Electron microscopy of α2-macroglobulin with a thiol ester bound ligand

In order to covalently bind the hydrolyzed thiol ester groups of the human α₂-macroglobulin (α2M) transformed by methylamine, the phospholipase A2 (PLA2), a small enzyme (M_r = 13 000) from Naja nigricollis snake venom was activated by succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC). Average images determined from electron micrographs of the methylamine-transformed α2M, with and without activated PLA2, were determined by image processing and compared. A localization of the PLA2 was achieved by subtracting the average image of α2M transformed by methylamine from that containing PLA2. The results are consistent with previous work showing the central localization of chymotrypsin trapped in α2M. They also suggest that the four thiol esters are located near the center of the α2M. molecule.     

Reaction of methylamine with human alpha 2-macroglobulin. Mechanism of inactivation

The human protease inhibitor alpha 2-macroglobulin (alpha 2 M) is inactivated by reaction with methylamine. The site of reaction is a protein functional group having the properties of a thiol ester. To ascertain the relationship between thiol ester cleavage and protein inactivation, the rates of methylamine incorporation and thiol release were measured. As expected for a concerted reaction of a nucleophile with a thiol ester, the rates were identical. Furthermore, both rates were first order with respect to methylamine and second order overall. The methylamine inactivation of alpha 2M was determined by measuring the loss of total protease-binding capacity. This rate was slower than the thiol ester cleavage and had a substantial initial lag. However, the inactivation followed the same time course as a conformational change in alpha 2M that was measured by fluorescent dye binding, ultraviolet difference spectroscopy, and limited proteolysis. Thus, the methylamine inactivation of alpha 2M is a sequential two-step process where thiol ester cleavage is followed by a protein conformational change. It is the latter that results in the loss of total protease-binding capacity. A second assay was used to monitor the effect of methylamine on alpha 2M. The assay measures the fraction of alpha 2M-bound protease (less than 50%) that is resistant to inactivation by 100 microM soybean trypsin inhibitor. In contrast to the total protease-binding capacity, this subclass disappeared with a rate coincident with methylamine cleavage of the thiol ester. alpha 2M-bound protease that is resistant to a high soybean trypsin inhibitor concentration may reflect the fraction of the protease randomly cross-linked to alpha 2M. Both the thiol ester cleavage and the protein conformational change rates were dependent on methylamine concentration. However, the thiol ester cleavage depended on methylamine acting as a nucleophile, while the conformational change was accelerated by the ionic strength of methylamine. Other salts and buffers that do not cleave the thiol ester increased the rate of the conformational change. A detailed kinetic analysis and model of the methylamine reaction with alpha 2M is presented. The methylamine reaction was exploited to study the mechanism of protease binding by alpha 2M. At low ionic strength, the protein conformational change was considerably slower than thiol ester cleavage by methylamine. Thus, at some time points, a substantial fraction of the alpha 2M had all four thiol esters cleaved, yet had not undergone the conformational change. This fraction (approximately 50%) retained full protease-binding capacity.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Inhibition and partial reversal of the methylamine-induced conversion of "slow" to "fast" electrophoretic forms of human alpha 2-macroglobulin by modification of the thiols.

It has been shown previously [Van Leuven, F., Marynen, P., Cassiman, J. J., & Van den Berghe, H. (1982) Biochem. J. 203, 405-411] that 2,4-dinitrophenyl thiocyanate (DNPSCN) can block the conversion of "slow" to "fast" electrophoretic forms of human alpha 2-macroglobulin (alpha 2M) normally resulting from reaction of alpha 2M with methylamine. The kinetics of reaction of DNPSCN with alpha 2M in the presence of methylamine are examined here and shown to approximate pseudo first order, reflecting the rate-limiting reaction of alpha 2M with methylamine [Larsson, L. J., & Bj?rk, I. (1984) Biochemistry 23, 2802-2807]. One mole of DNPS is liberated per mole of free thiol in alpha 2M, consistent with cyanylation of the thiol liberated upon scission of the internal thiol esters by methylamine. I3(-) can also react with the methylamine-generated thiol groups of alpha 2M with a stoichiometry consistent with conversion of the thiol to a sulfenyl iodide. Reaction of the thiol groups with either DNPSCN or I3(-) inhibits the conversion of alpha 2M from the "slow" to the "fast" electrophoretic form. Furthermore, DNPSCN added after the conformational change can partially reverse the change. A similar reversal can be effected by cyanylation, with NaCN, of methylamine-treated alpha 2M in which the liberated thiols have first been converted to mixed disulfides by reaction with dithiobis(nitrobenzoic acid). Differential scanning calorimetry shows nearly identical properties for the methylamine-treated "fast" form and the cyanylated "slow" form of alpha 2M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid sequence around the thiolester of alpha 2-macroglobulin from plasma of the crayfish, Pacifastacus leniusculus

Alpha 2-Macroglobulin (alpha 2 M) was isolated from plasma of the freshwater crayfish, Pacifastacus leniusculus, using ultracentrifugation, ion-exchange chromatography and gel filtration techniques. The Pacifastacus alpha 2 M molecule (P alpha 2 M) was radio-actively labeled in the thiol ester structure with iodo [14C]acetic acid in the presence of methylamine. After reduction and carboxymethylation of the protein, it was digested with trypsin. A 14C-labeled tryptic peptide was sequenced and contained an amino acid sequence very similar to other known thiol ester sequences from human alpha 2 M and related proteins. The N-terminal sequence of P alpha 2 M was related to that recently determined for lobster alpha 2 M [(1987) J. Biol. Chem. 262, 14606-14611].     

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".     

The structure of the C949S mutant human alpha(2)-macroglobulin demonstrates the critical role of the internal thiol esters in its proteinase-entrapping structural transformation

A three-dimensional reconstruction of a protein-engineered mutant alpha(2)-macroglobulin (alpha(2)M) in which a serine residue was substituted for the cysteine 949 (C949S), making it unable to form internal thiol ester moieties, was compared with native and methylamine-transformed alpha(2)Ms. The native alpha(2)M structure consists of two oppositely oriented Z-shaped strands. Thiol ester cleavage following an encounter with a proteinase or a nucleophilic attack by methylamine causes a structural transformation in which the strands assume an opposite handedness and a significant portion of the protein density migrates from the distal ends of the molecule toward the center. The C949S mutant showed a protein density distribution very similar to that of transformed alpha(2)M, with a compact central region of protein density connected to two receptor-binding arms on each end of the molecule. Since no particle shapes characteristic of native or half-transformed alpha(2)Ms were seen in electron micrographs and the C949S mutant and alpha(2)M-methylamine structures are highly similar, we conclude that the intact thiol esters maintain native alpha(2)M in a quasi-stable state. In their absence, alpha(2)M folds into the more stable transformed structure, which displays the functionally important receptor-binding domains and contains the proteinase-entrapping internal cavity.     

Three-dimensional reconstruction of a complex of human alpha 2-macroglobulin with monomaleimido Nanogold (Au1.4nm) embedded in ice.

Cysteine 949 and glutamine 952 are known to be part of the thiol ester site of each of the four subunits of human alpha 2-macroglobulin (alpha 2M). The hydrolysis of this thiol ester bound to methylamine results in the incorporation of the amine and liberation of a free sulfhydryl group that can be specifically labeled. Therefore, a high-resolution marker specific for the sulfhydryl groups, the monomaleimido Nanogold (Au1.4nm) cluster was used to bind this amino acid. After cryoelectron microscopy, a three-dimensional reconstruction of the alpha 2M-Nanogold conjugates (alpha 2M-Au1.4nm) was achieved, revealing the internal location of the thiol ester sites in the transformed alpha 2M molecules. From this study we propose three possible locations for the cysteine 949.     

Differences in the proteinase inhibition mechanism of human alpha 2-macroglobulin and pregnancy zone protein.

Different conformational states of human alpha 2-macroglobulin (alpha 2M) and pregnancy zone protein (PZP) were investigated following modifications of the functional sites, i.e. the 'bait' regions and the thiol esters, by use of chymotrypsin, methylamine and dinitrophenylthiocyanate. Gel electrophoresis, mAb (7H11D6 and alpha 1:1) and in vivo plasma clearance were used to describe different molecular states in the proteinase inhibitors. In alpha 2M, in which the thiol ester is broken by binding of methylamine and the 'trap' is closed, cyanylation of the liberated thiol group from the thiol ester modulates reopening of the 'trap' and the 'bait' regions become available for cleavage again. The trapping of proteinases in the cyanylated derivative indicates that the trap functions as in native alpha 2M. In contrast, cyanylation has no effect on proteinase-treated alpha 2M. As demonstrated by binding to mAb, the methylamine and dinitrophenylthiocyanate-treated alpha 2M exposes the receptor-recognition site, but the derivative is not cleared from the circulation in mice. The trap is not functional in PZP. In native PZP and PZP treated with methylamine, the conformational states seem similar. The receptor-recognition sites are not exposed and removal from the circulation in vivo is not seen for these as for the PZP-chymotrypsin complex. Tetramers are only formed when proteinases can be covalently bound to the PZP. Conformational changes are not detected in PZP derivatives in which the thiol ester is treated with methylamine and dinitrophenylthiocyanate. The results suggest that the conformational changes in alpha 2M are generated by mechanisms different to these in PZP. The key structure gearing the conformational changes in alpha 2M is the thiol ester, by which the events 'trapping' and exposure of the receptor-recognition site can be separated. In PZP, the crucial step for the conformational changes is the cleavage of the 'bait' region, since cleavage of the thiol ester does not lead to any detectable conformational changes by the methods used.     

Effect of methylamine on the reaction of alpha 2-macroglobulin with enzymes.

The kinetics of reaction of alpha 2-macroglobulin (alpha 2M) with thrombin and with trypsin were studied in the presence and absence of methylamine. The rate of enzyme-induced thiol release was found to be the same whether or not amine was present. The result suggests that covalent bond formation and enzyme-catalyzed amine incorporation proceed via a common (enzyme-dependent) rate-determining step. The reaction of lysyl-modified enzymes (which show poor covalent binding with alpha 2M) was similarly unaffected by amine, indicating that enzyme-catalyzed steps were also rate determining for hydrolysis of the thiol ester. The products of the reactions were analyzed by native and denaturing gel electrophoresis. Methylamine did not affect the total binding of enzyme to alpha 2M but did cause a substantial decrease in covalent binding. Surprisingly, not all covalent complexes were affected by the presence of amine: complexes in which enzyme was covalently bound to one half-molecule increased compared to the reaction with no amine; complexes in which two half-molecules are cross-linked by two bonds to a single enzyme were substantially reduced, however. The results are consistent with a mechanism of reaction in which an enzyme-dependent step is rate determining. This step is accompanied by activation of two thiol esters. One of these reacts immediately with the bound enzyme (or may be hydrolyzed if the enzyme amine groups are blocked). The other activated center is capable of reaction with external nucleophiles such as methylamine.     

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)     

The human alpha 2-macroglobulin receptor: identification of a 420-kD cell surface glycoprotein specific for the activated conformation of alpha 2-macroglobulin

Ligand affinity chromatography was used to purify a cell surface alpha 2-macroglobulin (alpha 2M) receptor. Detergent extracts of human placenta were applied to an affinity matrix consisting of alpha 2M, previously reacted with methylamine, coupled to Sepharose. Elution with EDTA specifically released polypeptides with apparent molecular masses of 420 and 39 kD. In some preparations, small amounts of a 90-kD polypeptide were observed. The 420- and 39-kD polypeptides appear specific for the forms of alpha 2M activated by reaction with proteinases or methylamine and do not bind to an affinity matrix consisting of native alpha 2M coupled to Sepharose. Separation of these two polypeptides was accomplished by anion exchange chromatography, and binding activity was exclusively associated with the 420-kD polypeptide. The purified 420-kD protein binds to the conformationally altered forms of alpha 2M that are known to specifically interact with alpha 2M receptors and does not bind to native alpha 2M. Binding of the 420-kD polypeptide to immobilized wheat germ agglutinin indicates that this polypeptide is a glycoprotein. The cell surface localization of the 420-kD glycoprotein was confirmed by affinity chromatography of extracts from surface radioiodinated fibroblasts. These properties suggest that the 420-kD polypeptide is a cell surface receptor for the activated forms of alpha 2M.     

Subunits of human alpha 2-macroglobulin produced by specific reduction of interchain disulfide bonds with thioredoxin

Disulfide bonds in alpha 2-macroglobulin (alpha 2M) were reduced with the thioredoxin system from Escherichia coli. Under the conditions selected, 3.5-4.1 disulfide bonds were cleaved in each alpha 2M molecule, as determined by the consumption of NADPH during the reaction and by the incorporation of iodo[3H]acetate into the reaction product. This extent of disulfide bond reduction, approximately corresponding to that expected from specific cleavage of all four interchain disulfide bonds of the protein, coincided with the nearly complete dissociation of the intact alpha 2M molecule to a species migrating as an alpha 2M subunit in gel electrophoresis, under both denaturing and nondenaturing conditions. The dissociation was accompanied by only small changes of the spectroscopic properties of the subunits, which thus retain a near-native conformation. Reaction of isolated subunits with methylamine or trypsin led to the appearance of approximately 0.55 mol of thiol group/mol of subunits, indicating that the thio ester bonds are largely intact. Moreover, the rate of cleavage of these bonds by methylamine was similar to that in the whole alpha 2M molecule. Although the bait region was specifically cleaved by nonstoichiometric amounts of trypsin, the isolated subunits had minimal proteinase binding ability. Reaction of subunits with methylamine or trypsin produced changes of farultraviolet circular dichroism and near-ultraviolet absorption similar to those induced in the whole alpha 2M molecule, although in contrast with whole alpha 2M no fluorescence change was observed. The methylamine- or trypsin-treated subunits reassociated to a tetrameric species, migrating as the "fast" form of whole alpha 2M in gradient gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Image processing of proteinase- and methylamine-transformed human alpha 2-macroglobulin. Localization of the proteinases

Comparative x-ray scattering experiments and electron microscopic observations have been performed on native S-form, and on different F-forms of human plasma alpha 2-macroglobulin (alpha 2M), obtained by proteinase (chymotrypsin, plasmin, and thrombin) or methylamine treatment. Image processing of electron micrographs of the alpha 2M molecules transformed by chymotrypsin, plasmin, and methylamine displayed average images which could be compared. The proteinase-complex alpha 2M molecules exhibited the usual H-like structure, but the methylamine-inactivated ones showed a different organization, with almost no stain-excluding material in the central region of the molecule, which therefore presented a central cavity filled with stain. By subtracting average images of alpha 2M-methylamine from alpha 2M-chymotrypsin or alpha 2M-plasmin, a putative localization of the proteinases inside the alpha 2M molecule, very close to its center was revealed. The values of the radii of gyration for the S- and F-forms obtained by x-ray scattering were very different (78 and 67.7 A, respectively). All four scattering curves of the F-forms were comparable in shape and showed maxima and minima different from that of the S-form alpha 2M. Image processing of electron micrographs and x-ray scattering have provided independent results which indicate that a large cavity exists in the alpha 2M-methylamine molecule and that the proteinases might be located in a very central position inside the alpha 2M-proteinase molecules.     

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.     

Relation of internal thioesters to conformational change and receptor-recognition site in alpha 2-macroglobulin complexes.

The unique steric type of inhibition of endopeptidases by human alpha 2-macroglobulin (alpha 2-M) and the inactivation of the latter by methylamine were examined in relation to the internal thioesters in alpha 2M. The present results confirm our previous findings that disruption of the internal thioesters, is not in itself sufficient to cause the conformational change of alpha 2M typical of alpha 2-M-proteinase complexes; the electrophoretically slow form of alpha 2M with [14C]methylamine incorporated was isolated. Moreover, this group is stabilized by derivatization of the exposed cysteine thiol groups. Cyanylation with 2,4-dinitrophenyl thiocyanate during the methylamine reaction was the most effective procedure, yielding essentially only slow-form alpha 2M. Other thiol-specific reagents were less effective. When allowed to react with trypsin the cyanylated derivative (slow-form alpha 2M with thioesters broken) produced anomalous complexes; only half the expected amount of trypsin was bound, whereas the complexes were fully inhibited by soya-bean trypsin inhibitor and were proteolytically active. Despite this, the anomalous complexes were recognized by two highly specific probes: the fibroblast alpha 2M-complex receptor and the monoclonal antibody (F2B2) directed against the receptor-recognition site on alpha 2M complexes. The results show that the internal thioesters in alpha 2M are necessary for the conformational change producing sterically inhibited endoproteinase complexes, but do not participate as such in receptor-mediated endocytosis of these complexes.     

Identification of alpha 2-macroglobulin as a cytokine binding plasma protein. Binding of interleukin-1 beta to "F" alpha 2-macroglobulin

Treatment of normal human plasma with methylamine resulted in the discovery of an interleukin-1 beta(IL-1 beta) binding protein. The protein was labeled with 125I-IL-1 beta and the relative molecular mass (Mr) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein-IL-1 beta complex had a Mr of approximately 400,000 in non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis but became dissociated when exposed to beta-mercaptoethanol. The 125I-IL-1 beta labeled protein complex could be immunoprecipitated from plasma by using an anti-alpha 2-macroglobulin (alpha 2M) antiserum. Similarly, a monoclonal antibody (mAb) specific for electrophoretically fast ("F")alpha 2M was able to adsorb the 125I-IL-1 beta labeled complex from plasma. The mAb was also capable of adsorbing "F" alpha 2M-125I-IL-1 beta complexes from binary reaction mixtures, but failed to adsorb free 125I-IL-1 beta. Experiments carried out with purified plasma alpha 2M established that IL-1 beta became bound to alpha 2M only upon reaction with trypsin or methylamine, which results in the appearance of free thiol groups in alpha 2M ("F" alpha 2M). There was no binding of IL-1 beta to the native form of alpha 2M (electrophoretically slow or "S" alpha 2M), which lacks free thiol groups. Pretreatment of "F" alpha 2M with N-ethylmaleimide or [ethylenebis(oxyethylenenitrilo)] tetraacetic acid prevented complex formation between "F" alpha 2M and IL-1 beta. In contrast, the yield of "F" alpha 2M IL-1 beta complex formation was increased severalfold in the presence of 2.5 mM Zn2+. These findings indicate that "F" alpha 2M interacts with IL-1 beta through a thiol-disulfide exchange reaction. Zn2+ may play a major role in bringing together the reactive domains of the adjoining peptide backbones into proper orientation. The ready complex formation between "F" alpha 2M and the pleiotropic cytokine IL-1 beta suggests a novel biological role for "F" alpha 2M, since "F" alpha 2M-IL-1 beta complexes, but not "F" alpha 2M alone, retained IL-1-like activity in the thymocyte costimulator bioassay.     

Proximity of thiol esters and bait region in human alpha 2-macroglobulin: paramagnetic mapping

The two key structural features of alpha 2-macroglobulin (alpha 2M) involved in inhibitory caging of proteases are the thiol ester and the bait region. This paper examines the environment of the hydrolyzed thiol ester in methylamine-treated human alpha 2M and the separation between the bait region and the thiol ester and between the four thiol esters in the tetramer to try to further our understanding of how bait region proteolysis triggers thiol ester cleavage. The sulfhydryl groups of Cys-949, formed upon cleavage of the thiol ester by methylamine, were specifically labeled with the nitroxide spin-labels 3-(2-iodoacetamido)-PROXYL (iodo-I) (PROXYL = 2,2,5,5-tetramethylpyrrolidine-1-oxyl), 3-[2-(2-iodoacetamido)acetamido]-PROXYL (iodo-II), and 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidine-1-oxyl (iodo-III). ESR spectra of these alpha 2M derivatives showed that label I is firmly held and label II has limited freedom of rotation consistent with location of the cysteine residue in a narrow cavity. Label III has much greater motional freedom. From the absence of dipole-dipole splittings in the ESR spectra, it is concluded that the four nitroxide groups in the tetramer are more than 20 A apart for both label I and label II. Label I broadens 1H NMR signals from one phenylalanyl, one tyrosyl, and four histidyl residues in the bait region. Separations of 11-17 A are estimated between the nitroxide of label I and these residues. Label II is further away and only broadens resonances from one of the histidines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of insulin incorporation into alpha 2-macroglobulin: implications for the study of peptide and growth factor binding.

In recent years, many studies have suggested a direct role for alpha 2-macroglobulin (alpha 2M), a plasma proteinase inhibitor, in growth factor regulation. When coincubated in the presence of either trypsin, pancreatic elastase, human neutrophil elastase, or plasmin, 125I-insulin rapidly formed a complex with alpha 2M which was greater than 80% covalent. The covalent binding was stable to reduction but abolished by competition with beta-aminopropionitrile. Neither native alpha 2M nor alpha 2M pretreated with proteinase or methylamine incorporated 125I-insulin. Experiments utilizing alpha 2M cross-linked with cis-dichlorodiammineplatinum(II) indicated that 125I-insulin must be present during alpha 2M conformational change to covalently bind. A maximum stoichiometry of 4 mol of insulin bound per mole of alpha 2M and the short half-life of the alpha 2M intermediate capable of covalent incorporation were consistent with thiol ester involvement. Protein sequence analysis of unlabeled insulin-alpha 2M complexes, together with results of beta-aminopropionitrile competition, confirmed that insulin incorporation occurs via the same gamma-glutamyl amide linkage responsible for covalent proteinase and methylamine binding to alpha 2M. Although intact insulin apparently incorporated through its sole lysine residue on the B chain, we found that isolated A chain also bound covalently to alpha 2M. Phenyl isothiocyanate derivatization of the N-terminus had no effect on A-chain binding, supporting the possibility of heretofore unreported gamma-glutamyl ester linkages to alpha 2M.     

Preparation and initial characterization of an intermediate, half-cleaved form of human alpha 2-macroglobulin

A form of human alpha 2-macroglobulin (alpha 2M) has been prepared that has properties intermediate to those of native alpha 2-macroglobulin and 2:1 protease-alpha 2 M ternary complex by using Sepharose-linked chymotrypsin. The intermediate form has mobility on native polyacrylamide gels between the fast and slow forms of alpha 2M and migrates as a diffuse band. Two bait regions and two thiol esters per alpha 2M tetramer are cleaved, although no chymotrypsin is detectable in the modified alpha 2-macroglobulin species. The remaining bait regions and thiol esters can be cleaved by further reaction with other proteases. Intermediate-form alpha 2M can trap 1.18 mol of chymotrypsin, 0.85 mol of trypsin, and 0.65 mol of thrombin. Although both thrombin and methylamine react with intermediate-form alpha 2M at rates not distinguishable within experimental error from those of their reactions with native alpha 2M, chymotrypsin-Sepharose reacts much more slowly with the intermediate form than with native alpha 2 M, indicating a nonequivalence of the two reactive sites on alpha 2M. This nonequivalence may be present initially or be induced by reaction at the first site. Comparison of ESR results obtained from spin-labeling methylamine-treated or protease-reacted alpha 2M with those from spin-labeling of the free SH groups in intermediate-form alpha 2M shows that trapped protease influences the mobility of the attached nitroxide either through direct contact or by producing a different conformation from that present in methylamine-treated or intermediate-form alpha 2M.