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Basic fibroblast growth factor (FGF-2) is important in development, wound healing and angiogenesis. The human plasma proteinase inhibitor alpha2-macroglobulin (alpha2M) binds to and regulates the biological activity of various growth factors, including FGF-2. FGF-2 binds specifically and saturably to native alpha2M and conformationally modified alpha2M (alpha2M*); however, the KD for FGF-2 binding to alpha2M* is 10-fold lower. This study investigates the biochemical nature of the interaction between FGF-2 and alpha2M* and localizes a possible FGF-2 binding site in the alpha2M subunit. FGF-2 binding to alpha2M* was not affected by shifts in pH between 6.5 and 10; however, increasing temperature decreased the KD for this interaction. The binding affinity of FGF-2 for alpha2M* also increased with increasing ionic strength. These results are consistent with the hypothesis that hydrophobic interactions predominate in promoting FGF-2 association with alpha2M*. Consistent with this hypothesis, FGF-2 bound to a glutathione S-transferase fusion protein containing amino acids 591-774 of the alpha2M subunit (FP3) and to a hydrophobic 16-amino-acid peptide (amino acids 718-733) within FP3. Specific binding of FGF-2 to the 16-amino-acid peptide was inhibited by excess transforming growth factor-beta1. When the 16-amino-acid peptide was chemically modified to neutralize the only two charged amino acids, FGF-2-binding activity was unaffected, supporting the predominant role of hydrophobic interactions. FGF-2 presentation to signalling receptors is influenced by growth factor binding to heparan sulphate proteoglycans (HSPGs), which is electrostatic in nature. Our results demonstrate that the interactions of FGF-2 with alpha2M* and HSPGs are biochemically distinct, suggesting that different FGF-2 sequences are involved.

Both plasminogen (Pg) activation and matrix metalloproteinases (MMPs) are involved in the proteolytic degradation of extracellular matrix components, a requisite event for malignant cell metastasis. The highly invasive 1-LN human prostate tumour cell line synthesizes and secretes large amounts of Pg activators and MMPs. We demonstrate here that the Pg type 2 (Pg 2) receptor in these cells is composed primarily of the membrane glycoprotein dipeptidyl peptidase IV (DPP IV). Pg 2 has six glycoforms that differ in their sialic acid content. Only the highly sialylated Pg 2gamma, Pg 2delta and Pg 2epsilon glycoforms bind to DPP IV via their carbohydrate chains and induce a Ca(2+) signalling cascade; however, Pg 2epsilon alone is also able to significantly stimulate expression of MMP-9. We further demonstrate that the Pg-mediated invasive activity of 1-LN cells is dependent on the availability of Pg 2epsilon. This is the first demonstration of a direct association between the expression of MMP-9 and the Pg activation system.

The alpha-macroglobulins are proteinase inhibitors that form part of a superfamily along with components of the complement system. Internal beta-cysteinyl-gamma-glutamyl thiol ester bonds are an important structural feature of most alpha-macroglobulins and several complement components. We have studied the reversibility of thiol ester cleavage caused by NH3 or CH3NH2 in tetrameric human alpha 2-macroglobulin (alpha 2M) and monomeric rat alpha 1-inhibitor-3 (alpha 1I3). When employing NH3 as the nucleophile, the thiol ester in alpha 1I3 re-formed spontaneously at room temperature after gel filtration to remove excess nucleophile, and an active proteinase inhibitor was regained. When CH3NH2 was employed as the nucleophile, thiol ester reversibility was more energy-demanding. With either nucleophile, alpha 2M once inactivated did not regain proteinase-inhibitory capacity at room temperature. At elevated temperatures, however, the reaction between alpha 2M and NH3 or CH3NH2 was reversible and the inhibitory capacity could be recovered. Modification of the cysteinyl groups from the thiol ester prevented its re-formation but did not prevent the heat-induced retrieval of inhibitory capacity, suggesting that conformational features rather than the thiol ester are essential for alpha 2M to function as an inhibitor. As demonstrated by non-denaturing PAGE, the conformation of native alpha 2M is restored when the proteinase-inhibitory capacity is recovered.

Streptokinase, an extracellular protein produced by Streptococci, is capable of activating the human fibrinolytic zymogen plasminogen. The rate of amidolytic activity of the plasminogen-streptokinase complex is greatly diminished by micromolar concentrations of ATP and heparin oligosaccharides. In addition, the plasminogen activator activity of the plasminogen-streptokinase complex is also inhibited by these effectors. ATP and heparin oligosaccharides show structural similarity, suggesting that the inhibition is caused by binding of these molecules to a common newly formed binding pocket in streptokinase, which appears after interaction with plasminogen. Addition of the bivalent cations Ca2+ and Mg2+ reverses the inhibition caused by ATP and heparin. In the presence of ATP and bivalent cations, the complex between plasminogen and streptokinase develops an autophosphorylating activity whose target is the sequence LTSRPAHG in the 4.5 kDa streptokinase N-terminal peptide, which is an early autolysis peptide. This streptokinase N-terminal peptide, which is essential for streptokinase activating activity, may serve, once phosphorylated, in mechanisms related to the pathogenicity of Streptococci. These studies suggest a critical role for plasminogen in regulating the activity of the streptokinase molecule.

We have recently described an alpha 2-macroglobulin (alpha 2M) signalling receptor which is distinct from the low-density lipoprotein-related protein/alpha 2M receptor (LRP/alpha 2MR). Ligation of the macrophage signalling receptor by alpha 2M-methylamine stimulates production of several second messengers and involves a pertussis toxin-insensitive G-protein. We now report that binding of alpha 2M-methylamine, or the cloned M(r) = 20,000 receptor-binding fragment from rat alpha 1M, to macrophage alpha 2M signalling receptors induces protein phosphorylation. By use of a monoclonal antibody to phospholipase C gamma l (PLC gamma l) we were able to identify it as one target for protein phosphorylation. Phosphorylation was time and concentration dependent, being optimal at about 60 s of incubation and a 100-200 nM ligand concentration. By use of a second monoclonal antibody directed against phosphotyrosine, we were able to demonstrate that at least a portion of the label was incorporated into one or more tyrosine residues. PLC gamma l phosphorylation was then studied in membrane preparations at 4 degrees C in order to minimize serine or threonine modification. Preincubation of macrophage membranes with genistein, a tyrosine kinase inhibitor, drastically reduced phosphorylation of PLC gamma l. Receptor-associated protein, which blocks alpha 2M binding to LRP/alpha 2MR but not to the alpha 2M signalling receptor, had no effect on alpha 2M-methylamine-induced tyrosine phosphorylation of PLC gamma l. Binding of lactoferrin to LRP/alpha 2MR failed to induce phosphorylation of PLC gamma l, further supporting the hypothesis that the alpha 2M signalling receptor and LRP/alpha 2MR are distinct entities. Growth factors which induce tyrosine phosphorylation typically cause a rise in cytosolic pH. Binding of a2M-methylamine to macrophages also gradually increased the intracellular pH in a concentration-dependent manner, being optimal at a 200 nM ligand concentration. The increase in pH was amiloride sensitive. We propose that receptor-recognized forms of a2M may function like growth factors with regard to macrophage regulation.

The present paper described interactions of urinary-type plasminogen activator (u-PA) with isolated protein components of the extracellular matrix (ECM) using kinetic and ligand-blotting analyses, as well as adhesion studies with u-PA-saturated U937 monocytic cells. Kinetic analyses showed that fibronectin and laminin were moderately effective at decreasing activation of plasminogen by u-PA (3-4-fold decrease in kcat/Km), while activation was stimulated slightly by collagen types I and IV (2-4-fold increase in kcat/Km). Ligand-blotting experiments using intact immobilized ECM proteins demonstrated that u-PA binds predominantly to vitronectin. This was supported by ELISA studies, which showed concentration dependent, saturable, reversible binding of u-PA to vitronectin (Kd,app. of 97 nM). Limited proteolysis of vitronectin followed by ligand-blotting analysis demonstrated u-PA binding to a specific vitronectin fragment (M(r) 49,000), and binding was shown to occur through the N-terminal fragment of u-PA. N-terminal sequence analysis indicated that this binding fragment of vitronectin originates with Thr-122 and comprises the hemopexin domain, including the heparin-binding region of the vitronectin molecule. Plasminogen activator inhibitor type I did not compete with u-PA for binding to vitronectin, suggesting both molecules may co-localize on vitronectin. In contrast, binding of u-PA to vitronectin was significantly inhibited by plasminogen, suggesting these molecules share a common binding site on vitronectin. In addition to in vitro studies, experiments were performed to assess the contribution of direct binding of u-PA to vitronectin on the adhesive behaviour of U937 cells. Binding of u-PA-saturated U937 cells to vitronectin was inhibited 66% by excess vitronectin, suggesting that direct binding of u-PA to vitronectin is the mechanism by which u-PA-dependent adhesion of U937 cells to vitronectin is mediated.

Human alpha 2-antiplasmin (alpha 2-AP) has previously been shown to possess overlapping inhibitory sites for trypsin and chymotrypsin [Potempa, Shieh and Travis (1988) Science 241, 699-700]. Since this is currently unique among active-site-directed inhibitors of proteinases, and difficult to explain in terms of accepted inhibitory mechanisms, we re-examined the claim. Initial characterization of purified alpha 2-AP revealed an additional 12 residues preceding the published N-terminus, prompting us to revise the previous numbering. We found that trypsin caused cleavage of the Arg376-Met377 bond in the reactive-site loop of the inhibitor, whereas chymotrypsin caused cleavage at two sites in approx. equal amounts at 37 degrees C: Met374-Ser375 (site 1) and Met377-Ser378 (site 2). At 0 degrees C alpha 2-AP became a more efficient inhibitor of chymotrypsin, and the proportion of cleavage at site 1 declined, indicating that chymotrypsin prefers to react with site 2 at 0 degrees C. Inhibitors of the alpha 2-AP type are inactivated when cleaved in their reactive-site loops by proteinases that they do not inhibit, so we conclude that site 1 is treated as a substrate by chymotrypsin. Site 2 is the inhibitory site for chymotrypsin. We confirm that alpha 2-AP does indeed have overlapping reactive sites for trypsin and chymotrypsin, and since the locations of chymotrypsin-interaction sites vary with temperature, we suggest that alpha 2-AP cannot have rigid reactive-site geometry. More likely, it has a mobile reactive-site loop of the type that has been recently demonstrated for eglin C.

Human plasma alpha 2-macroglobulin (alpha 2M) is a tetrameric proteinase inhibitor, which undergoes a conformational change upon reaction with either a proteinase or methylamine. As a result, a receptor recognition site is exposed on each subunit of the molecule enabling it to bind to its receptors on macrophages. We have used Fura-2-loaded murine peritoneal macrophages and digital video fluorescence microscopy to examine the effects of receptor binding on second messenger levels. alpha 2M-methylamine caused a rapid 2-4-fold increase in intracellular Ca2+ concentration ([Ca2+]i) within 5 s of binding to receptors. The agonists induced a focal increase in [Ca2+]i that spread out to other areas of the cell. The increase in [Ca2+]i was dependent on the alpha 2M-methylamine concentration and on the extracellular [Ca2+]. Both sinusoidal and transitory oscillations were observed, which varied from cell to cell. Neither alpha 2M nor boiled alpha 2M-methylamine, forms that are not recognized by the receptor, affected [Ca2+]i in peritoneal macrophages under identical conditions of incubation. The alpha 2M-methylamine-induced rise in [Ca2+]i was accompanied by a rapid and transient increase in macrophage inositol phosphates, including inositol tris- and tetrakis-phosphates. Native alpha 2M did not stimulate a rise in inositol phosphates. Finally, binding of alpha 2M-methylamine to macrophages increased cyclic AMP transiently. Thus receptor-recognized alpha-macroglobulins behave as agonists whose receptor binding causes stimulation of signal transduction pathways.

The alpha-macroglobulins are classified as broad-spectrum inhibitors because of their ability to entrap proteinases of different specificities and catalytic class. Tetrameric and dimeric alpha-macroglobulins have been identified in a wide variety of organisms including those as primitive as the mollusc Octopus vulgaris; however, monomeric alpha-macroglobulin proteinase inhibitors have been previously identified only in rodents. The monomeric alpha-macroglobulin proteinase inhibitors are believed to be analogous to the evolutionary precursor of the multimeric members of this family exemplified by the tetrameric human alpha 2-macroglobulin. Until now, monomeric alpha-macroglobulin proteinase inhibitors have only been identified in rodents and have therefore been considered an evolutionary anomaly. However, in this report we have utilized several sensitive assays to screen various plasmas and sera for the presence of monomeric alpha-macroglobulins, and our results suggest that monomeric alpha-macroglobulin proteinase inhibitors are present in organisms belonging to the avian, reptilian, amphibian and mammalian classes of the chordate phylum. This indicates that these proteins are more widespread than previously recognized and that their presence in rodents is not an anomaly. To demonstrate further that the identified proteins were indeed monomeric alpha-macroglobulin proteinase inhibitors, we purified the monomeric alpha-macroglobulin from the American bullfrog Rana catesbeiana. We conclude that this protein is a monomer of 180 kDa on the basis of its behaviour on (i) pore-limit gel electrophoresis, (ii) non-reducing and reducing SDS/PAGE and (iii) gel-filtration chromatography. In addition, we demonstrate that this protein is an alpha-macroglobulin proteinase inhibitor by virtue of (i) its ability to inhibit proteinases of different catalytic class, (ii) the presence of a putative internal beta-cysteinyl-gamma-glutamyl thioester and (iii) an inhibitory mechanism characterized by steric protection of the proteinase active site and by sensitivity to small primary amines. The frog monomeric alpha-macroglobulin is structurally and functionally similar to the well-characterized monomeric alpha-macroglobulin proteinase inhibitor rat alpha 1-inhibitor-3.

Plasmin inhibition by alpha 2-antiplasmin (alpha 2AP) is regulated by the vascular components fibrin(ogen) fragments, plasminogen and lipoprotein (a). Kinetic analysis demonstrates that CNBr-derived fibrinogen fragments completely protect plasmin from alpha 2AP. Plasminogen and 6-aminohexanoic acid decrease the rate of inhibition by 5- and 10-fold respectively. These studies show that CNBr-derived fibrinogen fragments and 6-aminohexanoic acid bind plasmin kringle(s) with binding constants of 2 micrograms/ml and 120 microM respectively, and that plasminogen binds to alpha 2AP with an affinity of 0.5 nM. The unmodulated inhibition is not effected by the presence of lipoprotein (a), but in the presence of protective CNBr-derived fibrinogen fragments the rate of inhibition is increased by the presence of the lipoprotein. The kinetics demonstrate that lipoprotein (a) binds to CNBr-derived fibrinogen fragments with an affinity of 4 nM, displacing plasmin from the protective surface. In addition, tissue-type plasminogen activator and trypsin inhibition by alpha 2AP is not slowed by the presence of CNBr-derived fibrinogen fragments or plasminogen (Pg), respectively. These kinetics suggest that the initial reversible interaction between plasmin and alpha 2AP is mediated by binding of the inhibitor to the kringle 1 domain of plasmin, with a reversible inhibition constant (Ki) of 5.0 x 10(-10) M. Under conditions where this kringle-inhibitor interaction is blocked, the reversible inhibition still occurs between the plasmin and alpha 2AP, but the initial Ki is increased to 5.0 x 10(-9) M. These data suggest that, in the circulation, plasmin inhibition by alpha 2AP may be down-regulated by fibrin, fibrin(ogen) fragments and Pg, but up-regulated by lipoprotein (a) in the presence of fibrin or fibrin(ogen) fragments. The lipoprotein (a)-mediated promotion of plasmin inhibition may provide an additional mechanism by which the lipoprotein impairs fibrinolysis and promotes atherosclerosis.

The cell-free haemolymph of the mollusc Octopus vulgaris inhibited the proteolytic activity of the thermolysin against the high-molecular-mass substrate hide powder azure. The purified inhibitor was a glycoprotein composed of two identical 180 kDa disulphide-linked subunits. In addition to the inhibition of the metalloproteinase thermolysin, the protein inhibited the serine proteinases human neutrophil elastase, pig pancreatic elastase, bovine chymotrypsin, bovine trypsin and the cysteine proteinase papain. A fraction of the proteinase-inhibitor complex resisted dissociation after denaturation indicating that some of the proteinase molecules became covalently bound. The nucleophile beta-aminopropionitrile decreased the covalent binding of proteinases to the Octopus vulgaris protein, suggesting that this interaction is mediated by an internal thiol ester; the reactivity and the amino acid sequence flanking the reactive residues of the putative thiol ester were consistent with this hypothesis. Bound trypsin remained active against the low-molecular-mass chromatogenic substrate H-D-Pro-Phe-Arg p-nitroanilide and was protected from inhibition by active-site-directed protein inhibitors of trypsin; however, the bound trypsin was readily inhibited by small synthetic inhibitors. This indicates that the inhibition of proteinases is accomplished by steric hindrance. The proteinase-inhibitory activity of this protein is characteristic of inhibition by mammalian alpha-macroglobulins and the presence of a putative thiol ester suggests that the Octopus vulgaris proteinase inhibitor is a homologue of human alpha 2-macroglobulin.

There are two major isoenzymes of plasminogen (Pg) in human plasma, designated Pg1 and Pg2. Both Pg forms have an identical primary structure, but differ in their extent of glycosylation. Removal of the oligosaccharide chains alters the normal physiological function of the zymogen and decreases the circulation time of both Pg glycoforms. Recent studies in our laboratory demonstrated that Pg2, with one carbohydrate chain, binds to the surface of U937 monocytoid cells considerably better than Pg1, with two carbohydrate chains, indicating a major role for the carbohydrate chains as determinants for differential binding to the cell surface [Gonzalez-Gronow, Grenett, Fuller & Pizzo (1990) Biochim. Biophys. Acta 1039, 269-276]. In this report we provide evidence that removal of terminal sialic acid from the Thr345-linked oligosaccharide chain of Pg2 is accompanied by the appearance of spontaneous amidolytic and fibrinolytic activity in the single-chain zymogen. Kinetic data demonstrate that asialo-Pg hydrolyses peptide substrates approximately 10% as efficiently as Pm. In addition, the change in carbohydrate content also alters Pg binding to U937 cells. Asialo-Pg binds to U937 cells with a decreased capacity but with a greater affinity than native Pg. Furthermore, asialo-Pg does not compete with native Pg for cell binding. These studies directly demonstrate that the oligosaccharide chains contribute to the heterogeneity observed in the physicochemical and biological properties of Pg1 and Pg2.

Plasminogen, the zymogen form of the serine proteinase plasmin, has been implicated in numerous physiological and pathological processes involving extracellular-matrix remodelling. We have previously demonstrated that the activation of plasminogen catalysed by tissue plasminogen activator is dramatically stimulated in the presence of basement-membrane-specific type IV collagen [Stack, Gonzalez-Gronow & Pizzo (1990) Biochemistry 29, 4966-4970]. The present paper describes the binding of plasminogen to type IV collagen. Plasminogen binds to both the alpha 1(IV) and alpha 2(IV) chains of basement-membrane collagen, with binding to the alpha 2(IV) chain preferentially inhibited by 6-aminohexanoic acid. This binding is specific and saturable, with Kd,app. values of 11.5 and 12.7 nM for collagen and gelatin respectively. Although collagen also binds to immobilized plasminogen, this interaction is unaffected by 6-aminohexanoic acid. Limited elastase proteolysis of plasminogen generated distinct collagen-binding fragments, which were identified as the kringle 1-3 and kringle 4 domains. No binding of collagen to mini-plasminogen was observed. These studies demonstrate a specific interaction between plasminogen and type IV collagen and provide further evidence for regulation of plasminogen activation by protein components of the extracellular matrix.

The glycosaminoglycans (GAGs) heparin, heparan sulphate and chondroitin 6-sulphate stimulate the rate of urokinase activation of human plasminogen. Kinetic analysis of plasminogen activation demonstrates that heparin, heparan sulphate and chondroitin 6-sulphate increased the catalytic rate (Kcat) by 5.3-, 3.5- and 2.5-fold respectively. These stimulatory GAGs had no effect on the affinity of urokinase for plasminogen, since the Km of the reaction is unaltered by the GAGs. The GAGs may enhance the rate of plasminogen activation through an interaction with the catalytic domain of the urokinase, with dissociation constants of approx. 30 nM. Additionally, the lipoproteins, lipoprotein (a) [Lp(a)] and low-density lipoprotein (LDL) inhibit heparin and heparan sulphate stimulation of plasmin formation. Lp(a) is a competitive inhibitor (Kic 20 nM) and LDL is a mixed inhibitor of heparin-enhanced urokinase-mediated plasminogen activation (Kic 24 nM and Kiu 60 nM). These inhibition constants correlate with physiological concentrations of these lipoproteins. These data suggest that these GAGs and lipoproteins may play an important role in vivo in regulating urokinase-mediated plasmin formation.

Human alpha 2-macroglobulin (alpha 2M) was eluted as a single nondispersed peak from a TSK-G4000SW size exclusion chromatography column equilibrated in 20 mM-sodium phosphate/100 mM-NaCl, pH 7.2 (PBS). The void volume and total accessible volume of the column were 6.08 ml and 14.42 ml. The elution volume (Ve) of native alpha 2M was 9.20 +/- 0.04 ml. The Ve was altered minimally by changing the ionic strength or adding ethanol to the equilibration buffer. Ternary alpha 2M-trypsin, containing 2 mol of proteinase/mol of inhibitor, and alpha 2M-methylamine failed to be eluted in well-defined peaks when the column was equilibrated in PBS. The majority of either preparation was recovered slowly at Ve values greater than 14.5 ml, reflecting significant nonideal interactions with the support structure. Addition of 10% ethanol or increased ionic strength in the equilibration buffer independently caused either form of reacted alpha 2M to be eluted in a distinct peak at decreased Ve, suggesting that the nonideal interactions included hydrophobic and electrostatic adsorption. When the equilibration buffer was 80 mM-sodium phosphate/320 mM-NaCl, pH 7.2, partial resolution of ternary alpha 2M-trypsin and alpha 2M-methylamine was obtained with a single column run. The Ve values of ternary alpha 2M-trypsin and alpha 2M-methylamine in this buffer were 13.15 +/- 0.08 ml and 11.94 +/- 0.14 ml, respectively. The Ve of native alpha 2M was 8.84 +/- 0.03 ml. The resolving capacity of TSK-G4000SW was exploited to purify native alpha 2M rapidly and efficiently from solutions that contained significant amounts of either ternary alpha 2M-trypsin or binary alpha 2M-trypsin (1 mol of proteinase/mol of inhibitor). This purification was complete within the limits of sensitivity of denaturing and nondenaturing polyacrylamide-gel electrophoresis. alpha 2M-plasmin was well resolved from native alpha 2M. The Ve of alpha 2M-plasmin was 12.88 +/- 0.32 ml in 80 mM-sodium phosphate/320 mM-NaCl, pH 7.2. A number of procedures were used to prepare solutions with up to 90% binary alpha 2M-trypsin. The Ve of binary alpha 2M-trypsin in these various solutions was intermediate between the values of native alpha 2M and ternary alpha 2M-trypsin. The conformations of binary and ternary complex, as reflected by mobility in nondenaturing electrophoresis, were identical, confirming previous results. Finally, in the binary alpha 2M-trypsin complex, the single trypsin cleaved more than two, and as many as all four alpha 2M subunits.

Human inter-alpha-trypsin inhibitor (I alpha I) is a plasma proteinase inhibitor active against cathepsin G, leucocyte elastase, trypsin and chymotrypsin. It owes its broad inhibitory specificity to tandem Kunitz-type inhibitory domains within an N-terminal region. Sequence studies suggest that the reactive-centre residues critical for inhibition are methionine and arginine. Reaction of I alpha I with the arginine-modifying reagent butane-2,3-dione afforded partial loss of inhibitory activity against both cathepsin G and elastase but complete loss of activity against trypsin and chymotrypsin. Reaction of I alpha I with the methionine-modifying reagent cis-dichlorodiammineplatinum(II) resulted in partial loss of activity against cathepsin G and elastase but did not affect inhibition of either trypsin or chymotrypsin. Employment of both reagents eliminated inhibition of cathepsin G and elastase. These findings suggest that both cathepsin G and elastase are inhibited at either of the reactive centres of I alpha I. Trypsin and chymotrypsin, however, appear to be inhibited exclusively at the arginine reactive centre.

Rat alpha 1-macroglobulin (alpha 1M), rat alpha 2-macroglobulin (alpha 2M) migrated as single bands on non-denaturing gels when purified by the methods described. All three proteins demonstrated increased mobility after reaction with trypsin. A single saturable pathway rapidly cleared complexes of trypsin and the alpha-macroglobulins of mouse, rat and human from the circulation of mice. None of the native alpha-macroglobulins competed for clearance with the trypsin complexes. [14C]Methylamine incorporation was 4.1, 3.9, 2.6 and 3.2 mol/mol of proteinase inhibitor for human alpha 2M, rat alpha 1M, rat alpha 2M and mouse alpha 2M, respectively. Only rat alpha 2M, the acute-phase alpha-macroglobulin studied, showed no evidence of conformational change when subjected to electrophoresis after reaction with methylamine. The clearance of rat alpha 2M-methylamine was comparable with that of the native molecule. The other alpha-macroglobulin-methylamine complexes cleared faster than the inhibitors that had not reacted. Rat alpha 2M and rat alpha 2M-methylamine bound equivalent quantities of 1251-labelled trypsin (1.01 and 0.96 mol/mol respectively). The soya-bean trypsin inhibitor-resistant esterolytic activity of trypsin bound to rat alpha 2M-methylamine was approx. 90% suppressed compared with proteinase bound to native rat alpha 2M. This suppression was not due to a change in the affinity of soya-bean trypsin inhibitor for the complex. Reaction of rat alpha 2M-methylamine with trypsin resulted in a 'slow' to 'fast' electrophoretic conversion of the proteinase inhibitor, and exposure of the signal on the alpha 2M that causes the complex to clear from the murine circulation.

alpha 2-Macroglobulin (alpha 2M)-methylamine that had been allowed to react with cis-dichlorodiammineplatinum(II) (cis-DDP) bound with greatly reduced affinity to specific alpha 2M receptors, as determined by macrophage binding studies in vitro and plasma-clearance experiments in vivo. Subsequent reaction with diethyl dithiocarbamate completely restored receptor recognition function. The optimal effect was obtained when the diethyl dithiocarbamate concentration was twice the total platinum concentration. alpha 2M-methylamine that was allowed to react with H2O2 competed less effectively for specific cell-surface binding sites, as demonstrated by studies both in vivo and in vitro. The apparent dissociation constant was increased nearly 7-fold by a 15 min exposure to H2O2. alpha 2M-methylamine was affected significantly less by the H2O2 exposure after pretreatment with cis-DDP. Amino acid analysis indicated that H2O2 treatment of alpha 2M modified 19 of the 25 methionine residues per alpha 2M subunit. Pretreatment with cis-DDP protected two to four of these methionine residues. The only other residue altered by H2O2 treatment of alpha 2M was histidine. A net decrease of two histidine residues per subunit was observed, but cis-DDP pretreatment did not alter this result. In order to rule out the slight possibility that histidine modification might account for the observed H2O2-induced loss in receptor recognition, diethyl pyrocarbonate was employed as a histidine-modifying reagent. This treatment modified 53 histidine residues in both native and fast-form alpha 2M. Fast-form alpha 2M was still recognized by the alpha 2M receptor, as determined by studies both in vivo and in vitro; however, a fraction of the modified protein now cleared via the acyl-low-density-lipoprotein receptor as well. Reaction of diethyl pyrocarbonate-treated alpha 2M with hydroxylamine reversed derivatization of 43 of the 53 histidine residues. Moreover, this treatment also resulted in an alpha 2M fast-form preparation that was recognized only by the alpha 2M receptor. It is concluded that cis-DDP and H2O2 modify a critical methionine residue in the primary sequence of the alpha 2M-receptor recognition site.

These studies explore the role of carbohydrate recognition systems and the direct involvement of terminal alpha 1-3-linked fucose in the clearance of lactoferrin from the murine circulation and in the specific binding of lactoferrin to receptors on murine peritoneal macrophages. As previously reported, radiolabelled lactoferrin cleared very rapidly (t1/2 less than 1 min) after intravenous injection into mice. However, competing levels of ligands specific for the hepatic galactose receptor (asialo-orosomucoid), the hepatic fucose receptor (fucosyl-bovine serum albumin), and the mononuclear-phagocyte system pathway recognizing mannose, N-acetylglucosamine and fucose (mannosyl-, N-acetylglucosaminyl- and fucosyl-bovine serum albumin) did not block radiolabelled lactoferrin clearance in vivo or binding to mouse peritoneal macrophage monolayers in vitro. Almond emulsin alpha 1-3-fucosidase was used to prepare defucosylated lactoferrin in which 88% of the alpha 1-3-linked fucose was hydrolysed. No difference in clearance or receptor binding was observed between radiolabelled native and defucosylated lactoferrin. Fucoidin, a fucose-rich algal polysaccharide, completely inhibits the clearance in vivo and macrophage binding in vitro of lactoferrin. This effect, however, is probably not the result of competition for binding to the fucose receptor, since gel-filtration studies demonstrated formation of a stable complex between lactoferrin and fucoidin. The present results indicate that the lactoferrin-clearance pathway is distinct from several pathways mediating glycoprotein clearance through recognition of terminal galactose, fucose, N-acetylglucosamine or mannose. Furthermore, alpha 1-3-linked fucose on lactoferrin is not essential for lactoferrin clearance in vivo or specific binding to macrophage receptors in vitro.