Covalent and non-covalent interaction of chymotrypsin with alpha 2-macroglobulin

The pattern of covalent crosslinking between human alpha 2-macroglobulin (alpha 2M) and chymotrypsin has been investigated by chromatography and polyacrylamide gel electrophoresis in denaturing medium. Reaction with a single mol of chymotrypsin per mol alpha 2M results in the formation of a 95% covalent 1:1 chymotrypsin-alpha 2M complex and in the proteolytic cleavage of both 180 kDa monomers in one alpha 2M subunit. Proteolytic cleavage in the other alpha 2M subunit requires the presence of a second mol of chymotrypsin; part (20%) of the protease in the 2:1 chymotrypsin-alpha 2M complex thus formed appears to be non-covalently bound to the alpha 2M chains. Covalent binding is abolished when the reaction of alpha 2M with the protease is carried out in the presence of hydroxylamine. A single mol of the protease is then able to cleave all four 180 kDa monomers in alpha 2M.     

Specific fluorescent labeling of alpha 2-macroglobulin-bound proteases: accessibility and localization of their active site within the complex

Pyrenebutylmethylphosphonofluoridate reacts with trypsin and elastase to yield a conjugate with a stoichiometry of one fluorescent label per enzyme molecule as already observed with chymotrypsin. The kinetics of inactivation indicate that the serine active center of the proteases is involved in the labeling reaction. The binding of the proteases to alpha 2-macroglobulin does not modify the specificity of the reaction but drastically diminishes the labeling rate which also depends upon alpha 2-macroglobulin protease binding ratio. Dynamic quenching of the conjugated pyrene moiety by acrylamide, and iodide ions is markedly reduced upon reaction of the protease with alpha 2-macroglobulin, indicating a reduced accessibility of the protease active center in the complex. Singlet--singlet energy transfer measurements from the donor pyrene labeled active center of the proteases to the alpha 2-macroglobulin acceptor labeled thiol groups which are liberated upon protease fixation, gave a rough estimate of the distance (about 25 A) between the active center of the two alpha 2-macroglobulin bound protease molecules.     

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.     

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.     

Platelet alpha2-macroglobulin and alpha1-antitrypsin.

Subcellular membrane and granule fractions derived from human platelets contain immunologically identifiable alpha2-macroglobulin and alpha1-antitrypsin. These platelet-derived inhibitors show a reaction of immunologic identity when compared to alpha2-macroglobulin and alpha1-antitrypsin purified from human plasma. Further, the platelet protease inhibitors possessed a similar subunit polypeptide chain structure to their plasma counterparts as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. Studies of the binding of radiolabeled trypsin to the various solubilized platelet subcellular fractions suggest that the granule-associated alpha2-macroglobulin and alpha1-antitrypsin, as well as membrane-associated alpha2-macroglobulin were functionally active. Quantitatively, circulating platelets contain relatively small concentrations of these inhibitors as compared to platelet-associated fibrinogen and factor VIIIAGN. Platelet protease inhibitors may modulate the protease-mediated events involved in the formation of hemostatic plugs and thrombi.     

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.     

A solid-phase immunosorbent assay to determine the proteinase binding capacity of alpha 2-macroglobulin using 125I-trypsin as indicator proteinase

Hyperimmune sera against human alpha 2 macroglobulin were raised in rabbits following immunization with 's' alpha 2-macroglobulin over half a year. Immunoglobulins were prepared by DEAE-Sephacel anion exchange chromatography. The immunoglobulin preparations showed a remarkably high and equal titer for 's' and 'f' alpha 2-macroglobulin (plasma alpha 2-macroglobulin fully saturated with pig pancreas trypsin), which amounted to 6.4 X 10(-6) as revealed by passive hemagglutination. Immunoimmobilization experiments revealed that at equilibrium, 's' alpha 2-macroglobulin and both 'f' alpha 2-macroglobulins (27 and 82% saturation of 's' alpha 2-macroglobulin with trypsin) had been bound to the same degree from the fluid phase to the monospecific antibodies that had been adsorbed to polystyrene tubes. Comparison of quantitative gel scans for disappearance of the intact alpha 2-macroglobulin subunit (Mr 182000) with 125I-labeled trypsin binding capacity of immunoimmobilized alpha 2-macroglobulin-trypsin complexes showed conspicuous agreement. Rocket immunoelectrophoresis did not give significant differences between 's' alpha 2-macroglobulin and 'f' alpha 2-macroglobulin. In the fluid phase, a binding ratio of 2.4 mol trypsin/mol alpha 2-macroglobulin was observed. Saturation of solid phase immunoimmobilized 's' alpha 2-macroglobulin with trypsin could be accomplished by incubation with a 100-200-fold molar excess of enzyme for 10 min. The solid-phase experiments showed a binding ratio of 2.0 mol trypsin/mol alpha 2-macroglobulin. The high molar excess of trypsin needed to saturate solid-phase immunoimmobilized alpha 2-macroglobulin, which binds 20% less trypsin than in the liquid phase, is partially explained by an enhancement of the negative cooperativity of trypsin binding to alpha 2-macroglobulin found in the liquid-phase system. Assessment of the trypsin-binding capacity of alpha 2-macroglobulin immunoadsorbed from synovial fluids (n = 19) of patients with seropositive rheumatoid arthritis yielded an inactive alpha 2-macroglobulin of 0-53% when compared to the trypsin-binding capacity of normal plasma alpha 2-macroglobulin.     

Studies on the influence of Trasylol on the partition of trypsin between the human plasma protease inhibitors in vitro.

The distribution of trypsin between the protease inhibitors of human serum with and without Trasylol was studied in vitro. 1) Trypsin was preferentially bound by alpha2-macroglobulin on addition of small amounts of the enzyme to normal serum in both the presence and absence of Trasylol in a molar concentration equal to that of alpha2-macroglobulin. 2) On saturation of alpha2-macroglobulin, a considerable amount of trypsin was bound by Trasylol even when most of the serum alpha1-antitrypsin was in a free form. 3) In reaction mixtures containing small amounts of trypsin, Trasylol was identified in a free form as well as in complex with trypsin-alpha2-macroglobulin complex and to a limited extent with trypsin. 4) With larger amounts of trypsin, sufficient to saturate alpha2-macroglobulin, increasing amounts of Trasylol were bound to trypsin. The relative amount of Trasylol bound to trypsin-alpha2-macroglobulin complexes was now smaller. This was explained by a higher affinity (or binding rate) of Trasylol for trypsin than for trypsin-alpha2-macroglobulin complexes. 5) Trypsin-Trasylol complexes showed no signs of dissociation after 5 h incubation at 37 degrees C in serum.     

Study of the complex between porcine plasmin and alpha2-macroglobulin (author's transl)]

Porcine plasmin (EC 3.4.21.7) is obtained from plasminogen activated by human urokinase. This enzyme can bind, in an equimolecular ratio, to an alpha2-macroglobulin isolated from porcine serum. The number of active sites of plasmin has been determined through a burst titration of nitroaniline during the presteady-state hydrolysis of an amide substrate (N-alpha-carbobenzoxy-L-arginine-p-nitroanilide). The kinetic constants relative to a very sensitive ester substrate (N-alpha-carbobenzoxy-L-lysine nitrophenylester) hydrolysis have been measured. The binding of plasmin to alpha2-macroglobulin results in a complete inhibition of proteolytic activity, a reduction of active sites number and a decrease of esterolytic activity towards this substrate. In the complex, the residual activity (about 60%) is protected from protein inhibitors. Absorbance difference spectra show that 1 mol of alpha2-macroglobulin binds 1 mol of plasmin and 2 mol of trypsin. When plasmin is first bound to alpha2-macroglobulin, only 1 mol of trypsin can gain access tothe second site without removing the plasmin, showing that a steric hindrance is implicated in the inhibition performed by alpha2-macroglobulin binding.     

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.     

Conformation and protease binding activity of binary and ternary human alpha 2-macroglobulin-protease complexes

Human alpha 2-macroglobulin (alpha 2M) undergoes a conformational change after reaction with proteases. In this report, it is shown that although two trypsin molecules may bind simultaneously to each alpha 2M, only one trypsin is necessary to induce alpha 2M conformational change. Ternary complexes of alpha 2M and either two radioiodinated trypsins or two nonradioiodinated trypsins were purified by gel filtration chromatography. The nonradioactive complex did not bind 125I-trypsin, even after incubation for 24 h with the free protease present at a large molar excess. Under comparable conditions, a large molar excess of nonradioactive trypsin did not cause significant dissociation of the complex prepared with radioiodinated protease. Equations are presented that distinguish between two separate models of protease binding and demonstrate that binary alpha 2M-trypsin complex retains no significant trypsin binding activity despite the presence of a vacant protease binding site. Purified alpha 2M-plasmin complex, with 1.10 mol of plasmin/mol of inhibitor, also retained no trypsin binding activity as assessed with radioiodinated protein binding experiments. These studies suggest that reactions of alpha 2M with proteases are accurately described by the "trap hypothesis" (Barrett, A. J., and Starkey, P. M. (1973) Biochem. J. 133, 709-724) independent of protease size or binding stoichiometry.     

Subunit and primary structure of a mouse alpha-macroglobulin, a human alpha 2-macroglobulin homologue

A mouse alpha-macroglobulin (AMG), a homologue of human alpha 2-macroglobulin (alpha 2 M), has been purified to homogeneity. In contrast to human and acute-phase rat alpha 2 M which contains subunits of about Mr 190 000, the mouse protein contains two major (Mr 163000 and 35000) and one minor (Mr 185000) subunits. Also unlike human alpha 2 M, which can be broken down into about 85000-dalton subunits when reacted with an endopeptidase, the native AMG is cleaved by trypsin into multiple components (Mr 86000, 63000, 61000 and 33000). Two-dimensional peptide map analysis of these various 125I-labeled subunit components reveals that the 185000- and 163000-dalton components are homologous proteins but only the 185000-dalton protein contains the 35000-dalton component. The 163000-dalton protein is cleaved by trypsin into 86000- and 63000-dalton components, and the 86-kDa component in turn can be broken down into 61000- and 33000-dalton fragments. Since the 35000-dalton component is serologically related to AMG but does not share any tryptic peptides with both the 163000- and 33000-dalton components, it is neither a copurified impurity nor a cleavage product of the major (163000-dalton) subunit. AMG, therefore, is composed of covalently linked subunits of Mr 163000 and 35000, and the 185000-dalton protein may be a variant subunit of AMG. Trypsin treatment of the [14C]methylamine-labeled AMG and alpha 2 M also sequentially generate subunit patterns indistinguishable from those of the unlabeled macroglobulins. The methylamine-sensitive site(s) of AMG is localized in the 63000-dalton peptide, which is rather resistant to trypsin digestion and to staining by Coomassie brillant blue. We conclude from this study that the mouse homologue has a subunit composition and primary structure distinctly different from those of human and rat alpha 2 M.     

NMR and ESR studies on human pregnancy zone protein. Comparison with human alpha 2-macroglobulin.

NMR and ESR spectroscopies have been used to examine the plasma protease inhibitor pregnancy zone protein (PZP) and its complex with chymotrypsin. The 1H NMR spectrum of PZP shows relatively few sharp resonances, which, by analogy with human alpha 2-macroglobulin, probably arise from the proteolytically sensitive bait region. Upon reaction with chymotrypsin to form a 1:1 protease.PZP tetramer complex, there is a large increase in the intensity of sharp resonances due to an increase in mobility of these residues. 35Cl NMR has been used to follow binding of zinc and manganese to apo-PZP. Zinc binding causes a linear broadening of the bulk Cl-, consistent with access of Cl- to PZP-bound zinc. Since zinc in the two highest affinity sites in human alpha 2-macroglobulin causes no broadening of Cl-, it is concluded that these zinc sites are absent from PZP. The mobility of chymotrypsin in the PZP.chymotrypsin complex was examined by covalently attaching a nitroxide spin label at the serine residue in the active site of the enzyme and examining the appearance of the ESR spectrum. The chymotrypsin is rigidly held by the PZP to which it is covalently bound. In an analogous experiment performed previously on alpha 2-macroglobulin, chymotrypsin, bound in the presence of methylamine and therefore largely noncovalently bound, was found to be free to rotate inside the cage formed by the protease inhibitor.     

A conserved region in alpha-macroglobulins participates in binding to the mammalian alpha-macroglobulin receptor

Efforts to characterize the receptor recognition domain of alpha-macroglobulins have primarily focused on human alpha 2-macroglobulin (alpha 2M). In the present work, the structure and function of the alpha-macroglobulin receptor recognition site were investigated by amino acid sequence analysis, plasma clearance, and cell binding studies using several nonhuman alpha-macroglobulins: bovine alpha 2M, rat alpha 1-macroglobulin (alpha 1M), rat alpha 1-inhibitor 3 (alpha 1I3), and proteolytic fragments derived from these proteins. Each alpha-macroglobulin bound to the murine peritoneal macrophage alpha-macroglobulin receptor with comparable affinity (Kd approximately 1 nM). A carboxyl-terminal 20-kDa fragment was isolated from each of these proteins, and this fragment bound to alpha-macroglobulin receptors with Kd values ranging from 10 to 125 nM. The amino acid identity between the homologous carboxyl-terminal 20-kDa fragments of human and bovine alpha 2M was approximately 90%, while the overall sequence homology between all carboxyl-terminal fragments studied was 75%. The interchain disulfide bond present in the human alpha 2M carboxyl-terminal 20-kDa fragment was conserved in bovine alpha 2M and rat alpha 1I3, but not in rat alpha 1M. The clearance of each intact alpha-macroglobulin-proteinase complex was significantly retarded following treatment with cis-dichlorodiammineplatinum(II) (cis-DDP). cis-DDP treatment, however, did not affect receptor recognition of purified carboxyl-terminal 20-kDa fragments of these alpha-macroglobulins. A carboxyl-terminal 40-kDa subunit, which can be isolated from rat alpha 1M, bound to the murine alpha-macroglobulin receptor with a Kd of 5 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human fibroblast collagenase-alpha-macroglobulin interactions. Localization of cleavage sites in the bait regions of five mammalian alpha-macroglobulins

The interaction between human fibroblast collagenase and five mammalian alpha-macroglobulins (human alpha 2-macroglobulin and pregnancy zone protein, rat alpha 1- and alpha 2-macroglobulin, and rat alpha 1-inhibitor 3) differing in primary and quaternary structure has been investigated. Complex formation with each of these alpha-macroglobulins follows the course identified for many other proteinases, i.e. specific limited proteolysis in their bait regions inducing a set of conformational changes resulting in activation of the internal beta-cysteinyl-gamma-glutamyl thiol esters and covalent complex formation. At collagenase: alpha-macroglobulin molar ratios of less than 1:1 3.2-3.6 mol of SH groups appear for 1 mol of collagenase bound to human and rat alpha 2-macroglobulin and to rat alpha 1-macroglobulin. For these alpha-macroglobulins it can be estimated that the overall rate constant of complex formation is greater than 1.10(6) M-1 s-1 while it is much lower for human pregnancy zone protein and rat alpha 1-inhibitor 3. More than 95% of the complexed collagenase is covalently bound, and sodium dodecyl sulfate gel electrophoresis shows the typical pattern of bands corresponding to reaction products of very high apparent molecular weight. The same pattern is also seen in the covalent (greater than 98%) complex very slowly formed from Clostridium histolyticum collagenase and human alpha 2-macroglobulin. The identification of the sites of specific limited proteolysis in the bait regions of the five alpha-macroglobulins shows that cleavage may take place in sequences that are not related to those identified earlier in the collagens. These results greatly expand the repertoire of sequences known to be cleaved by fibroblast collagenase and suggest that this proteinase has a primary substrate specificity resembling that of the microbial proteinase thermolysin, as it preferentially cleaves at the NH2-terminal side of large hydrophobic residues. In addition, the results highlight the unique structure of the flexible alpha-macroglobulin bait region in that it can accommodate a conformation required by the highly restrictive fibroblasts collagenase. It is suggested that alpha-macroglobulins may play an important role in locally controlling the activity of collagenases and perhaps other proteinases of the extracellular matrix.     

Interaction of hemorrhagic metalloproteinases with human alpha 2-macroglobulin.

The interaction between four Crotalus atrox hemorrhagic metalloproteinases and human alpha 2-macroglobulin was investigated. The proteolytic activity of the hemorrhagic toxins Ht-c, -d, and -e against the large molecular weight protein substrates, gelatin type I and collagen type IV, was completely inhibited by alpha 2-macroglobulin. The proteolytic activity of Ht-a against the same substrates was not significantly inhibited. Each mole of alpha 2-macroglobulin bound maximally 2 mol of Ht-e and 1.1 mol of Ht-c and Ht-d. These proteinases interacted with alpha 2-macroglobulin rapidly at 22 degrees C. Rate constants based on intrinsic fluorescence measurements were 0.62 X 10(5) M-1 s-1 for interaction of alpha 2-macroglobulin with Ht-c and -d and 2.3 X 10(5) M-1 s-1 for the interaction of alpha 2-macroglobulin with Ht-e. Ht-a interacted with alpha 2-macroglobulin very slowly at 22 degrees C. Increasing the temperature to 37 degrees C and prolonging the time of interaction with alpha 2-macroglobulin resulted in the formation of Mr 90,000 fragments and high molecular weight complexes (Mr greater than 180,000), in which Ht-a is covalently bound to the carboxy-terminal fragment of alpha 2-M. The identification of the sites of specific proteolysis of alpha 2-macroglobulin shows that the cleavage sites for the four metalloproteinases are within the bait region of alpha 2-macroglobulin. Ht-c and -d cleave only at one site, the Arg696-Leu697 peptide bond, which is also the site of cleavage for plasmin, thrombin, trypsin, and thermolysin. Ht-a cleaves alpha 2-macroglobulin primarily at the same site, but a secondary cleavage site at the His694-Ala695 peptide bond was also identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of alpha 2-macroglobulin-bound thrombin with hirudin

The human thrombin bound to alpha 2-macroglobulin (alpha 2 M) in a 1:1 stoichiometry is still able to interact with one of its specific inhibitors, hirudin. The dissociation constant of the complex hirudin--alpha 2M-bound thrombin is 1 X 10(-7) M, whatever the mode of thrombin binding, covalent or non-covalent.     

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.     

Covalent binding of proteinases in their reaction with alpha 2-macroglobulin.

Although it is known that most of the plasma proteinase inhibitors form complexes with proteinases that are not dissociated by SDS (sodium dodecyl sulphate), there has been disagreement as to whether this is true for alpha 2M (alpha 2-macroglobulin). We have examined the stability to SDS with reduction of complexes between alpha 2M and several 125I-labelled proteinases (trypsin, plasmin, leucocyte elastase, pancreatic elastase and papain) by gel electrophoresis. For each enzyme, some molecules were separated from the denatured alpha 2M chains, but amounts ranging from 8.3% (papain) to 61.2% (trypsin) were bound with a stability indicative of a covalent link. Proteolytic activity was essential for the covalent binding to occur, and the proteinase molecules became attached to the larger of the two proteolytic derivatives (apparent mol.wt. 111 000) of the alpha 2M subunit. We take this to mean that cleavage of the proteinase-susceptible site sometimes leads to covalent-bond formation between alpha 2M and proteinase. Whatever the nature of this bond, it does not involve the active site of the proteinase, as bound serine-proteinase molecules retain the ability to react with the active-site-directed reagent [3H]Dip-F (di-isopropyl phosphorofluoridate). Our conclusion is that the ability to form covalent links is not essential for the inhibitory capacity of alpha 2M. It may, however, help to stabilize the complexes against dissociation or proteolysis.     

Partial characterization of the polyisoprenoid carrier of N-acetylglucosamine in Glycine max (soya bean).

Radiolabelled anhydrotrypsin was bound by alpha 2M (alpha 2-macroglobulin) sufficiently tightly to resist separation during gel electrophoresis; 2 mol of anhydrotrypsin were bound/mol of alpha 2M, but the interaction differed in important respects from that between active proteinases and alpha 2M. Anhydrotrypsin was bound by the electrophoretically 'fast' form of alpha 2M, although much less effectively than by the 'slow' form. The inactive enzyme was displaced from alpha 2M by trypsin inhibitor, the order of effectiveness being aprotinin > soya-bean trypsin inhibitor > benzamidine. Saturation of alpha 2M with anhydrotrypsin did not prevent subsequent binding and inhibition of active trypsin by the alpha 2M, and the anhydrotrypsin was not displaced during this reaction. Anhydrotrypsin bound by alpha 2M retained its ability to react with antibodies against trypsin, whereas bound trypsin did not.