The conformational changes of alpha 2-macroglobulin induced by methylamine or trypsin. Characterization by extrinsic and intrinsic spectroscopic probes.

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].     

Changes of the proteinase binding properties and conformation of bovine alpha 2-macroglobulin on cleavage of the thio ester bonds by methylamine

Cleavage of the thio ester bonds of human alpha2-macroglobulin (alpha 2M) by methylamine leads to an extensive conformational change and to inactivation of the inhibitor. In contrast, cleavage of these bonds in bovine alpha 2M only minimally perturbs the hydrodynamic volume of the protein [Dangott, L. J., & Cunningham, L. W. (1982) Biochem. Biophys. Res. Commun. 107, 1243-1251], as well as its spectroscopic properties, as analyzed by ultraviolet difference spectroscopy, circular dichroism, and fluorescence in this work. A conformational change analogous to that undergone by human alpha 2M thus does not occur in the bovine inhibitor. However, changes of several functional properties of bovine alpha 2M are induced by the amine. The apparent stoichiometry of inhibition of trypsin thus is reduced from about 1.2 to about 0.7 mol of enzyme/mol of inhibitor. In spite of this decrease, the interaction with the proteinase induces similar conformational changes in methylamine-treated alpha 2M as in intact alpha 2M, as revealed by spectroscopic analyses, indicating that the mode of binding of the proteinase to the inhibitor is essentially unperturbed by thio ester bond cleavage. The reaction with methylamine also greatly increases the sensitivity of bovine alpha 2M to proteolysis by trypsin at sites other than the "bait" region. Moreover, the second-order rate constant for the reaction with thrombin is reduced by about 10-fold. These results indicate that the thio ester bonds of bovine alpha 2M, although not required per se for the binding of proteinases, nevertheless are responsible for maintaining certain structural features of the inhibitor that are of importance for full activity.     

The primary structures of Pseudomonas AM1 amicyanin and pseudoazurin. Two new sequence classes of blue copper proteins.

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.     

Characterization of alpha 2-macroglobulin-plasmin complexes: complete subunit cleavage alters receptor recognition in vivo and in vitro

When human alpha 2-macroglobulin (alpha 2M) binds proteinases, it undergoes subunit cleavage. Binding of small proteinases such as trypsin results in proteolysis of each of the four subunits of the inhibitor. By contrast, previous studies suggest that reaction of plasmin with alpha 2M results in cleavage of only two or three of the inhibitor subunits. In this paper, we demonstrate that the extent of subunit cleavage of alpha 2M is a function of plasmin concentration. When alpha 2M was incubated with a 2.5-fold excess of plasmin, half of the subunits were cleaved; however, at a 20-fold enzyme to inhibitor ratio, greater than 90% of the subunits were cleaved with no additional plasmin binding. This increased cleavage was catalyzed by free rather than bound plasmin. It is concluded that this "nonproductive" subunit cleavage is dependent upon the molar ratio of proteinase to inhibitor. The consequence of complete subunit cleavage on receptor recognition of alpha 2M-plasmin (alpha 2M-Pm) complexes was studied. Preparations of alpha 2M-Pm with only two cleaved subunits bound to the murine macrophage receptor with a Kd of 0.4 nM and 60 fmol of bound complex/mg of cell protein. When preparations of alpha 2-M-Pm with four cleaved subunits were studied, the Kd was unaltered but ligand binding increased to 140 fmol/mg of cell protein. The receptor binding behavior of the latter preparation is equivalent to that observed when alpha 2M is treated with small proteinases such as trypsin. This study suggests that receptor recognition site exposure is not complete in the alpha 2M-Pm complex with half of the subunits cleaved. Proteolytic cleavage of the remaining subunits of the inhibitor results in a further conformational change exposing the remaining receptor recognition sites.     

Thrombin-induced conformational changes of human alpha 2-macroglobulin: evidence for two functional domains

The interaction of thrombin with alpha 2-macroglobulin (alpha 2M) was characterized by monitoring conformational changes and measuring the increase of free sulfhydryl groups during the reaction. Under experimental conditions where [thrombin] greater than [alpha 2M], the conformational change, measured by increases in the fluorescence of 6-(p-toluidino)-2-naphthalenesulfonate, and thiol group appearance displayed biphasic kinetics. The initial rapid phase results in the formation of a stable complex, the appearance of two sulfhydryl groups, the cleavage of approximately half of the Mr 180 000 subunits, and a conformational change that is not as extensive as that which occurs with trypsin. The slower phase is associated with the appearance of two additional sulfhydryl groups, increased cleavage of the Mr 180 000 subunit, and additional conformational changes. The available evidence suggests that the slow phase results from hydrolysis of the Mr 180 000 subunit(s) due to proteolysis of the alpha 2M-thrombin complex by free thrombin. Experiments with 125I-thrombin document the binding of 1 mol of thrombin/mol of alpha 2M that is not dissociated upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the complex. At higher ratios of thrombin to alpha 2M, a second mole of thrombin will reversibly associate with the 1:1 alpha 2M-thrombin complex. Under conditions where [thrombin] less than [alpha 2M], biphasic kinetics were not observed, and the conformational change, sulfhydryl appearance, and hydrolysis of the Mr 180 000 subunit were found to follow second-order kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunoelectron microscopy studies with a monoclonal antibody directed against a receptor recognition site epitope in human alpha 2-macroglobulin.

Alpha 2-Macroglobulin (alpha 2M) is a plasma proteinase inhibitor that binds up to 2 mole of proteinase per mole of inhibitor. Proteinase binding or reaction with small primary amines causes a major conformational change in alpha 2M. As a result of this conformational change, a new epitope recognized by monoclonal antibody 7H11D6 is exposed. The association of alpha 2M-proteinase or alpha 2M-methylamine with alpha 2M cellular receptors is prevented by 7H11D6. In this investigation, the binding of 7H11D6 to alpha 2M was studied by electron microscopy. 7H11D6 bound to alpha 2M-methylamine and alpha 2M-trypsin but not to native alpha 2M. The structure of alpha 2M after conformational change resembled the letter "H." 7H11D6 epitopes were identified near the apices of the four arms in the alpha 2M "H" structure. 7H11D6 that was adducted to colloidal gold (7HAu) retained the specificity of the free antibody (binding to alpha 2M-trypsin but not to native alpha 2M). alpha 2M conformational change intermediates prepared by sequential reaction with a protein crosslinker and trypsin also bound 7HAu. These results suggest that a complete alpha 2M conformational change is not necessary for 7H11D6 epitope exposure and may not be required for receptor recognition. 7HAu was used to isolate a preparation consisting primarily of binary alpha 2M-trypsin (1 mole trypsin per mole alpha 2M instead of 2). Structures resembling the letter "H" were most common; however, each field showed some atypical molecules with arms that were compacted instead of thin and elongated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidized caprine alpha-2-macroglobulin: damaged but not completely dysfunctional

Caprine alpha-2-macroglobulin (alpha2M) is a broad-spectrum, homotetrameric proteinase inhibitor that can maximally bind a single molecule of proteinase. Inhibition of proteinases by caprine alpha2M results from a series of conformational changes that are initiated by the proteinase and results in physical sequestration of the proteinase within the closed cage-like structure of conformationally altered alpha2M. In a previous study, uric acid-generated superoxide anion was identified as one of the physiologically relevant inactivators of alpha2M S.A. Khan, F.H. Khan [Free. Radic. Res. 34 (2001) 113]. We now demonstrate that hypochlorous acid (HOCl) and, to lesser extent, hydrogen peroxide (H2O2) destroy the antiproteolytic potential of caprine alpha2M. At physiologically attainable concentration, we found that HOCl significantly compromised functional integrity of the inhibitor. High concentrations of H2O2 also partially diminished proteinase inhibitory capacity of alpha2M by a mechanism not involving formation of hydroxyl radicals. For hydrogen peroxide, catalase completely protected alpha2M activity while the ability to protect the inhibitor from HOCl-induced inactivation was limited by availability of albumin. Structure function analysis demonstrated that oxidized caprine inhibitor, unlike its human counterpart, retained its tetrameric configuration as well as its characteristic ability to undergo major conformational change upon trypsinization. It is proposed that inhibition of alpha2M activity may be due to oxidation of essential residues of the inhibitor and/or structural rearrangement of the subunits.     

Intersubunit cross-linking by cis-dichlorodiammineplatinum(II) stabilizes an alpha 2-macroglobulin "nascent" state: evidence that thiol ester bond cleavage correlates with receptor recognition site exposure

Treatment of human alpha 2-macroglobulin (alpha 2M) with proteinase results in cleavage of the alpha 2M subunits and subsequently in a conformational change in the inhibitor. This change irreversibly traps the proteinase and is accompanied by the generation of four thiol groups as well as exposure of receptor recognition sites. cis-Dichlorodiammineplatinum(II) (cis-DDP) causes extensive intersubunit cross-linking of alpha 2M. Incubation of alpha 2M or cis-DDP-treated alpha 2M with trypsin results in complete subunit cleavage; however, trypsin treatment of cis-DDP-alpha 2M does not result in a conformational change as determined by nondenaturing polyacrylamide gel electrophoresis (PAGE), receptor recognition site exposure, or appearance of thiol groups from the inhibitor. These results are in marked contrast to previous studies which demonstrated that incubation of cis-DDP-treated alpha 2M with CH3NH2 resulted in thiol ester bond cleavage and receptor recognition site exposure. cis-DDP-treated alpha 2M bound only 0.13 mol of 125I-trypsin/mol of cis-DDP-alpha 2M. Incubation of trypsin-treated cis-DDP-alpha 2M with diethyldithiocarbamate (DDC), a potent chelator of platinum compounds, results in the removal of the intersubunit cross-links and completion of the alpha 2M conformational change as determined by nondenaturing PAGE. Complete receptor recognition site exposure and the appearance of 3.3 thiol groups/mol of alpha 2M also occur following this treatment. These results demonstrate that cross-linking of alpha 2M by cis-DDP prevents a conformational change in the inhibitor which is necessary for thiol ester bond activation and cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reaction of proteinases with alpha 2-macroglobulin: rapid-kinetic evidence for a conformational rearrangement of the initial alpha 2-macroglobulin-trypsin complex.

The kinetics of the reaction of trypsin with alpha 2M were examined under pseudo-first-order conditions with excess inhibitor. Initial studies indicated that the fluorescent dye TNS is a suitable probe for monitoring the reaction over a wide concentration range of reactants. Titration experiments showed that the conformational changes associated with the binding of trypsin to alpha 2M result in an increased affinity of the inhibitor for TNS. Two distinct phases were observed when this dye was used to monitor the progress of the reaction. Approximately half of the fluorescence signal was generated during a rapid phase, with the remainder generated during a second, slower phase. The observed pseudo-first-order rate constant of the first phase varied linearly with the concentration of alpha 2M up to the highest concentration of inhibitor used, whereas the rate constant of the second phase was independent of alpha 2M concentration. The data fit a mechanism in which the association of trypsin with alpha 2M occurs in two consecutive, essentially irreversible steps, both leading to alterations in TNS fluorescence. The initial association occurs with a second-order rate constant of (1.0 +/- 0.1) X 10(7) M-1 s-1 and is followed by a slower, intramolecular conformational rearrangement of the initial complex with a rate constant of 1.4 +/- 0.2 s-1. The data are consistent with a previously proposed model for the reaction of proteinases with alpha 2M [Larsson et al. (1989) Biochemistry 28, 7636-7643].2+ this model, once an initial 1:1 alpha 2M-proteinase     

Probing the stability of native and activated forms of alpha2-macroglobulin

alpha2-Macroglobulin (alpha2M) is a 718 kDa homotetrameric proteinase inhibitor which undergoes a large conformational change upon activation. This conformational change can occur either by proteolytic attack on an approximately 40 amino acid stretch, the bait region, which results in the rupture of the four thioester bonds in alpha2M, or by direct nucleophilic attack on these thioesters by primary amines. Amine activation circumvents both bait region cleavage and protein incorporation, which occurs by proteolytic activation. These different activation methods allow for examination of the roles bait region cleavage and thioester rupture play in alpha2M stability. Differential scanning calorimetry and urea gel electrophoresis demonstrate that both bait region cleavage and covalent incorporation of protein ligands in the thioester pocket play critical roles in the stability of alpha2M complexes.     

Further characterization of the covalent linking reaction of alpha 2-macroglobulin.

It is shown that non-proteolytic proteins can become covalently linked to alpha 2M (alpha 2-macroglobulin) during its reaction with proteinases, and that this probably occurs by the mechanism that leads to the covalent linking of proteinases described previously [Salvesen & Barrett (1980) Biochem. J. 187, 695-701]. The covalent linking of trypsin was at least partly dependent on the presence of unblocked lysine side chains on the protein. The covalent linking of proteinases was inhibited by nucleophiles of low Mr, and these compounds were themselves linked to alpha 2M in a molar ratio approaching one per quarter subunit. Peptide "mapping" indicated that the site of proteinase-mediated incorporation of the amines was the same as that at which methylamine is incorporated in the absence of a proteinase. The nucleophile-reactive site revealed in alpha 2M after reaction with a proteinase was shown to decay with a t1/2 of 112 s, at pH 7.5. After the reaction with a proteinase or with methylamine, a free thiol group was detectable on each subunit of alpha 2M. We propose that the site for incorporation of methylamine in each subunit is a thiol ester, which in S-alpha 2M (the electrophoretically "slow" form) is sterically shielded from reaction with large nucleophiles, but is revealed as a highly reactive group, free from steric hindrance, after the proteolytic cleavage. We have designated the activated species of the molecule "alpha 2M".     

Identification of a monoclonal antibody specific for a neoantigenic determinant on alpha 2-macroglobulin: use for the purification and characterization of binary proteinase-inhibitor complexes

A monoclonal antibody was obtained from the fusion of spleen cells of mice, immunized with methylamine-treated alpha 2-macroglobulin (alpha 2M), with the myeloma cell line P3-X63-Ag8.653. A competitive binding assay demonstrated that the antibody was specific for a neoantigen expressed on alpha 2M when the inhibitor reacts with proteinases or with methylamine. When immobilized, the monoclonal antibody retained its ability to specifically bind alpha 2M-proteinase complexes or methylamine-treated alpha 2M, both of which could be quantitatively recovered from the immunoaffinity column by lowering the pH to 5.0. Binary alpha 2M-proteinase complexes of trypsin, plasmin, and thrombin, prepared by incubating large amounts of alpha 2M with a small amount of enzyme, were isolated by immunoaffinity chromatography. Each purified complex was characterized with regard to proteinase content, extent of alpha 2M subunit cleavage, extent of thiol ester hydrolysis, and extent of conformational change. Each complex contained 0.8-0.9 mol of proteinase/mol of inhibitor. In the alpha 2M-thrombin, alpha 2M-plasmin, and alpha 2M-trypsin complexes, approximately 50%, 60%, and 75% of the subunits are cleaved, respectively. Titration of sulfhydryl groups revealed that all purified binary complexes contained 2 +/- 0.5 mol of thiol/mol of complex, suggesting that each complex retains two intact thiol ester bonds. When the purified complexes were incubated with excess trypsin or with methylamine, an additional 1-2 mol of sulfhydryl/mol of complex could be titrated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Independent analysis of bait region cleavage dependent and thiolester bond cleavage dependent conformational changes by cross-linking of alpha 2-macroglobulin with cis-dichlorodiammineplatinum(II) and dithiobis(succinimidyl propionate)

Treatment of the human plasma proteinase inhibitor alpha 2-macroglobulin (alpha 2M) with proteinase results in conformational changes in the inhibitor and subsequent activation and cleavage of the internal thiolester bonds of alpha 2M. Previous studies from this laboratory have shown that cross-linking the alpha 2M subunits with cis-dichlorodiammineplatinum(II) (cis-DDP) prevents the proteinase-induced conformational changes which lead to the activation and cleavage of the internal thiolester bonds of alpha 2M. In addition, cis-DDP treatment prevents the proteinase- or CH3NH2-induced conformational changes in alpha 2M which lead to a "slow" to "fast" change in nondenaturing polyacrylamide gel electrophoresis. In this paper, we demonstrate that treatment of alpha 2M with dithiobis(succinimidyl propionate) (DSP) also results in cross-linking of the subunits of alpha 2M with concomitant loss of proteinase inhibitory activity. Although proteinase is not inhibited by DSP-treated alpha 2M, bait region specific proteolysis of the alpha 2M subunits still occurs. Unlike cis-DDP-treated alpha 2M, however, incubation of DSP-treated alpha 2M with proteinase does not prevent the bait region cleavage dependent conformational changes which lead to activation and cleavage of the internal thiolester bonds in alpha 2M. On the other hand, cross-linking of alpha 2M with DSP does prevent the conformational changes which trigger receptor recognition site exposure following cleavage of the alpha 2M thiolester bonds by CH3NH2. These conformational changes, however, occur following incubation of the CH3NH2-treated protein with proteinase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescent probes as a measure of conformational alterations induced by nucleophilic modification and proteolysis of bovine alpha 2-macroglobulin

Conformational alterations occurring in bovine alpha 2-macroglobulin (alpha 2M) resulting from proteolysis and nucleophilic modification have been monitored by UV difference spectra, circular dichroism, and changes in the fluorescence of 6-(p-toluidino)-2-naphthalenesulfonate (TNS) and bis(8-anilino-1-naphthalenesulfonate) (Bis-ANS). The results of this study indicate that these two dyes appear capable of differentiating between conformational changes induced by proteolysis and those induced by methylamine treatment. It appears that TNS is a sensitive probe for monitoring protease-induced but not methylamine-induced conformational changes in bovine alpha 2M. Bis-ANS, on the other hand, appears suitable for monitoring conformational changes induced by methylamine treatment or proteolysis of the molecule and was used as a probe to monitor the kinetics of the conformational change induced by methylamine treatment. It was found that the conformational change did not occur simultaneously with cleavage of the thiol ester bonds by the nucleophile, measured by titration of free sulfhydryl groups with 5,5'-dithiobis(2-nitrobenzoate). The data are consistent with a model in which initial nucleophilic attack results in exposure of sulfhydryl groups, resulting in a conformational change measured by an increase in fluorescence. This event is followed by a unimolecular step representing a conformational change in the protein that results in a further increase in the fluorescence signal. The second-order rate constant for hydrolysis of the thiol ester bonds was determined to be 3.4 +/- 1.0 M-1 s-1, while the rate constant for the conformational change was (4.4 +/- 0.8) X 10(-4) s-1.     

The role of histidyl residues in zinc-induced precipitation of alpha 2-macroglobulin-proteinase complexes

When alpha 2-macroglobulin (alpha 2M) is reacted with proteinases including trypsin, plasmin, alpha-thrombin, or with CH3NH2, each resulting alpha 2M derivative is precipitated by Zn2+ in a similar manner. By contrast, unreacted alpha 2M is not precipitated over the same Zn2+ concentration range. Zn2+-induced precipitation of alpha 2M-CH3NH2 or alpha 2M-trypsin is prevented by acylation of the protein employing the histidine-specific reagent diethylpyrocarbonate (DEP). The Zn2+-induced precipitation of alpha 2M-trypsin is prevented by acylation of the preformed alpha 2M-trypsin complex or by the reaction of acylated native alpha 2M with trypsin. Acylation of alpha 2M by treatment with DEP results in the modification of 13.5 histidyl residues per subunit of either native alpha 2M or alpha 2M-CH3NH2. Subsequent treatment with hydroxylamine reverses the modification of 10.5 histidyl residues per subunit in each protein preparation. These results indicate that histidyl residues are involved in the Zn2+-induced precipitation of alpha 2M-proteinase or alpha 2M-CH3NH2 complexes, and that these residues are accessible to extensive protein-metal interactions only after alpha 2M has undergone a major conformational change. These appear to be the same histidyl residues which, upon acylation by DEP, are responsible for recognition of alpha 2M-proteinase complexes by the acyl-low-density-lipoprotein cell surface receptor (S. V. Pizzo, P. A. Roche, S. R. Feldman, and S. L. Gonias (1986) Biochem. J. 238, 217-225).     

Analysis of thiolester bond cleavage-dependent conformational changes in binary alpha 2-macroglobulin-proteinase complexes

The structures of the two proteinase-binding sites in human alpha 2-macroglobulin (alpha 2M) were probed by treatment of alpha 2M with the serine proteinases thrombin and plasmin. Each proteinase forms an equimolar complex with alpha 2M (a binary alpha 2M-proteinase complex) which results in the activation and cleavage of two internal thiolester bonds in alpha 2M. Binary alpha 2M-proteinase complexes demonstrated an incomplete conformational change as determined by nondenaturing polyacrylamide gel electrophoresis and incomplete receptor recognition site exposure as determined by in vivo plasma elimination studies. Treatment of binary alpha 2M-proteinase complexes with CH3NH2, trypsin, or elastase resulted in cleavage of an additional one or two thiolester bonds in alpha 2M and complete receptor recognition site exposure, demonstrating that a limited conformational change had occurred. Treatment of the alpha 2M-thrombin complex with elastase resulted in the incorporation of approximately 0.5 mol proteinase/mol alpha 2M and completion of the conformational change in the complex. Similar treatment of the alpha 2M-plasmin complex resulted in the incorporation of less than 0.1 mol proteinase/mol alpha 2M. Unlike the alpha 2M-thrombin complex, the alpha 2M-plasmin complex did not undergo a complete conformational change following treatment with CH3NH2 or trypsin. Incubation of this complex with elastase resulted in proteolysis of the kringle 1-4 region of the alpha 2M-bound plasmin heavy chain, and following this treatment the alpha 2M-plasmin complex underwent a complete conformational change. The results of this investigation demonstrate that binary alpha 2M-proteinase complexes retain a relatively intact proteinase-binding site. In the case of the alpha 2M-plasmin complex, however, the heavy chain of alpha 2M-bound plasmin protrudes from the proteinase-binding site and prevents a complete conformational change in the complex despite additional thiolester bond cleavage.     

Structural and functional characterization of the inhibition of urokinase by alpha 2-macroglobulin

We have investigated the interaction of alpha 2-macroglobulin (alpha 2M) with the serine proteinase urokinase, an activator of plasminogen. Urokinase formed sodium dodecyl sulfate stable complexes with purified alpha 2M and with alpha 2M in plasma. These complexes could be visualized after polyacrylamide gel electrophoresis by protein blots using 125I-labeled anti-urokinase antibody or by fibrin autography, a measure of fibrinolytic activity. According to gel electrophoretic analyses under reducing conditions, urokinase cleaved alpha 2M subunits and formed apparently covalent complexes with alpha 2M. Urokinase cleaved only about 60% of the alpha 2M subunits maximally at a mole ratio of 2:1 (urokinase: alpha 2M). Binding of urokinase to alpha 2M protected the urokinase active site from inhibition by antithrombin III-heparin and inhibited, to a significant extent, plasminogen activation by urokinase. Reaction of urokinase with alpha 2M caused an increase in intrinsic protein fluorescence and, thus, induced the conformational change in alpha 2M that is characteristic of its interactions with active proteinases. Our results indicate that both in plasma and in a purified system the alpha 2M-urokinase reaction is functionally significant.     

Localization of the proteinases in the human alpha 2-macroglobulin-chymotrypsin complex by image processing of electron micrographs.

Human alpha 2-macroglobulin (alpha 2M), a large tetrameric plasma glycoprotein, inhibits a wide spectrum of proteinases by a particular "trapping" mechanism resulting from the proteolysis of peptide bonds at specific "bait" regions. This induces the hydrolysis of four thiol esters triggering both the possible covalent bonding of the proteinases and a considerable structural change in the alpha 2M molecule, also observed following direct cleavage of the thiol esters by methylamine. By subtracting average images of electron micrographs from two populations of alpha 2M molecules in the same biochemical state (with both the four cleaved bait regions and thiol esters), but containing either two or zero chymotrypsins, we are able to demonstrate the position of the two proteinases inside the tetrameric alpha 2M molecule. The comparison of the alpha 2M molecules transformed either by immobilized chymotrypsin or methylamine shows that the proteolysis of the bait regions seems of minimal importance for the general shape of the molecule and provides a direct visualization of the actual role of the thiol esters in the conformational change.     

Local and global ligand-induced changes in the structure of the GABA(A) receptor

Ligand-gated channels mediate synaptic transmission through conformational transitions triggered by the binding of neurotransmitters. These transitions are well-defined in terms of ion conductance, but their structural basis is poorly understood. To probe these changes in structure, GABA(A) receptors were expressed in Xenopus oocytes and labeled at selected sites with environment-sensitive fluorophores. With labels at two different residues in the alpha1 subunit in loop E of the GABA-binding pocket, GABA elicited fluorescence changes opposite in sign. This pattern of fluorescence changes is consistent with a closure of the GABA-binding cavity at the subunit interface. The competitive antagonist SR-95531 inverted this pattern of fluorescence change, but the noncompetitive antagonist picrotoxin failed to elicit optical signals. In response to GABA (but not SR-95531), labels at the homologous residues in the beta2 subunit showed the same pattern of fluorescence change as the alpha1-subunit labels, indicating a global transition with comparable movements in homologous regions of different subunits. Incorporation of the gamma2 subunit altered the fluorescence changes of alpha1-subunit labels and eliminated them in beta2-subunit labels. Thus, the ligand-induced structural changes in the GABA(A) receptor can extend over considerable distances or remain highly localized, depending upon subunit composition and ligand.     

Image analysis and three-dimensional model of chymotrypsin-transformed human alpha 2-macroglobulin complexed with a monoclonal antibody specific for this conformation

Alpha 2-macroglobulin (alpha 2M) is a plasma inhibitor of proteinases, the steric mechanism of which is based on a considerable conformational change. The typical and distinct H-like shape of alpha 2M-chymotrypsin (alpha 2M-chy) complexes seen by electron microscopy led us to an ultrastructural study of the binding of a monoclonal antibody (Mab) specific for this conformation of alpha 2M. The epitope of this Mab is located near the extremities of the 4 arms of the H-like alpha 2M-chy, at a site that is not accessible on the native molecule. The identical binding of the Mab on the 4 arms of the tetrameric molecule demonstrates that these arms are equivalent portions of the 4 monomers. Various types of immune complexes between alpha 2M and IgG are described, and images of individual immune complexes were processed by correspondence analysis. This extracts new information concerning the organization of chymotrypsin-transformed alpha 2M. The molecule appears asymmetrical, presents 2 conformational states (which we describe as relaxed and twisted), and has flexible arms. These intramolecular motions are supposed to be related to IgG binding. The results are discussed in comparison with previously published models of proteinase-transformed alpha 2M.