Alpha-macroglobulin from Limulus polyphemus exhibits proteinase inhibitory activity and participates in a hemolytic system.

Significant primary sequence homology between the alpha-macroglobulin family of proteinase inhibitors and the complement components C3, C4, and C5 implies that these proteins arose from a common ancestor. Hemolymph from the ancient invertebrate Limulus polyphemus contains both complement-like and proteinase inhibitory activity. In this report, we present evidence that L. polyphemus alpha-macroglobulin not only possesses proteinase inhibitory activity, but it also participates in the lytic system of the horseshoe crab. The protein is a disulfide-linked dimer of subunits of molecular mass 185 kDa. Upon reaction with proteinase or methylamine, L. polyphemus alpha-macroglobulin underwent a major conformational change and no proteinase-associated multimerization was detected. L. polyphemus alpha-macroglobulin is the only detectable inhibitor of a number of proteinases in L. polyphemus hemolymph. Proteinase inhibition follows the general "trapping" mechanism shared by most alpha-macroglobulins; however, no covalent linking of proteinases to the inhibitor was detected despite the presence of a functional thiolester. Moreover, the inhibitor demonstrated thiolester-mediated binding to sheep erythrocytes, a property also observed with complement components such as C3. Depletion of functional protein by treatment of hemolymph with methylamine destroyed the proteinase inhibitory capacity and the lytic activity of the hemolymph. Both activities were restored by adding purified protein to depleted hemolymph. Studies with purified L. polyphemus alpha-macroglobulin demonstrated that the thiolester incorporates glycerol as well as methylamine, a property shared by human C3. The data support the hypothesis that L. polyphemus alpha-macroglobulin is both a proteinase inhibitor and part of a lytic system, providing a link between the two distinct sides of the alpha-macroglobulin family. Because both properties are contained in one molecule, we propose the name "limac" to describe this Limulus alpha-macroglobulin complement-like protein.     

Purification and characterization of frog alpha-macroglobulin: receptor recognition of an amphibian glycoprotein

Frog alpha-macroglobulin was purified to apparent homogeneity by Ni2+ chelate affinity chromatography. Frog alpha-macroglobulin migrated as an alpha 1-globulin in cellulose acetate electrophoresis. A molecular weight of 730 000 was obtained by equilibrium sedimentation, and in sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), the protein migrated as a single band of Mr approximately 360 000 before reduction and Mr approximately 180 000 after reduction. Treatment with trypsin resulted in subunit cleavage to yield a fragment of Mr approximately 90 000. After being heated, the protein fragmented, migrating in SDS-PAGE as two bands of Mr approximately 120 000 and 60 000. This fragmentation was inhibited by prior reaction of the protein with methylamine. In native pore-limit electrophoresis the protein exhibited the characteristic "slow" to "fast" conformational change of protease-treated alpha-macroglobulins. In contrast, typical "slow" to "fast" conformational change was not observed in native PAGE with this preparation. Moreover, the protein incorporated approximately 2 mol of [14C]methylamine/mol of inhibitor without demonstrating a change in mobility in native PAGE. In circular dichroism studies, the protein exhibited a spectrum similar to that of human alpha 2M. Reaction with trypsin resulted in a broadening and decrease in the magnitude of the spectrum. Reaction with methylamine resulted in similar changes, but of smaller magnitude. The inhibitor bound approximately 0.7 mol of trypsin in both radiolabeled protease binding and amidolytic titration studies. 125I-Labeled native frog alpha 1M was removed slowly from the circulation of mice with a t1/2 greater than 2h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circular dichroic spectroscopy of non-human alpha-macroglobulins

Bovine, chicken and frog alpha-macroglobulins and ovomacroglobulin were studied by circular dichroic spectroscopy over the region 205-250 nm. All four spectra exhibited negative ellipticity with minima at about 215 nm similar to that reported for human alpha 2-macroglobulin. On reaction of the alpha-macroglobulins with trypsin, the spectrum of each of the four changed similarly. However, these proteins exhibited different conformational changes when treated with methylamine. These differences were exploited to determine which characteristics of alpha-macroglobulins correlate with changes in circular dichroic spectroscopy.     

Isolation and characterization of an alpha-macroglobulin from the gastropod mollusc Helix pomatia with tetrameric structure and preserved activity after methylamine treatment

A proteinase inhibitor with M(r) 697000 and 20.3% (w/w) carbohydrate was isolated from the haemolymph of the snail Helix pomatia and characterized. It was shown to have a tetrameric structure with subunits disulphide linked by two. It inhibited the activity of several types of proteinases against large substrates but not that of trypsin against N-alpha-benzoyl-DL-arginine-4-nitroanilide. This indicated a nonspecific and steric hindrance mode of inhibition. The ratio of trypsin molecules inactivated per inhibitor amounted to 1.5. This interaction led to a cleavage of the subunits into two equal fragments and to a slow to fast conformational change of the whole molecules. Experiments with 125I-labelled trypsin indicated that the proteinase had become covalently linked to one of the fragments. Heating of the inhibitor led to autolytic cleavage products but not when methylamine treated. Thiol titration after trypsin or methylamine treatment indicated the presence of one thiol ester bond per subunit. These facts are all indicative of an alpha-macroglobulin type of inhibitor. However, unlike for most of them the methylamine treatment did not induce a conformational change nor suppress its proteinase inhibitory activity. Moreover, invertebrate alpha-macroglobulins are mostly dimeric in structure but tetramers likewise do occur in Biomphalaria glabrata.     

Physical properties of human alpha 2-macroglobulin following reaction with methylamine and trypsin

Circular dichroism spectroscopy, sedimentation velocity and ultraviolet difference spectroscopy were used to compare alpha 2-macroglobulin, alpha 2-macroglobulin-trypsin complex and alpha 2-macroglobulin-methylamine complex. The circular dichroic spectrum of native alpha 2-macroglobulin is significantly changed in shape and magnitude following reaction with either trypsin or methylamine. The spectra of alpha 2-macroglobulin-trypsin and alpha 2-macroglobulin-methylamine are, however, indistinguishable. The ultraviolet difference spectrum between alpha 2-macroglobulin-methylamine and native alpha 2-macroglobulin displays a tyrosine blue shift consistent with the exposure of several tyrosine residues to solvent. The conformational change which occurs in alpha 2-macroglobulin during reaction with methylamine follows pseudo-first-order kinetics. T 1/2 was 10.5 min for the reaction with 200 mM methylamine at pH 8.0 and 45 min for the reaction with 50 mM methylamine, also at pH 8.0. Reaction of methylamine with alpha 2-macroglobulin results in loss of trypsin-binding activity which appears to be a direct consequence of the conformational change induced by methylamine. A sedimentation coefficient (S0(20),W) of 20.5 was determined for alpha 2-macroglobulin-methylamine compared to a value of 18.5 for unreacted alpha 2-macroglobulin. This increase in sedimentation velocity is attributed to a 10% decrease in alpha 2-macroglobulin Stokes radius. alpha 2-Macroglobulin-trypsin complex prepared by reaction of the protease at a 2-fold molar excess with the inhibitor was a S0(20),W of 20.3. Although this sedimentation coefficient does reflect compacting of the alpha 2-macroglobulin structure compared to native alpha 2-macroglobulin, it is not large enough to rule out significant protrusion of the proteases from pockets in the alpha 2-macroglobulin structure.     

Murinoglobulin, a novel protease inhibitor from murine plasma. Isolation, characterization, and comparison with murine alpha-macroglobulin and human alpha-2-macroglobulin

Two glycoproteins having trypsin-protein esterase activity were purified to apparent homogeneity from murine plasma. One was alpha-macroglobulin, a homologue of human alpha-2-macroglobulin, while the other, tentatively named murinoglobulin, did not correspond to any of the known plasma protease inhibitors that have been well characterized in men or other mammals. Murinoglobulin contained about 7.6% carbohydrate and was composed of a single-polypeptide chain of Mr = 180,000 as judged by the equilibrium sedimentation analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Murinoglobulin did not cross-react immunologically with mouse alpha-macroglobulin nor with human alpha-2-macroglobulin. Protease-inhibiting properties of murinoglobulin were compared with those of mouse alpha-macroglobulin and human alpha-2-macroglobulin. All the three proteins inhibited trypsin, papain, and thermolysin, although they differed considerably in both the degree of inhibition and the binding stoichiometry of protease-inhibitor complexes. The two macroglobulins inhibited pepsin at pH 5.5, whereas murinoglobulin was inactivated at this pH. Murinoglobulin was more sensitive to methylamine than the two macroglobulins. No protein corresponding to murinoglobulin was detected in human plasma.     

Comparison of the binding of chicken alpha-macroglobulin and ovomacroglobulin to the mammalian alpha 2-macroglobulin receptor

Chicken alpha-macroglobulin (alpha M) and ovomacroglobulin were purified by Ni+2 chelate chromatography. These proteins had similar subunit structure as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chicken alpha M bound 1.0 mol and ovomacroglobulin bound 0.8 mol 125I-trypsin per mol inhibitor, respectively. Ovomacroglobulin cleared rapidly from the circulation of mice, and the clearance was inhibited by asialoorosomucoid, but native chicken alpha M cleared slowly (t 1/2 greater than 1 h). After reaction with trypsin, this alpha-macroglobulin cleared rapidly (t 1/2 = 3 min), and this clearance was inhibited by a 1000-fold molar excess of human alpha 2M-methylamine. Ovomacroglobulin-trypsin did not inhibit the binding of 0.2 nM 125I-labeled human alpha 2M-methylamine to mouse peritoneal macrophages in vitro, but chicken alpha M reacted with trypsin inhibited the binding by 50% at 1.9 nM. A kappa I of 1.1 nM was calculated for the binding of chicken alpha M-trypsin to the mammalian alpha-macroglobulin receptor. This affinity is comparable to that obtained with human and bovine alpha 2M.     

Isolation and characterization of alpha-macroglobulin from guinea pig plasma

Guinea pig alpha-macroglobulin was purified to apparent homogeneity by sequential chromatography on Sephacryl S-300, DEAE-cellulose, and hydroxyapatite. A molecular weight of 780,000 was obtained by equilibrium sedimentation. The preparation migrated as a single band of Mr = 180,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Rabbit antiserum raised against the final preparation partially cross-reacted with human and rat alpha-2-macroglobulins but not with rat alpha-1-macroglobulin. Guinea pig alpha-macroglobulin stimulated the amidolytic activity of trypsin towards a small substrate, but inhibited the proteolytic activity of trypsin towards remazol brilliant blue hide powder. When treated with trypsin or methylamine, four thiol groups per molecule were newly generated. The reaction with trypsin proceeded with at least at two different rates: half of the thiol groups were generated in a fast reaction and the remaining half in a slower reaction. On the other hand, such a two-step reaction was not detected in the reaction with methylamine. The methylamine-treated alpha-macroglobulin retained half the capacity to bind trypsin and its mobility in polyacrylamide gel under nondenaturing conditions remained virtually unchanged. These properties are in marked contrast to those reported for human alpha-2-macroglobulin, but resemble those of rat alpha-2- and mouse alpha-macroglobulins. The amidase activity of trypsin bound to guinea pig alpha-macroglobulin was impaired by soybean trypsin inhibitor to a much greater degree than that of trypsin bound to human or rat alpha-2-macroglobulin.     

Nucleophilic scission of thioester linkages and conformational changes in human plasma low density lipoproteins.

A comparison between conformations of native and methylamine modified human plasma low density lipoproteins (hydrated density 1.032-1.043 g/ml) has been presented. Near UV circular dichroism and difference absorption spectra of modified low density lipoprotein have suggested substantial differences in the local environments of several aromatic amino acid side chains. Relatively lower ellipticity at 222 nm of modified lipoprotein indicated alterations in the secondary structures of its protein moiety. Nucleophilic reaction of methylamine did not cause the peptide bond scission but it brought conformational transition such that some of the buried hydrophobic domains of the protein moiety got exposed to the aqueous environment.     

Unusual properties of crocodilian ovomacroglobulin shown in its methylamine treatment and sulfhydryl titration

The inhibitory activity of chicken and crocodilian ovomacroglobulins against trypsin was measured before and after their incubation with methylamine. The result for crocodilian ovomacroglobulin showed that methylamine treatment destroyed half of its activity, in unique contrast to human alpha 2-macroglobulin and chicken ovomacroglobulin for which methylamine either destroys the inhibitory activity of the former completely or does not affect that of the latter at all. Free sulfhydryl groups of chicken and crocodilian ovomacroglobulins were titrated with 5,5'-dithiobis(2-nitrobenzoic acid) before and after incubation with trypsin. Prior to the incubation with trypsin the chicken and crocodilian proteins respectively had 0 and 1 titratable sulfhydryl per molecule of Mr 720,000. After treatment with trypsin the crocodilian protein had 3.5-4 titratable sulfhydryls, whereas there were no titratable sulfhydryls in the chicken protein. After denaturation of the crocodilian protein in sodium dodecyl sulfate at 100 degrees C the number of titratable sulfhydryls was 4. Chicken ovomacroglobulin again did not have an appreciable number of titratable sulfhydryls under similar denaturing conditions. Incubation of crocodilian protein with [14C]methylamine showed an incorporation of at least 2 mol of methylamine per molecule. The result indicated the presence of three intramolecular thiol ester bonds in crocodilian ovomacroglobulin with differential stability against external perturbations.     

Proteinase binding and inhibition by the monomeric alpha-macroglobulin rat alpha 1-inhibitor-3

The inhibitory capacity of the alpha-macroglobulins resides in their ability to entrap proteinase molecules and thereby hinder the access of high molecular weight substrates to the proteinase active site. This ability is thought to require at least two alpha-macroglobulin subunits, yet the monomeric alpha-macroglobulin rat alpha 1-inhibitor-3 (alpha 1I3) also inhibits proteinases. We have compared the inhibitory activity of alpha 1I3 with the tetrameric human homolog alpha 2-macroglobulin (alpha 2M), the best known alpha-macroglobulin, in order to determine whether these inhibitors share a common mechanism. alpha 1I3, like human alpha 2M, prevented a wide variety of proteinases from hydrolyzing a high molecular weight substrate but allowed hydrolysis of small substrates. In contrast to human alpha 2M, however, the binding and inhibition of proteinases was dependent on the ability of alpha 1I3 to form covalent cross-links to proteinase lysine residues. Low concentrations of proteinase caused a small amount of dimerization of alpha 1I3, but no difference in inhibition or receptor binding was detected between purified dimers or monomers. Kininogen domains of 22 and 64 kDa were allowed to react with alpha 1I3- or alpha 2M-bound papain to probe the accessibility of the active site of this proteinase. alpha 2M-bound papain was completely protected from reaction with these domains, whereas alpha 1I3-bound papain reacted with them but with affinities several times weaker than uncomplexed papain. Cathepsin G and papain antisera reacted very poorly with the enzymes when they were bound by alpha 1I3, but the protection provided by human alpha 2M was slightly better than the protection offered by the monomeric rat alpha 1I3. Our data indicate that the inhibitory unit of alpha 1I3 is a monomer and that this protein, like the multimeric alpha-macroglobulins, inhibits proteinases by steric hindrance. However, binding of proteinases by alpha 1I3 is dependent on covalent crosslinks, and bound proteinases are more accessible, and therefore less well inhibited, than when bound by the tetrameric homolog alpha 2M. Oligomerization of alpha-macroglobulin subunits during the evolution of this protein family has seemingly resulted in a more efficient inhibitor, and we speculate that alpha 1I3 is analogous to an evolutionary precursor of the tetrameric members of the family exemplified by human alpha 2M.     

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.     

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].     

The detection of streptococcal cell-wall proteins that form complexes with human macroglobulins]

The composition of the extracts of the cultures of individual streptococcal strains, studied by immunoblotting techniques, has been shown to contain proteins with a molecular weight of 70-80 KD. These proteins have pronounced affinity to human macroglobulins: alpha-macroglobulin, alpha-glycoprotein associated with pregnancy and protein A. The significance of this phenomenon on the cellular and somatic levels is discussed.     

Structure of a rat alpha 1-macroglobulin receptor-binding domain dimer

Alpha-macroglobulin inhibits a broad spectrum of proteinases by forming macromolecular cages inside which proteinases are cross-linked and trapped. Upon formation of a complex with proteinase, alpha-macroglobulin undergoes a large conformational change that results in the exposure of its receptor-binding domain (RBD). Engagement of this domain by alpha-macroglobulin receptor permits clearance of the alpha-macroglobulin: proteinase complex from circulation. The crystal structure of rat alpha1-macroglobulin RBD has been determined at 2.3 A resolution. The RBD is composed of a nine-stranded beta-sandwich and a single alpha-helix that has been implicated as part of the receptor binding site and that lies on the surface of the beta-sandwich. The crystallographic asymmetric unit contains a dimer of RBDs related by approximate twofold symmetry such that the putative receptor recognition sites of the two monomers are contiguous. By gel filtration and ultracentrifugation, it is shown that RBD dimers form in solution with a dissociation constant of approximately 50 microM. The structure of the RBD dimer might mimic a conformation of transformed alpha-macroglobulin in which the proposed receptor binding residues are exposed on one face of the dimer. A pair of phenylalanine residues replaces a cystine that is conserved in other members of the macroglobulin family. These residues participate in a network of aromatic side-chain interactions that appears to stabilize the dimer interface.     

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 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.     

Internal structure of ovomacroglobulin studied by electron microscopy.

As a model for the molecular structure of proteins belonging to the alpha 2-macroglobulin family, ovomacroglobulin of reptilian origin was studied by electron microscopy in the original tetrameric form as well as in the dissociated forms into half- and quarter molecules. The following aspects of the molecular internal structure which had previously not been known for the homologous human alpha 2-macroglobulin or chicken ovomacroglobulin were revealed. First, the negatively stained tetrameric native protein gave an appearance of a collection of four semi-circular strings placed on the four corners of a molecule. They were connected to each other in the center of a molecule through a set of globular domains which formed a cross-figured subunit contact region. Second, two kinds of active half-molecules prepared either by the reduction of intersubunit disulfide bonds or by the disruption of noncovalent subunit interface had similarly elongated forms having semi-circular units on the two ends, indicating quasi-equivalent subunit arrangement in the two kinds of half-molecules. We thus concluded that the structure of native ovomacroglobulin can be represented by four circular strings each equipped with an extra domain to form the central intersubunit contact region. The results may also be adapted to the internal structure of human alpha 2-macroglobulin because it was sometimes possible to observe similar ring-like internal structure in the human protein.     

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.     

Purification and characterization of a "half-molecule" alpha 2-macroglobulin from the southern grass frog: absence of binding to the mammalian alpha 2-macroglobulin receptor

An alpha-macroglobulin (alpha 2M), which is a dimer consisting of two non-disulfide-bonded subunits, was identified and purified from frog plasma by Ni2+ chelate affinity chromatography. This frog "half-molecule" alpha-macroglobulin migrated as an alpha 2-globulin in cellulose-acetate electrophoresis rather than as the previously described frog alpha 1M, which exists as a tetramer formed by the noncovalent association of disulfide-bonded pairs. A molecular weight of approximately 380 000 was obtained by gel-filtration high-pressure liquid chromatography, and in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the protein migrated as a single band of Mr approximately 180 000 before and after reduction. No evidence was obtained for association of this protein to a higher molecular weight species. After the preparation was heated, additional bands were obtained in SDS-PAGE with Mr approximately 60 000 and 12 000. The additional bands were not obtained after heating methylamine-treated preparations. The circular dichroic spectrum of frog alpha 2M exhibits negative ellipticity over the region 205-250 nm with a minimum at 216 nm. After reaction with proteinase, a decrease in the absolute mean residue rotation was obtained. Amino acid analysis demonstrated that frog alpha 2M and alpha 1M are similar in composition to avian and mammalian alpha-macroglobulins; however, there are sufficient differences in the composition of these two amphibian alpha-macroglobulins to support the conclusion that they are distinct proteins. Frog alpha 2M bound approximately 0.5 mol of trypsin/mol of inhibitor. This binding was abolished by pretreatment with methylamine.(ABSTRACT TRUNCATED AT 250 WORDS)