochemistry 24, 2653-2660]. These results indicate that the thioester bonds of human and bovine alpha 2M are not required as such for the stability of the gross conformation of the protein or for the binding of proteinases. Nevertheless they participate directly in maintaining certain structural features, similar in the two inhibitors, that are necessary for full proteinase-binding ability. Disruption of these structures leads to a slower and less efficient trapping of the enzymes.

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“…This "slow to fast" conversion is frequently used as a diagnostic for this major conformational change, with the result that ␣ 2 M conformations are often referred to as the slow form or fast form. In addition, there is an intermediate mobility species in which conformational change has occurred in only two of the four subunits (6,23). We tested the ability of the four ␣ 2 M variants to undergo this slow to intermediate or slow to fast form interconversion upon reaction with either methylamine or proteinase.…”

Section: Electrophoretic Mobility Of Variants On Nondenaturingmentioning

confidence: 99%

“…Cleavage anywhere within the bait region also results in activation of an internal thiol ester toward cleavage by nucleophiles. From studies on the kinetics of cleavage of the thiol ester by nucleophiles and of the subsequent conformational change within the ␣ 2 M, it has been shown that the conformational change occurs cooperatively after both thiol esters within one half of the ␣ 2 M tetramer have been cleaved (5,6).…”

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confidence: 99%

Bait Region Involvement in the Dimer-Dimer Interface of Human α2-Macroglobulin and in Mediating Gross Conformational Change

Bowen¹,

Gettins

1998

Journal of Biological Chemistry

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We have characterized four human ␣ 2 -macroglobulin (␣ 2 M) bait region variants (G679C, M690C, V700C, and T705C) to test the hypothesis that the bait regions are involved in the interface between noncovalently associated dimers. All four variants folded correctly as judged by many normal properties. However, the presence of a cysteine resulted in disulfide formation between otherwise noncovalently associated dimers in all four variants. The extent of disulfide cross-linking varied with the location of the cysteine and gave a mixture of species that probably contained two, one, or zero interdimer disulfides in the tetramer. This was reflected in heterogeneity of conformational change upon thiol ester cleavage by methylamine, with the presence of crosslinks correlating with blockage of conformational change. The stoichiometry of trypsin inhibition was less in all cases than for wild-type ␣ 2 M. The M690C variant also showed evidence of some species with an intramolecular disulfide between bait regions of monomers within the same dimer. Taken together, the results are consistent with a location of the four bait regions in contact with, or in very close proximity to, one another. This suggests that they form all or part of the "cavity body" seen in the low resolution x-ray structure of transformed ␣ 2 M. Inhibition of proteinases by humanresults from a series of conformational changes that are initiated by the proteinase and that result in the physical sequestration of the proteinase within the closed cage-like structure of the conformationally altered ␣ 2 M (1, 2). The initiating event is a proteolytic cleavage by the attacking proteinase near the center of the ␣ 2 M polypeptide in a region termed the bait region (3, 4). Cleavage anywhere within the bait region also results in activation of an internal thiol ester toward cleavage by nucleophiles. From studies on the kinetics of cleavage of the thiol ester by nucleophiles and of the subsequent conformational change within the ␣ 2 M, it has been shown that the conformational change occurs cooperatively after both thiol esters within one half of the ␣ 2 M tetramer have been cleaved (5, 6).Knowledge of the structural relationship between the thiol ester and the bait region and hence of the details of the activation mechanism is limited by the absence of a high resolution x-ray structure of either native or conformationally altered human ␣ 2 M. From fluorescence resonance energy transfer measurements, it was shown that the four cysteines that form the four thiol esters of the human ␣ 2 M tetramer are centrally located and are about 35 Å apart (7). This was subsequently confirmed by a low resolution (ϳ10 Å) x-ray structure of conformationally altered ␣ 2 M (8). NMR measurements on ␣ 2 M showed that the bait region of each monomer was unusually flexible (9, 10) and that it lies close (10 -17 Å) to the cysteine of the thiol ester in the transformed protein (11). However, the location of the four bait regions is still not known. Studies from this laboratory on ␣ 2 M varian...

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Kinetics of conformational changes and inactivation of human .alpha.2-macroglobulin on reaction with methylamine

Cited by 21 publications

References 43 publications

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

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

Binding of proteinases to human α2-macroglobulin with its thioester bonds cleaved by methylamine in the presence of a thiol-group-cyanylating reagent

Bait Region Involvement in the Dimer-Dimer Interface of Human α2-Macroglobulin and in Mediating Gross Conformational Change

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