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 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].     

Functional modifications of α2-macroglobulin by primary amines. Kinetics of inactivation of α2-macroglobulin by methylamine, and formation of anomalous complexes with trypsin

The unique steric inhibition of endopeptidases by human alpha(2)M (alpha(2)-macroglobulin) and the inactivation of the latter by methylamine were examined in relation to each other. Progressive binding of trypsin by alpha(2)M was closely correlated with the loss of the methylamine-reactive sites in alpha(2)M: for each trypsin molecule bound, two such sites were inactivated. The results further showed that, even at low proteinase/alpha(2)M ratios, no unaccounted loss of trypsin-binding capacity occurred. As alpha(2)M is bivalent for trypsin binding and no trypsin bound to electrophoretic slow-form alpha(2)M was observed, this indicates that the two sites must react (bind trypsin) in rapid succession. Reaction of [(14)C]methylamine with alpha(2)M was biphasic in time; in the initial rapid phase complex-formation with trypsin caused a largely increased incorporation of methylamine. In the subsequent slow phase trypsin had no such effect. These results prompted further studies on the kinetics of methylamine inactivation of alpha(2)M with time of methylamine treatment. It was found that conformational change of alpha(2)M and decrease in trypsin binding (activity resistant to soya-bean trypsin inhibitor) showed different kinetics. The latter decreased rapidly, following pseudo-first-order kinetics. Conformational change was much slower and followed complex kinetics. On the other hand, binding of (125)I-labelled trypsin to alpha(2)M did follow the same kinetics as the conformational change. This discrepancy between total binding ((125)I radioactivity) and trypsin-inhibitor-resistant binding of trypsin indicated formation of anomalous complexes, in which trypsin could still be inhibited by soya-bean trypsin inhibitor. Further examination confirmed that these complexes were proteolytically active towards haemoglobin and bound (125)I-labelled soya-bean trypsin inhibitor to the active site of trypsin. The inhibition by soya-bean trypsin inhibitor was slowed down as compared with reaction with free trypsin. The results are discussed in relation to the subunit structure of alpha(2)M and to the mechanism of formation of the complex.     

Evolution of α2-macroglobulin. The structure of a protein homologous with human α2-macroglobulin from plaice (Pleuronectes platessa L.) plasma

The plaice (Pleuronectes platessa L.) papain-binding protein previously demonstrated to be homologous with human alpha(2)-macroglobulin, and designated plaice alpha(2)-macroglobulin homologue or alphaMh, was shown to be a glycoprotein of s(20,w) 11.86S. In polyacrylamide-gel pore-limit electrophoresis under non-denaturing conditions plaice alphaMh migrated to the same position as half-molecules of human alpha(2)-macroglobulin, and treatment with methylamine or a proteinase caused no change in its electrophoretic properties. Either denaturation in urea (4m) or mild reduction by dithiothreitol (1mm) partially dissociated plaice alphaMh into half-molecules. Denaturation with reduction further dissociated the protein into quarter-subunits. In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reducing conditions plaice alphaMh dissociated into subunits of M(r) 105000 (I) and 90000 (II). Approximately equal amounts of each subunit were formed, and peptide ;mapping' showed subunits I and II to be distinct polypeptide chains. Under alkaline denaturing conditions, a proportion of the I chains of alphaMh were cleaved into fragments of M(r) about 60000 and 40000. This cleavage was favoured by reducing conditions and prevented by prior inactivation of the alphaMh with methylamine. [(14)C]Methylamine allowed to react with alphaMh became covalently linked to subunit I. These properties suggested the existence of an autolytic site on subunit I analogous to the autolytic site of human alpha(2)-macroglobulin. Reaction of alphaMh with a proteinase resulted in cleavage of a fragment of M(r) 10000-15000 from subunit I. A proportion of the proteinase molecules trapped by alphaMh became covalently linked to the inhibitor. A scheme is proposed for the evolution of human alpha(2)-macroglobulin and plaice alphaMh from a common ancestral protein, which may also have been an ancestor of complement components C3 and C4.     

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)     

Characterization of human alpha 2-macroglobulin monomers obtained by reduction with dithiothreitol.

We compared the physicochemical characteristics of alpha 2-macroglobulin (alpha 2M) monomers produced by limited reduction and carboxamidomethylation to those of the naturally occurring monomeric alpha-macroglobulin homologue rat alpha 1-inhibitor 3 (alpha 1 I3). Unlike alpha 1 I3, alpha 2 M monomers fail to inhibit proteolysis of the high molecular weight substrate hide powder azure by trypsin. In contrast to alpha 1 I3, which remains monomeric after reacting with proteinase, alpha 2 M monomers reassociate to higher molecular weight species (dimers, trimers, and tetramers) after reacting with proteinase. Reaction of alpha 2 M monomers at molar ratios of proteinase to alpha 2M monomers as low as 0.3:1 leads to extensive reassociation and is accompanied by complete bait-region and thiolester bond cleavage. During the reaction of alpha 2M monomers with proteinases, the proteinase binds to the reassociating alpha 2M subunits but is not inhibited. Of significance, all the bound proteinase was covalently linked to the reassociated alpha 2M species. Treatment of alpha 2M monomers with methylamine results in thiolester bond cleavage but minimal reassociation. Treatment of alpha 2M monomers with methylamine followed by proteinase results in complete bait-region cleavage and is accompanied by marked reassociation of alpha 2M monomers to higher molecular weight species. However, no proteinase is associated with these higher molecular weight forms. We infer that bait-region cleavage is more important than thiolester bond cleavage in driving alpha 2M monomers to reassociate. Despite many similarities between alpha 1I3 and alpha 2M monomers, significant differences must exist with respect to proteinase orientation within the inhibitor to account for the failure of alpha 2M monomers to protect large molecular weight substrates from proteolysis by bound proteinase, in contrast to the naturally occurring monomeric homologue rat alpha 1 I3.     

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)     

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.     

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)     

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)     

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.     

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)     

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)     

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.     

Proteolysis of the 85-kilodalton crystalline cysteine proteinase inhibitor from potato releases functional cystatin domains.

The protein crystals found in potato (Solanum tuberosum L.) tuber cells consist of a single 85-kD polypeptide. This polypeptide is an inhibitor of papain and other cysteine proteinases and is capable of binding several proteinase molecules simultaneously (P. Rodis, J.E. Hoff [1984] Plant Physiol 74: 907-911). We have characterized this unusual inhibitor in more detail. Titrations of papain activity with the potato papain inhibitor showed that there are eight papain binding sites per inhibitor molecule. The inhibition constant (Ki) value for papain inhibition was 0.1 nM. Treatment of the inhibitor with trypsin resulted in fragmentation of the 85-kD polypeptide into a 32-kD polypeptide and five 10-kD polypeptides. The 32-kD and 10-kD fragments all retained the ability to potently inhibit papain (Ki values against papain were 0.5 and 0.7 nM, respectively) and the molar stoichiometries of papain binding were 2 to 3:1 and 1:1, respectively. Other nonspecific proteinases such as chymotrypsin, subtilisin Carlsberg, thermolysin, and proteinase K also cleaved the 85-kD inhibitor polypeptide into functional 22-kD and several 10-kD fragments. The fragments obtained by digestion of the potato papain inhibitor with trypsin were purified by reverse-phase high-performance liquid chromatography, and the N-terminal amino acid sequence was obtained for each fragment. Comparison of these sequences showed that the fragments shared a high degree of homology but were not identical. The sequences were homologous to the N termini of members of the cystatin superfamily of cysteine proteinase inhibitors. Therefore, the inhibitor appears to comprise eight tandem cystatin domains linked by preteolytically sensitive junctions. We have called the inhibitor potato multicystatin (PMC). By immunoblot analysis and measurement of papain inhibitory activity, PMC was found at high levels in potato leaves (up to 0.6 microgram/g fresh weight tissue), where it accumulated under conditions that induce the accumulation of other proteinase inhibitors linked to plant defense. PMC may have a similar defensive role, for example in protecting the plant from phytophagous insects that utilize cysteine proteinases for dietary protein digestion.     

A functional, thioester-containing alpha 2-macroglobulin homologue isolated from the hemolymph of the American lobster (Homarus americanus)

An alpha 2-macroglobulin-like protease inhibitor was isolated from the cell-free hemolymph of the american lobster (Homarus americanus) by ion-exchange chromatography and gel filtration. Whereas the undissociated molecule has a molecular weight of 342,000 as determined by ultracentrifugation studies, under reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein has a subunit molecular weight of 180,000. On the basis of this and other evidence, we conclude that the lobster protein is a dimer consisting of two disulfide-bonded monomers. The purified protein inhibits proteolytic enzymes but protects the esterolytic activity of trypsin toward low molecular weight substrates from inactivation by soybean trypsin inhibitor. The methylamine sensitivity of this activity suggests the presence of an internal thioester bond. This was confirmed by the covalent incorporation of [14C]methylamine, by the formation of Mr 55,000 and 125,000 autolytic cleavage fragments in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and, more directly, by the amino acid sequence of a tryptic peptide containing the putative thioester region. Whereas the N-terminal amino acid sequence (22 residues) of the protein revealed an overall identity of only 18% when compared with the human protein, the sequence of the thioester-containing peptide was highly conserved, both with respect to human alpha 2-macroglobulin and to other proteins having a thioester bond. The protein showed the "slow to fast" conformational change typical in alpha 2-macroglobulins in nondenaturing gel electrophoresis after treatment with trypsin, but not after incubation with methylamine.     

Evolution of alpha 2-macroglobulin. The purification and characterization of a protein homologous with human alpha 2-macroglobulin from plaice (Pleuronectes platessa L.) plasma.

A papain-binding protein (PB-protein) was purified to homogeneity from the plasma of plaice (Pleuronectes platessa L.). PB-protein inhibited the activity of trypsin and pancreatic elastase (serine proteinases), thermolysin (a metalloproteinase) and papain (a cysteine proteinase). Presaturation of PB-protein with trypsin prevented the subsequent inhibition of thermolysin, and vice versa. Only catalytically active endopeptidases were bound by PB-protein. The catalytic activity of trypsin bound by PB-protein was inhibited by 95% against an insoluble protein substrate, but only by 38% against a low-molecular-weight synthetic substrate. The remaining activity of the bound trypsin was partially protected against further inhibition by soya-bean trypsin inhibitor. Trypsin bound by PB-protein showed a decrease of 67% in its reactivity with antibodies. The inhibitory activity of PB-protein was inactivated at pH 8.0 by methylamine (0.2M) or dithiothreitol (1 mM). The inhibition of proteinases by plaice PB-protein shows the distinctive characteristics of inhibition by human alpha 2-macroglobulin, and it is concluded that the plaice protein is a homologue of the human macroglobulin.     

Inhibition of Growth of L5178Y Leukemia Cells by a Fungal Folate-deaminating Enzyme

Reactive sites of adzuki bean proteinase inhibitor II were determined by limited hydrolyses with catalytic amounts of trypsin [EC 3.4.21.4] and chymotrypsin [EC 3.4.21.1] at pH 3.0. Treatment of the trypsin-modified inhibitor with carboxypeptidase B [EC 3.4.12.3] released lysine from the inhibitor and led to complete loss of the activity for trypsin, virtually, without affecting the chymotrypsin-inhibitory activity. Limited hydrolysis with chymotrypsin resulted in a selective cleavage of a single tyrosyi peptide bond in the inhibitor, and treatment of this modified inhibitor with carboxypeptidase A [EC 3.4.12.2] abolished the chymotrypsininhibitory activity, having no effect on the trypsin-inhibitory activity. After reduction and S-carboxymethylation, the trypsin- and the chymotrypsin-modified inhibitors both could be separated into two components by gel-filtration on Sephadex G–50 and DEAE-cellulose chromatography. Amino acid and end group analyses of these components indicated that the reactive sites of inhibitor II are the Lys²⁷-Ser²⁸ bond against trypsin and the Tyr⁵⁴-Ser⁵⁵ against chymotrypsin.

Chemical modification of inhibitor II with cyanogen bromide had a fatal effect on the inhibitory activity against trypsin but no effect against chymotrypsin.     

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.     

Inactivation of the plasma protease inhibitor alpha 2-macroglobulin by the antitumor drug cis-dichlorodiamineplatinum(II)

The plasma protease inhibitor alpha 2-macroglobulin (alpha 2M) was reacted in vitro with cis-dichlorodiamineplastinum(II) (cis-DDP). Following the reaction, alpha 2M demonstrated a significantly decreased ability to bind trypsin as determined by esterase activity assays in the presence of soybean trypsin inhibitor and studies with radiolabeled trypsin. Inactivation of alpha 2M by cis-DDP was not associated with a conversion to the "fast" electrophoretic form, as determined on nondenaturing gels, in contrast to the inactivation of alpha 2M by proteases and certain amine salts. The extent of reaction increased with the elevation of temperature within the thermal stability range of the protein; however, variation of pH within the range 6.82-8.55 had little effect. Binding of [14C]methylamine to alpha 2M was not affected by cis-DDP. The conformational change, however, which normally accompanies this reaction did not occur. It is concluded that the alpha 2M thiolesters are most likely not reactive sites for cis-DDP. cis-DDP-treated alpha 2M failed to dissociate into quarter subunits under denaturing and reducing conditions, suggesting cross-linking of subunits. This cross-linking may be responsible for locking the alpha 2M quarternary structure into the "slow conformation."