Alpha 2-macroglobulin is the primary inhibitor of miniplasmin in vitro and in vivo in the mouse. Comparison with alpha 2-antiplasmin in simultaneous reaction experiments.

Miniplasmin reacted rapidly with purified human alpha 2-macroglobulin (alpha 2M). More than 98% of the complexes were stabilized by at least one covalent bond. The second-order rate constant for the reaction of alpha 2M with miniplasmin at 4 degrees C was 5.1 x 10(5) M-1.s-1. This value was determined by measuring the formation of covalent alpha 2M-125I-miniplasmin complex; however, the rate constant most likely reflects the bait-region cleavage step in the reaction mechanism. Miniplasmin bound primarily to alpha 2M when incubated at 37 degrees C with various mixtures of alpha 2-antiplasmin (alpha 2AP) and alpha 2M. A 2.4-fold molar excess of alpha 2AP was required to yield an equal distribution of proteinase between the two inhibitors. alpha 2M was the primary miniplasmin inhibitor in human and murine plasma (4 degrees C and 37 degrees C). The extent of covalent-bond formation with murine alpha 2M was approx. 96%. Intravenously injected miniplasmin cleared rapidly from the circulation of mice and was recovered principally in the liver. The catabolic pathway was distinctly different from that of miniplasminogen, which was sequestered mainly in the kidneys. The rate of miniplasmin clearance was much faster than that of purified alpha 2AP-miniplasmin complex, suggesting reaction with alpha 2M in vivo. This was confirmed in clearance competition experiments with alpha 2M-methylamine.     

Subunit structure of the rat alpha-macroglobulin proteinase inhibitors

Rats produce 2 alpha-macroglobulin (alpha M) proteinase inhibitors, the alpha 1 M, normally found in the plasma, and the alpha 2 M, an acute phase protein. The alpha-macroglobulins were purified from the plasma of rats with adjuvant arthritis by polyethylene glycol precipitation, chromatography on a Zn2+ affinity column, and filtration on Sephacryl S-300 superfine. Comparison of the purified proteins on sodium dodecyl sulfate polyacrylamide gel electrophoresis following reduction reveals a 185 000 Da subunit for rat alpha 2 M identical to the human alpha 2 M, but a 167 000 plus a 38 000 Da subunit for rat alpha 1 M. Heat/alkali treatment (pH 11, 37 degrees C for 45 min) prior to reduction results in the appearance of 125 000 Da and 60 000 Da components from rat alpha 2 M analogous to the pattern of human alpha 2 M. In contrast, alpha 1 M showed in addition to the 125 000 Da band (and the unaltered 38 000 Da band), two bands of approx. 25 000 Da. Incubation with trypsin (approximately 1 mol/mol alpha M) prior to reduction causes formation of approximately 90 000 Da components from both rat inhibitors and the human alpha 2 M. The data suggests that only rat alpha 2 M and not rat alpha 1 M is structurally homologous to human alpha 2 M.     

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.     

Structural and functional characterization of the inhibition of urokinase by alpha 2-macroglobulin

We have investigated the interaction of alpha 2-macroglobulin (alpha 2M) with the serine proteinase urokinase, an activator of plasminogen. Urokinase formed sodium dodecyl sulfate stable complexes with purified alpha 2M and with alpha 2M in plasma. These complexes could be visualized after polyacrylamide gel electrophoresis by protein blots using 125I-labeled anti-urokinase antibody or by fibrin autography, a measure of fibrinolytic activity. According to gel electrophoretic analyses under reducing conditions, urokinase cleaved alpha 2M subunits and formed apparently covalent complexes with alpha 2M. Urokinase cleaved only about 60% of the alpha 2M subunits maximally at a mole ratio of 2:1 (urokinase: alpha 2M). Binding of urokinase to alpha 2M protected the urokinase active site from inhibition by antithrombin III-heparin and inhibited, to a significant extent, plasminogen activation by urokinase. Reaction of urokinase with alpha 2M caused an increase in intrinsic protein fluorescence and, thus, induced the conformational change in alpha 2M that is characteristic of its interactions with active proteinases. Our results indicate that both in plasma and in a purified system the alpha 2M-urokinase reaction is functionally significant.     

Interaction of a trypsin-like enzyme of Porphyromonas gingivalis W83 with antithrombin III

Abstract We have previously observed that trypsin-like activity in Porphyromonas gingivalis culture supernatants is inhibitable by the plasma arg-serpin antithrombin III (ATIII). This report demonstrates that a partially purified P. gingivalis trypsin-like enzyme ( M _r 47 000) is inhibited by ATIII with an association rate constant ( k _ass) of 5.65 × 10⁴ M⁻¹ s⁻¹ but does not form SDS-stable complexes. Heparin enhances the k _ass and stabilizes the complexes but in either case such inhibition is temporary and results in ATIII inactivation by reactive centre proteolysis between R³⁹³-S³⁹⁴. In the absence of heparin this is accompanied by N-terminal cleavage between K³⁹-I⁴⁰.     

Highly efficient inhibition of human chymase by alpha(2)-macroglobulin.

The inhibition of human chymase by the protease inhibitor alpha(2)-macroglobulin (alpha2M) was investigated. Titration of chymase hydrolytic activity with purified alpha2M showed that approximately 1 mol of alpha2M tetramer inhibits 1 mol of chymase. Inhibition was associated with cleavage of the alpha2M bait region and formation of a 200-kDa covalent complex. NH(2)-terminal sequencing of chymase-treated alpha2M revealed cleavage at bonds Phe684-Tyr685 and Tyr685-Glu686 of the bait region. alpha2M pretreated with methylamine, an inactivator of alpha2M, did not inhibit chymase. The apparent second-order rate constant for inhibition (k(ass)) was 5 x 10(6) M(-1) s(-1), making alpha2M the most efficient natural protein protease inhibitor of chymase so far described. The k(ass) value for inhibition was decreased approximately 10-fold by addition of heparin, a glycosaminoglycan produced by mast cells that binds to chymase. Heparin did not change significantly the stoichiometry of inhibition or block covalent complex formation. These results indicate that alpha2M is an important inhibitor to consider in the regulation of human chymase.     

Interaction of human plasma kallikrein and its light chain with alpha 2-macroglobulin

Human plasma kallikrein participates in the contact activation system of plasma. The light chain of kallikrein contains the enzymatic active site; the heavy chain is required for binding to high molecular weight kininogen and for surface-dependent activation of coagulation. This study has examined the functional contributions of the heavy chain of kallikrein and of high molecular weight kininogen in the inactivation of kallikrein and of its isolated light chain by alpha 2-macroglobulin (alpha 2M). Irreversible inhibition was observed for both kallikrein and its light chain, with the initial formation of a reversible enzyme-inhibitor complex. The second-order rate constants for these reactions were 3.5 X 10(5) and 4.8 X 10(5) M-1 min-1 for kallikrein and its light chain, respectively. When present in excess, high molecular weight kininogen decreased the rate of kallikrein inactivation by alpha 2M, whereas the rate of inactivation of the light chain was unaffected by high molecular weight kininogen. Although at a drastically reduced rate, high molecular weight kininogen was cleaved by alpha 2M-bound kallikrein. Sodium dodecyl sulfate gradient polyacrylamide gel electrophoresis was used to study complex formation between alpha 2M and kallikrein or its light chain. Under reducing conditions, four kallikrein-alpha 2M complexes were observed. Three of these complexes consisted of alpha 2M and the light chain of kallikrein (Mr 123 000, 235 000, and 330 000). Two alpha 2M-kallikrein light chain complexes incorporated [3H]diisopropyl fluorophosphate ( [3H]DFP) whereas the Mr 330 000 complex did not react with [3H]DFP.(ABSTRACT TRUNCATED AT 250 WORDS)     

IGF-1-dependent subunit communication of the IGF-1 holoreceptor: interactions between alpha beta heterodimeric receptor halves

Examination of 125I-IGF-1 affinity cross-linking and beta-subunit autophosphorylation has indicated that IGF-1 induces a covalent association of isolated alpha beta heterodimeric IGF-1 receptors into an alpha 2 beta 2 heterotetrameric state, in a similar manner to that observed for the insulin receptor [Morrison, B.D., Swanson, M.L., Sweet, L.J., & Pessin, J.E. (1988) J. Biol. Chem. 263, 7806-7813]. The formation of the alpha 2 beta 2 heterotetrameric IGF-1 receptor complex from the partially purified alpha beta heterodimers was time dependent with half-maximal formation in approximately 30 min at saturating IGF-1 concentrations. The IGF-1-dependent association of the partially purified alpha beta heterodimers into an alpha 2 beta 2 heterotetrameric state was specific for the IGF-1 receptors since IGF-1 was unable to stimulate the protein kinase activity of the purified alpha beta heterodimeric insulin receptor complex. Incubation of the alpha 2 beta 2 heterotetrameric IGF-1 holoreceptor with the specific sulfhydryl agent iodoacetamide (IAN) did not alter 125I-IGF-1 binding of IGF-1 stimulation of protein kinase activity. In addition, IAN did not affect the Mn/MgATP-dependent noncovalent association of IGF-1 receptor alpha beta heterodimers into an alpha 2 beta 2 heterotetrameric state. However, IAN treatment of the alpha beta heterodimeric IGF-1 receptors inhibited the IGF-1-dependent covalent formation of the disulfide-linked alpha 2 beta 2 heterotetrameric complex. These data indicate that IGF-1 induces the covalent association of isolated alpha beta heterodimeric IGF-1 receptor complexes into a disulfide-linked alpha 2 beta 2 heterotetrameric state whereas Mn/MgATP induces a noncovalent association.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha 2-macroglobulin is a binding protein for basic fibroblast growth factor

After incubation with human serum or plasma, 125I-basic fibroblast growth factor (bFGF) (molecular mass 18.5 kDa) exhibits molecular mass forms greater than 200 kDa as determined by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. These high molecular mass forms of bFGF are immunoprecipitable with antiserum raised against alpha 2-macroglobulin (alpha 2M). Purified alpha 2M and 125I-bFGF form a covalent complex in a specific, saturable manner. Excess unlabeled bFGF competes with 125I-bFGF for complex formation. Complex formation is complete after 4 h and is inhibited by pretreating alpha 2M with dithiothreitol, iodoacetamide, iodoacetic acid, and N-ethylmaleimide. The complex is resistant to acidic conditions and denaturants such as urea. Heparin, which binds bFGF, has no effect on complex formation. Methylamine, which blocks protease binding to alpha 2M, increases the amount of 125I-bFGF that can be bound 2-fold. Plasmin and trypsin treatment of alpha 2M has no effect on 125I-bFGF binding. The ability of growth factors to compete for binding is specific, as aFGF and TGF-beta compete for binding to alpha 2M, whereas platelet-derived growth factor does not. 125I-bFGF.alpha 2M complexes do not bind to low affinity bFGF binding sites and bind poorly to high affinity bFGF binding sites on BHK-21 cells. In addition, 125I-bFGF bound to alpha 2M has decreased ability to stimulate plasminogen activator production in bovine capillary epithelial cells.     

In vivo metabolism of inter-alpha-trypsin inhibitor and its proteinase complexes: evidence for proteinase transfer to alpha 2-macroglobulin and alpha 1-proteinase inhibitor

Inter-alpha-trypsin inhibitor was purified by a modification of published procedures which involved fewer steps and resulted in higher yields. The preparation was used to study the clearance of the inhibitor and its complex with trypsin from the plasma of mice and to examine degradation of the inhibitor in vivo. Unlike other plasma proteinase inhibitor-proteinase complexes, inter-alpha-trypsin inhibitor reacted with trypsin did not clear faster than the unreacted inhibitor. Studies using 125I-trypsin provided evidence for the dissociation of complexes of proteinase and inter-alpha-trypsin inhibitor in vivo, followed by rapid removal of proteinase by other plasma proteinase inhibitors, particularly alpha 2-macroglobulin and alpha 1-proteinase inhibitor. Studies in vitro also demonstrated the transfer of trypsin from inter-alpha-trypsin inhibitor to alpha 2-macroglobulin and alpha 1-proteinase inhibitor but at a much slower rate. The clearance of unreacted 125I-inter-alpha-trypsin inhibitor was characterized by a half-life ranging from 30 min to more than 1 h. Murine and human inhibitors exhibited identical behavior. Multiphasic clearance of the inhibitor was not due to degradation, aggregation, or carbohydrate heterogeneity, as shown by competition studies with asialoorosomucoid and macroalbumin, but was probably a result of extravascular distribution or endothelial binding. 125I-inter-alpha-trypsin inhibitor cleared primarily in the liver. Analysis of liver and kidney tissue by gel filtration chromatography and sodium dodecyl sulfate gel electrophoresis showed internalization and limited degradation of 125I-inter-alpha-trypsin inhibitor in these tissues. No evidence for the production of smaller proteinase inhibitors from 125I-inter-alpha-trypsin inhibitor injected intravenously or intraperitoneally was detected, even in casein-induced peritoneal inflammation. No species of molecular weight similar to that of urinary proteinase inhibitors, 19,000-70,000, appeared in plasma, liver, kidney, or urine following injection of inter-alpha-trypsin inhibitor.     

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.     

Inhibition of human factor Xa by various plasma protease inhibitors

The inhibitory effects of the plasma protease inhibitors antithrombin III, alpha 2-macroglobulin and alpha 1-antitrypsin on the activity of human factor Xa have been studied using purified proteins. The rate of inhibition was determined by measuring the residual factor Xa activity at timed intervals utilizing the synthetic peptide susbtrate Bz-Ile-Glu(piperidyl)-Gly-Arg-pNA. Kinetic analysis with varying molar concentrations of inhibitors demonstrated that the inhibition of factor Xa by antithromin III, alpha 2-macroglobulin and alpha 1-antitrypsin followed second-order kinetics. Calculated values of the rate constants for the inhibition of factor Xa by antithrombin III, alpha 2-macroglobulin and alpha 1-antitrypsin were 5.8 . 10(4), 4.00 . 10(4) and 1.36 . 10(4) M -1 . min -1, respectively. The plasma concentrations of the inhibitors can be used to assess their potential relative effectiveness against factor Xa. In plasma this was found as alpha 1-antitrypsin greater than antithrombin III greater than alpha 2-macroglobulin in the ratio 4.64: 2.08: 1.0. Cephalin was shown to inhibit the rate of reaction between factor Xa and antithrombin III.     

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.     

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

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

Partial purification and characterization of a new fast-acting plasmin inhibitor from human platelets. Evidence for non-identity with the known plasma proteinase inhibitors.

An inhibitor of the plasma proteinase plasmin (EC 3.4.21.7) was partially purified from washed and lysed human blood platelets by (NH4)2SO4 fractionation and affinity chromatrography on Sepharose-linked purified plasminogen. The material contained none of the known plasma proteinase inhibitors when studied by crossed-immunoelectrophoresis and electroimmunoassay, but inhibited a clot-lysis-time assay and an esterolytic assay that used the synthetic substrate S-2251 (D-Val-Leu-Lys-p-nitroanilide). The inhibitory activity had the same mobility as the alpha 2-plasma proteins on preparative agarose-gel electrophoresis. Titration of the inhibitor preparation by active-site-titrated plasmin demonstrated a dissociation constant of approx. 0.1 nM. The inhibition was complete within 1 min. The inhibitor increased the mobility in agarose-gel electrophoresis of purified activator-free plasmin or 125I-labelled plasmin, as demonstrated by crossed-immunoelectrophoresis against specific immunoglobulins against plasminogen or by radioautography. The results strongly suggest the presence in platelets of a plasmin inhibitor different from the known plasma proteinase inhibitors.     

Alpha 2-macroglobulin restricts plasminogen activation to the surface of RC2A leukemia cells.

Human RC2A myelomonocytic leukemia cells are able to activate the prourokinase (pro-u-PA) they secrete so that active u-PA is present both in serum-free conditioned medium from these cells, as well as on the cell surface. When the cells are grown in serum-containing medium, no u-PA activity can be found in the medium but active u-PA is found bound to the cell surface where it can generate bound plasmin. This distribution of u-PA activity was shown to be, first, the net result of slow inactivation of free active u-PA by serum inhibitor(s) and simultaneous rapid uptake of u-PA onto the cell surface. Binding to cells was at least six times faster than inactivation by 10% serum. The principal serum inhibitor of u-PA was identified as alpha 2-macroglobulin (alpha 2M), and prior inactivation of u-PA by purified human alpha 2M was also shown to prevent uptake of u-PA activity onto cells. Second, although endogenous u-PA could form covalent complexes with purified alpha 2M in the culture medium of RC2A cells, covalent alpha 2M complexes were not formed by u-PA on the cell surface; the u-PA taken up in this compartment was protected against alpha 2M inhibition. u-PA anchored to plastic surfaces via monoclonal antibodies to the amino-terminal region of u-PA was also protected against alpha 2M, suggesting that the protection of cell surface u-PA results from a steric effect. These results provide evidence as to how the active u-PA produced by leukemia cells can contribute to proteolytic activity on their cell surface in the presence of serum inhibitors.     

Identification of the high affinity binding site in transforming growth factor-beta involved in complex formation with alpha 2-macroglobulin. Implications regarding the molecular mechanisms of complex formation between alpha 2-macroglobulin and growth factors, cytokines, and hormones

The biological activities of transforming growth factor-beta isoforms (TGF-beta(1,2)) are known to be modulated by alpha(2)-macroglobulin (alpha(2)M). alpha(2)M forms complexes with numerous growth factors, cytokines, and hormones, including TGF-beta. Identification of the binding sites in TGF-beta isoforms responsible for high affinity interaction with alpha(2)M many unravel the molecular basis of the complex formation. Here we demonstrate that among nine synthetic pentacosapeptides with overlapping amino acid sequences spanning the entire TGF-beta(1) molecule, the peptide (residues 41-65) containing Trp-52 exhibited the most potent activity in inhibiting the formation of complexes between (125)I-TGF-beta(1) and activated alpha(2)M (alpha(2)M*) as determined by nondenaturing polyacrylamide gel electrophoresis and by plasma clearance in mice. TGF-beta(2) peptide containing the homologous sequence and Trp-52 was as active as the TGF-beta(1) peptide, whereas the corresponding TGF-beta(3) peptide lacking Trp-52, was inactive. The replacement of the Trp-52 with alanine abolished the inhibitory activities of these peptides. (125)I-TGF-beta(3), which lacks Trp-52, bound to alpha(2)M* with an affinity lower than that of (125)I-TGF-beta(1). Furthermore, unlabeled TGF-beta(3) and the mutant TGF-beta(1)W52A, in which Trp-52 was replaced with alanine, were less potent than unlabeled TGF-beta(1) in blocking I(125)-TGF-beta(1) binding to alpha(2)M*. TGF-beta(1) and TGF-beta(2) peptides containing Trp-52 were also effective in inhibiting I(125)-nerve growth factor binding to alpha(2)M*. Tauhese results suggest that Trp-52 is involved in high affinity binding of TGF-beta to alpha(2)M*. They also imply that TGF-beta and other growth factors/cytokines/hormones may form complexes with alpha(2)M* via a common mechanism involving the interactions between topologically exposed Trp and/or other hydrophobic residues and a hydrophobic region in alpha(2)M*.     

Kinetics of the reaction of thrombin and alpha 2-macroglobulin.

The kinetics of the reaction of alpha 2-macroglobulin (alpha 2M) with human thrombin were studied by recording the appearance of thiol groups spectrophotometrically and by measuring the distribution of protein species by denaturing non-reducing gel electrophoresis. The goals were to study the relation between the formation of various covalent enzyme-inhibitor complex species and the appearance of free thiol, and from the kinetic analysis, to try to characterize the chemical nature of the protein complexes. The kinetics of thiol-group release were observed to be biphasic, the early phase showing second-order behaviour, results consistent with previous reports in the literature. The observed second-order rate constant for thiol-group release was found to be faster than the second-order rate constant for the disappearance of the band corresponding to native alpha 2M on gel electrophoresis. This may be a reflection of the multiple products formed from the thioester. Alternatively, it is possible that covalent-bond formation is slower than some enzyme-induced change in the thioester centre, and this may be suggestive evidence for a reactive alpha 2M centre that does not contain an intact thioester. The kinetics of covalent-bond formation were found to be consistent with the internal cross-link of several alpha 2M chains by the bound proteinase, providing further evidence that the very-high-Mr species seen on gels may arise from dimers of the alpha 2M molecule held together by covalent bonds to the enzyme.     

Inhibition of human pancreatic proteinases by human plasma alpha2-antiplasmin and antithrombin

Human plasma alpha1-antitrypsin inhibits human pancreatic trypsin, chymotrypsin and elastase, which are massively released into the blood stream during acute pancreatitis. To examine whether the plasma proteins of individuals with genetic deficiency of alpha1-antitrypsin are protected against the deleterious action of these enzymes by other inhibitors, we have tested their inhibition by alpha2-antiplasmin and antithrombin. We have determined the inhibition rate constants kass and calculated d(t), the in vivo inhibition time. Surprisingly, trypsin is inhibited faster by alpha2-antiplasmin [kass=2.5 x 10(6) M(-1)S(-1), d(t)=2.3 s] and antithrombin [kass=1.7 x 10(5) M(-1)s(-1), d(t)=5.8 s] than by alpha1-antitrypsin [d(t)=17 s or 116 s in alpha1-antitrypsin-sufficient or alpha1-antitrypsin-deficient individuals, respectively]. Low molecular weight heparin accelerates the inhibition of trypsin by antithrombin by a factor of 16 [d(t)=0.36 s]. Antithrombin and alpha2-antiplasmin are not physiological inhibitors of chymotrypsin and elastase. These enzymes are, however, physiologically inhibited by alpha1-antitrypsin and alpha1-antichymotrypsin even in alpha1-antitrypsin-deficient individuals. We conclude that (i) low molecular weight heparin may be helpful in the management of acute pancreatitis, and (ii) genetically determined alpha1-antitrypsin deficiency probably does not lead to a significantly increased risk of plasma protein degradation during this disease.     

Novel complex formed between a nonproteolytic cell wall protein of group A streptococci and alpha 2-macroglobulin.

Binding of 125I-labeled alpha 2-macroglobulin (alpha 2M) to streptococci belonging to serological groups A, B, C, and G was studied. Streptococci of groups A and G interacted only with native alpha 2M, and those of group C reacted only with alpha 2M-trypsin complex. Binding of alpha 2M to group A streptococci was saturable and reversible. The dissociation constant was 2.02 X 10(-7) M, and the number of binding sites was calculated to be 18,000 per streptococcus. The alpha 2M-binding protein could be solubilized by treatment of group A streptococci with a murolytic enzyme and subsequently purified by affinity chromatography and high-pressure liquid chromatography. The purified protein was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a molecular weight of 78,000. It possessed no proteolytic activity and interacted with native alpha 2M in Western blots (immunoblots). Interaction of purified binding protein with alpha 2M led to a change in the conformation of alpha 2M similar to that obtained by alpha 2M-protease complexes. Reversible binding of a nonproteolytic streptococcal component of alpha 2M is thus a novel feature of alpha 2M reactivity.