The interaction of α2-macroglobulin with proteinases. Characteristics and specificity of the reaction, and a hypothesis concerning its molecular mechanism

1. alpha(2)-Macroglobulin is known to bind and inhibit a number of serine proteinases. We show that it binds thiol and carboxyl proteinases, and there is now reason to believe that alpha(2)-macroglobulin can bind essentially all proteinases. 2. Radiochemically labelled trypsin, chymotrypsin, cathepsin B1 and papain are bound by alpha(2)-macroglobulin in an approximately equimolar ratio. Equimolar binding was confirmed for trypsin by activesite titration. 3. Pretreatment of alpha(2)-macroglobulin with a saturating amount of one proteinase prevented the subsequent binding of another. We conclude that each molecule of alpha(2)-macroglobulin is able to react with one molecule of proteinase only. 4. alpha(2)-Macroglobulin did not react with exopeptidases, non-proteolytic hydrolases or inactive forms of endopeptidases. 5. The literature on binding and inhibition of proteinases by alpha(2)-macroglobulin is reviewed, and from consideration of this and our own work several general characteristics of the interaction can be discerned. 6. A model is proposed for the molecular mechanism of the interaction of alpha(2)-macroglobulin with proteinases. It is suggested that the enzyme cleaves a peptide bond in a sensitive region of the macroglobulin, and that this results in a conformational change in the alpha(2)-macroglobulin molecule that traps the enzyme irreversibly. Access of substrates to the active site of the enzyme becomes sterically hindered, causing inhibition that is most pronounced with large substrate molecules. 7. The possible physiological importance of the unique binding characteristics of alpha(2)-macroglobulin is discussed.     

Plasma proteinase inhibitors in Morris hepatoma-bearing rats: changes in the blood level and synthesis in tissue slices

The antiproteinase activities against trypsin, chymotrypsin, elastase, papain and rat leucocyte proteinases were determined in plasma from control and Morris hepatoma-bearing rats. Bovine trypsin and chymotrypsin were similarly inhibited by the two types of plasma whereas porcine pancreatic elastase, papain and rat leucocyte neutral proteinases were more efficiently inhibited by plasma from tumour-bearing rats. The increased plasma concentrations of some proteinase inhibitors, as determined by rocket immunoelectrophoresis, are suggested to be responsible for the observed differences in inhibition. The highest increases in plasma of tumour-bearing rats were observed for alpha 2-macroglobulin and alpha 1-acute-phase globulin. The synthesis and secretion of six proteinase inhibitors: antithrombin III, alpha 1-proteinase inhibitor, alpha 1-macroglobulin, alpha 2-macroglobulin, alpha 1-acute-phase globulin and haptoglobin, as well as albumin, were measured in tissue slices from rat liver and Morris hepatoma after incubation with [14C]leucine. Local inflammation inflicted upon the tumour-bearing rats increased formation of acute-phase proteins in liver slices but not in hepatoma slices.     

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.     

Some consequences of the covalent and non-covalent binding modes of plasmin with alpha 2-macroglobulin

Analysis of plasmin-alpha 2-macroglobulin interactions by polyacrylamide gel electrophoresis showed that both the light and heavy chains of the proteinase have covalent links with the inhibitor. This covalent binding occurs with a 95 +/- 5% yield and can be abolished in the presence of hydroxylamine without modification of the plasmin-alpha 2-macroglobulin stoichiometry, the extent of the 180-kDa peptide chain cleavage and the generation of the -SH groups. However, these two different binding modes greatly influence the enzymatic properties of the proteinase as well as the occupancy by an other proteinase molecule of the free binding site of the (1:1) plasmin-alpha 2-macroglobulin complex. Non-covalently bound plasmin is more active on synthetic substrates and interacts more tightly with the basic pancreatic trypsin inhibitor than the covalently bound enzyme. Furthermore, the former complex incorporates significantly more chymotrypsin than the latter. The incorporation of chymotrypsin influences the catalytic properties of plasmin within the ternary complex.     

Uptake of proteinase-alpha-macroglobulin complexes by macrophages.

Complexes of labelled proteinases (subtilopeptidase A, trypsin) with serum alpha 1-macroglobulin or alpha 2-macroglobulin are rapidly taken up in vitro by rabbit alveolar macrophages and peritoneal macrophages but not by mixed rabbit peripheral blood leukocytes. Enzyme, not bound to alpha 1- or alpha 2-macroglobulin, does not become associated with alveolar macrophages. Chemically inactivated subtilopeptidase A does not bind to alpha 1- or alpha 2-macroglobulin; chemically inactivated subtilopeptidase A in mixtures with alpha 1 - or alpha 2-microglobulin, does not interact with alveolar macrophages. Blocking experiments confirmed that the interaction of proteinase with alveolar macrophages is complex specific; uptake of labelled complex was prevented by the simultaneous addition of macroglobulin complexes formed with non-labelled subtilopeptidase A, subtilopeptidase B, trypsin or chymotrypsin but not by macroglobulin alone. The findings demonstrate a complex-specific interaction between proteinase-alpha-macroglobulin complexes and macrophages.     

The role of inter-alpha-trypsin inhibitor and other proteinase inhibitors in the plasma clearance of neutrophil elastase and plasmin

The plasma clearance of neutrophil elastase, plasmin, and their complexes with human inter-alpha-trypsin inhibitor (I alpha I) was examined in mice, and the distribution of the proteinases among the plasma proteinase inhibitors was quantified in mixtures of purified inhibitors, in human or murine plasma, and in murine plasma following injection of purified proteins. The results demonstrate that I alpha I acts as a shuttle by transferring proteinases to other plasma proteinase inhibitors for clearance, and that I alpha I modulates the distribution of proteinase among inhibitors. The clearance of I alpha I-elastase involved transfer of proteinase to alpha 2-macroglobulin and alpha 1-proteinase inhibitor. The partition of elastase between these inhibitors was altered by I alpha I to favor formation of alpha 2-macroglobulin-elastase complexes. The clearance of I alpha I-plasmin involved transfer of plasmin to alpha 2-macroglobulin and alpha 2-plasmin inhibitor. Results of distribution studies suggest that plasmin binds to endothelium in vivo and reacts with I alpha I before transfer to alpha 2-macroglobulin and alpha 2-plasmin inhibitor. Evidence for this sequence of events includes observations that plasmin in complex with I alpha I cleared faster than free plasmin, that plasma obtained after injection of plasmin contained a complex identified as I alpha I-plasmin, and that a murine I alpha I-plasmin complex remained intact following injection into mice. Plasmin initially in complex with I alpha I more readily associated with alpha 2-plasmin inhibitor than did free plasmin.     

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.     

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.     

The interaction of α2-macroglobulin with proteinases. Binding and inhibition of mammalian collagenases and other metal proteinases

1. Experiments were performed to determine whether the specific collagenases and other metal proteinases are bound and inhibited by alpha(2)-macroglobulin, as are endopeptidases of other classes. 2. A specific collagenase from rabbit synovial cells was inhibited by human serum. The inhibition could be attributed entirely to alpha(2)-macroglobulin; alpha(1)-trypsin inhibitor was not inhibitory. alpha(2)-Macroglobulin presaturated with trypsin or cathepsin B1 did not inhibit collagenase, and pretreatment of alpha(2)-macroglobulin with collagenase prevented subsequent reaction with trypsin. The binding of collagenase by alpha(2)-macroglobulin was not reversible in gel chromatography. 3. The collagenolytic activity of several rheumatoid synovial fluids was completely inhibited by incubation of the fluids with alpha(2)-macroglobulin. 4. The collagenase of human polymorphonuclear-leucocyte granules showed time-dependent inhibition by alpha(2)-macroglobulin. 5. The collagenolytic metal proteinase of Crotalus atrox venom was inhibited by alpha(2)-macroglobulin. 6. The collagenase of Clostridium histolyticum was bound by alpha(2)-macroglobulin, and inhibited more strongly with respect to collagen than with respect to a peptide substrate. 7. Thermolysin, the metal proteinase of Bacillus thermoproteolyticus, was bound and inhibited by alpha(2)-macroglobulin. 8. It was shown by polyacrylamidegel electrophoresis of reduced alpha(2)-macroglobulin in the presence of sodium dodecyl sulphate that synovial-cell collagenase, clostridial collagenase and thermolysin cleave the quarter subunit of alpha(2)-macroglobulin near its mid-point, as do serine proteinases. 9. The results are discussed in relation to previous work, and it is concluded that the characteristics of interaction of the metal proteinases with alpha(2)-macroglobulin are the same as those of other proteinases.     

In vitro binding and in vivo clearance of human alpha 2-macroglobulin after reaction with endoproteases from four different classes

The binding of human alpha 2-macroglobulin complexed with trypsin, papain, thermolysin and cathepsin-D to murine macrophages was studied at 4 degrees C. Similar dissociation constants (0.4 nM) were determined for all of the complexes except alpha 2-macroglobulin-cathepsin-D (0.7 nM). Radioiodinated alpha 2-macroglobulin-protease complexes were injected into mice, and the clearance studied. Native alpha 2-macroglobulin cleared slowly, as previously reported, while greater than 50% of the complexes formed with trypsin, papain and thermolysin cleared in less than 5 min. The clearance of alpha 2-macroglobulin-cathepsin-D was biphasic, suggesting that only about half the alpha 2-macroglobulin was present in a reacted complex.     

Localization of epsilon-lysyl-gamma-glutamyl cross-links in five human alpha 2-macroglobulin-proteinase complexes. Nature of the high molecular weight cross-linked products.

Covalent binding of proteinases by human alpha 2-macroglobulin (alpha 2M) results primarily from the formation of stable epsilon-Lys-gamma-Glu isopeptide bonds. Cross-linking engages 12, 13, and 10 of the 14, 14, and 11 Lys residues in chymotrypsin, trypsin, and subtilisin, respectively, and reaction with the alpha-amino group of the C-chain of chymotrypsin and the B-chain of beta-trypsin is also seen. In contrast, cross-linking engages only 6 of the 11 Lys residues in thermolysin. In each of these proteinases, a few residues react to the greatest extent: Lys36, Lys79, Lys87, and Lys93 in chymotrypsin; Lys87, Lys109, Lys222, and Lys239 in trypsin; Lys12, Lys43, and Lys141 in subtilisin; and Lys210 and Lys219 in thermolysin. In elastase, 1 of the 3 Lys residues (Lys87) is tentatively identified as being cross-linked. Formation of unstable bonds judged to be mainly p-tyrosyl-gamma-glutamyl esters can also be significant for some proteinases. In each of the proteinases, several of the strongly reacting Lys residues are located relatively close to each other, presumably reflecting steric constraints within the alpha 2M-proteinase complexes as they form. Proteinases are covalently bound to alpha 2M to one or two of its COOH-terminal bait region-cleaved half-subunits. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern of the high molecular weight cross-linked species indicates that binding of a proteinase through two cross-links occurs not only within the 360-kDa disulfide-bridged alpha 2M dimer but also between the two dimers in the alpha 2M tetramer.     

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.     

Characterization of thrombin binding to alpha 2-macroglobulin

The formation and structural characteristics of the human alpha 2-macroglobulin (alpha 2M)-thrombin complex were studied by intrinsic protein fluorescence, sulfhydryl group titration, electrophoresis in denaturing and nondenaturing polyacrylamide gel systems, and in macromolecular inhibitor assays. The interaction between alpha 2M and thrombin was also assessed by comparison of sodium dodecyl sulfate-gel electrophoretic patterns of peptides produced by Staphylococcus aureus V-8 proteinase digests of denatured alpha 2M-125I-thrombin and alpha 2M-125I-trypsin complexes. In experiments measuring fluorescence changes and sulfhydryl group exposure caused by methylamine, we found that thrombin produced its maximum effects at a mole ratio of approximately 1.3:1 (thrombin:alpha 2M). Measurements of the ability of alpha 2M to bind trypsin after prior reaction with thrombin indicated that thrombin binds rapidly at one site on alpha 2M, but occupies the second site with some difficulty. Intrinsic fluorescence studies of trypsin binding to alpha 2M at pH 5.0, 6.5, and 8.0 not only revealed striking differences in trypsin's behavior over this pH range, but also some similarities between the behavior of thrombin and trypsin not heretofore recognized. Structural studies, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to measure alpha 2M-125I-thrombin covalent complex formation, indicated that covalency reached a maximum at a mole ratio of approximately 1.5:1. At this ratio, only 1 mol of thrombin is bound covalently per mol of alpha 2M. These gel studies and those of proteolytic digests of denatured alpha 2M-125I-trypsin and alpha 2M-125I-thrombin complexes suggest that proteinases form covalent bonds with uncleaved alpha 2M subunits. The sum of our results is consistent with a mechanism of proteinase binding to alpha 2M in which the affinity of the proteinase for alpha 2M during an initial reversible interaction determines its binding ratio to the inhibitor.     

Functional alpha 2-macroglobulin half-molecules induced by cadmium

Human alpha 2-macroglobulin can be reversibly dissociated by Cd2+ at low ionic strength in half-molecules which retain their ability to bind tightly plasmin and chymotrypsin. The steady state kinetic parameters of these proteinases towards chromogenic substrates when bound to half-molecules are not greatly different from those determined for these enzymes linked to whole alpha 2M molecules. Cd2+ can also induce the dissociation of plasmin- and chymotrypsin - alpha 2M complexes into proteinase-alpha 2M half-molecule conjugates. These results, taken with the fact that monomeric units of alpha 2M cannot bind these proteinases, strongly suggest that each active site of alpha 2M consists in a specific arrangement of two monomeric units linked by disulfide bridges.     

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)     

Localization of the proteinase-induced thiol groups in alpha 2-macroglobulin

Free thiol groups released on proteolytic attack of alpha 2-macroglobulin by trypsin or chymotrypsin bind covalently to thiopropyl-Sepharose, indicating that they are located at the surface of the complexes. These cysteine sulfhydryl groups appear to be in contact with the alpha 2M-bound proteases from singlet-singlet energy transfer measurements between fluorescein isothiocyanate-labeled proteinases and N-(iodoacetylaminoethyl)-5-naphtylamine-1-sulfonic acid-labeled thiols in alpha 2-macroglobulin.     

Enzymatic properties of alpha 2-macroglobulin-proteinase complexes. Apparent discrimination between covalently and noncovalently bound trypsin by reaction with soybean trypsin inhibitor

The binding of trypsin to alpha 2-macroglobulin, the appearance of free beta-cysteinyl thiol groups of the formed complexes, the steady-state kinetics of their enzymic hydrolysis of carbobenzoxy-L-valyl-glycyl-L-arginyl-4-nitroanilide and finally their reactions with soybean trypsin inhibitor leading to the formation of ternary alpha 2-macroglobulin-trypsin-soybean trypsin inhibitor complexes were investigated. Each alpha 2-macroglobulin molecule binds two trypsin tightly; the dissociation constants were found to be unmeasureably small, but the extent of formation of 1:1 and 1:2 complexes at different molar ratios of alpha 2-macroglobulin to trypsin as determined from the appearance of thiol groups clearly indicated that binding of trypsin to alpha 2-macroglobulin shows negative cooperativity. Binding of the first trypsin makes the access of the second less easy. The kinetic results showed a decrease of the kc/Km value of hydrolysis of the tripeptide substrate by approx. 4-fold compared to that of free trypsin for each alpha 2-macroglobulin-bound trypsin. Here no differences were seen between the bound trypsins. The analysis of the reactions between the alpha 2-macroglobulin-trypsin complexes and soybean trypsin inhibitor shows that ternary complexes do form, although slowly, and that two processes occur, not only when 1:2 complexes but also when 1:1 complexes react with soybean trypsin inhibitor. Soybean trypsin inhibitor apparently discriminates between two distinct binding modes of trypsin to alpha 2-macroglobulin, the covalently and the noncovalently alpha 2-macroglobulin-bound trypsins.     

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

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

Ovostatin: a novel proteinase inhibitor from chicken egg white. II. Mechanism of inhibition studied with collagenase and thermolysin

The inhibition mechanism of ovostatin was studied using rabbit synovial collagenase and thermolysin. When enzymes were complexed with ovostatin, only the proteolytic activity towards high molecular weight substrates was inhibited. Activity towards low molecular weight substrates was partially modified: the catalytic activity of collagenase bound to ovostatin was inhibited by only 40% towards 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg and that of thermolysin bound to ovostatin was activated about 2.6-fold towards benzyloxycarbonyl-Gly-Leu-NH2 and benzyloxycarbonyl-Gly-Phe-NH2. Collagenase-ovostatin complexes failed to react with anti-(collagenase) antibody. Saturation of ovostatin with thermolysin prevented the subsequent binding of collagenase. Ovostatin-proteinase complexes ran faster than free ovostatin on 5% polyacrylamide gel electrophoresis. Complexing ovostatin with either collagenase or thermolysin resulted in the cleavage of the quarter-subunit of ovostatin (Mr = 165,000) into two fragments with Mr = 88,000 and 78,000. On the other hand, when the inhibitory capacity of ovostatin was tested with trypsin, chymotrypsin, and papain, only partial inhibition of their proteolytic activities was observed towards azocasein. Stronger inhibition was noted when Azocoll was a substrate, however. Analyses of ovostatin-enzyme complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the quarter-subunit of ovostatin was cleaved into several fragments by those enzymes. These results led us to propose that ovostatin inhibits metalloproteinases in preference to proteinases of other classes in a manner similar to alpha 2-macroglobulin; hydrolysis of a peptide bond by a proteinase in the susceptible region of the ovostatin polypeptide chain triggers a conformational change in the ovostatin molecule and the enzyme becomes bound to ovostatin in such a way that the proteinase is sterically hindered from access to large protein substrates and yet is accessible to small synthetic substrates. A kinetic study of collagenase binding to ovostatin gave the value of k2/Ki = 6.3 X 10(5) M-1 min-1. The results indicate that ovostatin is equally as good a substrate for collagenase as type I collagens.     

The interaction between some serine proteinases and horse leucocyte inhibitor

Horse blood leucocyte cytosol exhibits a broad inhibitory activity against serine proteinases. The purified inhibitor was exposed to investigated enzymes (trypsin, chymotrypsin, elastases and serine proteinase from S. aureus) for variable time and the products were analyzed by gradient polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molar ratio I:E, association rate constants k on and inhibition constants Ki for the enzymes and inhibitor were determined. The examined elastases form stable, stoichiometric complexes with the inhibitor (Ki less than 10(-10) M), and do not undergo proteolytic degradation during 30 min incubation at 20 degrees C even at the 2-fold molar excess of the proteinases. The reactions with elastases are extremely rapid (k on greater than 10(7) M-1 s-1) and are completed within one second whereas similar reactions with chymotrypsin and trypsin are much slower (k on = 3 X 10(5) M-1 s-5 and 5 X 10(2) M-1 s-1, respectively). Serine proteinase from S. aureus neither react nor inactivates the investigated inhibitor. The complexes of the inhibitor with trypsin and chymotrypsin are digested even at a molar ratio I:E = 2:1. All these observations point out that the inhibitor from horse leucocyte cytosol is a specific and effective inhibitor of elastases.