Murinoglobulin, a novel protease inhibitor from murine plasma. Isolation, characterization, and comparison with murine alpha-macroglobulin and human alpha-2-macroglobulin

Two glycoproteins having trypsin-protein esterase activity were purified to apparent homogeneity from murine plasma. One was alpha-macroglobulin, a homologue of human alpha-2-macroglobulin, while the other, tentatively named murinoglobulin, did not correspond to any of the known plasma protease inhibitors that have been well characterized in men or other mammals. Murinoglobulin contained about 7.6% carbohydrate and was composed of a single-polypeptide chain of Mr = 180,000 as judged by the equilibrium sedimentation analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Murinoglobulin did not cross-react immunologically with mouse alpha-macroglobulin nor with human alpha-2-macroglobulin. Protease-inhibiting properties of murinoglobulin were compared with those of mouse alpha-macroglobulin and human alpha-2-macroglobulin. All the three proteins inhibited trypsin, papain, and thermolysin, although they differed considerably in both the degree of inhibition and the binding stoichiometry of protease-inhibitor complexes. The two macroglobulins inhibited pepsin at pH 5.5, whereas murinoglobulin was inactivated at this pH. Murinoglobulin was more sensitive to methylamine than the two macroglobulins. No protein corresponding to murinoglobulin was detected in human plasma.     

Amidolytic activity of porcine trypsin bound to human plasma alpha-1-antiproteinase

Human plasma alpha-1-antiproteinase interacted with porcine trypsin in two different manners. One was a well known interaction, which resulted in inhibition of the proteolytic activity of the trypsin. The other has not been described to date, and resulted in retention of the amidolytic activity of the trypsin towards benzoyl-L-arginine p-nitroanilide in the presence of soybean trypsin inhibitor. The latter, so-called trypsin-protein amidase, activity is essentially the same as that observed with vertebrate alpha-macroglobulin and rodent murinoglobulin under similar conditions. All attempts to separate the two different activities as well as to abolish either activity by means of chemical or physical modifications were unsuccessful. The proteolysis-inhibiting interaction, which was virtually completed within 5 min, was predominant over the amidolysis-retaining interaction, when the inhibitor/trypsin molar ratio was less than 1. On the other hand, the amidolysis-retaining interaction, which proceeded much more slowly, became evident when the molar ratio was greater than 1.     

Changes in trypsin-binding properties and conformation of rabbit alpha-2-macroglobulin on reaction with methylamine

Reactions of rabbit alpha-2-macroglobulin with methylamine and trypsin were studied and the results were compared with those obtained for previously described 2-macroglobulins from other species. Rabbit alpha-2-macroglobulin was cleaved by trypsin at a number of sites, whereas the human homologue was split essentially only in the "bait" region into two fragments of similar sizes. Reaction of native or methylamine-treated rabbit alpha-2-macroglobulin with trypsin resulted in a substantial decrease in the intensity of fluorescence induced by binding of 6-(p-toluidino)-2-naphthalenesulfonate or bis(8-anilino-1-naphthalenesulfonate). Under the same conditions, the fluorescence of the human protein increased. The time course of the reaction of rabbit alpha-2-macroglobulin with methylamine was studied by measuring (i) the generation of thiol groups, (ii) the decrease in trypsin-inhibiting activity with remazol brilliant blue hide powder as the substrate, and (iii) the decrease in trypsin-protein amidase activity. The thiol appearance reaction exhibited a multiphasic time course. The initial phase was found to follow second-order kinetics with an apparent rate constant of 1.2 M-1.s-1. Under the same conditions, the human protein showed monophasic kinetics with a rate constant of 12 M-1.s-1. Both the trypsin-inhibiting activity and the trypsin-protein amidase activity concurrently decreased at a slower rate than the thiol appearance. These results indicate that rabbit alpha-2-macroglobulin is more stable to nucleophilic attack by methylamine but less resistant to proteolysis by trypsin than the human homologue, and that the final conformation induced by methylamine differs considerably from that induced by trypsin.     

Trypsin inhibitors in guinea pig plasma: isolation and characterization of contrapsin and two isoforms of alpha-1-antiproteinase and acute phase response of four major trypsin inhibitors

Contrapsin and two isoforms, F (fast) and S (slow), of alpha-1-antiproteinase (also called alpha-1-proteinase inhibitor) were isolated in an apparently homogeneous state from plasma of inflamed guinea pigs. Contrapsin inactivated trypsin, but did not significantly affect chymotrypsin, pancreatic elastase, or pancreatic kallikrein. On the other hand, both isoforms of alpha-1-antiproteinase inhibited trypsin, chymotrypsin, and elastase, but not plasma or pancreatic kallikrein. The S isoform of alpha-1-antiproteinase was present in barely detectable amounts in healthy animals, but increased markedly when the acute-phase reaction was induced by subcutaneous injection of turpentine. On the other hand, the plasma levels of the F isoform, contrapsin, and alpha-macroglobulin showed moderate (1.5 to 2.3-fold) elevation during the acute-phase reaction. In contrast to the previous findings that rats and rabbits contain two different alpha-macroglobulins, one of which is an acute-phase reactant while the other is not, inflamed guinea pigs contained only one species of alpha-macroglobulin. Murinoglobulin, the most prominent acute-phase negative protein in both mice and rats, showed no significant change in guinea pigs. These results indicate that guinea pig plasma contains four major trypsin inhibitors, i.e., contrapsin, alpha-1-antiproteinase, alpha-macroglobulin, and murinoglobulin, the properties of which are very similar to those of the respective mouse homologues, but that the acute-phase response of these inhibitors differs greatly from that of the homologous proteins in rats or mice.     

Isolation and characterization of alpha-macroglobulin from guinea pig plasma

Guinea pig alpha-macroglobulin was purified to apparent homogeneity by sequential chromatography on Sephacryl S-300, DEAE-cellulose, and hydroxyapatite. A molecular weight of 780,000 was obtained by equilibrium sedimentation. The preparation migrated as a single band of Mr = 180,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Rabbit antiserum raised against the final preparation partially cross-reacted with human and rat alpha-2-macroglobulins but not with rat alpha-1-macroglobulin. Guinea pig alpha-macroglobulin stimulated the amidolytic activity of trypsin towards a small substrate, but inhibited the proteolytic activity of trypsin towards remazol brilliant blue hide powder. When treated with trypsin or methylamine, four thiol groups per molecule were newly generated. The reaction with trypsin proceeded with at least at two different rates: half of the thiol groups were generated in a fast reaction and the remaining half in a slower reaction. On the other hand, such a two-step reaction was not detected in the reaction with methylamine. The methylamine-treated alpha-macroglobulin retained half the capacity to bind trypsin and its mobility in polyacrylamide gel under nondenaturing conditions remained virtually unchanged. These properties are in marked contrast to those reported for human alpha-2-macroglobulin, but resemble those of rat alpha-2- and mouse alpha-macroglobulins. The amidase activity of trypsin bound to guinea pig alpha-macroglobulin was impaired by soybean trypsin inhibitor to a much greater degree than that of trypsin bound to human or rat alpha-2-macroglobulin.     

Guinea pig plasma murinoglobulin. Purification and some properties

Murinoglobulin, a newly identified mouse plasma protein resembling alpha-macroglobulins [Saito, A. & Sinohara, H. (1985) J. Biol. Chem. 260, 775-781], was also found in guinea pig plasma, and purified to homogeneity. Guinea pig murinoglobulin consisted of a single 180-kDa polypeptide chain containing about 18% carbohydrate. It inhibited the proteolytic activities of trypsin and thermolysin towards Remazol brilliant blue hide powder, but stimulated the amidolytic activities of trypsin and Staphylococcus aureus V8 protease towards small synthetic substrates. Heat treatment of murinoglobulin completely abolished the former activities, but partially retained the latter activities. The ability of guinea pig murinoglobulin to inhibit the proteolysis was much weaker than that of the mouse homologue. On interaction with trypsin, murinoglobulin underwent cleavage of one susceptible bond with concomitant unmasking of one thiol group. Methylamine treatment also released one thiol group per molecule.     

Effects of alpha-macroglobulins and murinoglobulins on the hemagglutination by influenza C virus

Rat alpha-1- and alpha-2-macroglobulins as well as rat murinoglobulins I and II were shown to inhibit hemagglutination by influenza C virus. In marked contrast, neither alpha-macroglobulins nor murinoglobulins from mouse or guinea pig plasma had the inhibitory activity. These results suggest that the hemagglutination-inhibiting activity of rat alpha-macroglobulins or murinoglobulins is not related to their protease-binding capacity.     

Characterization of an alpha-macroglobulin-like glycoprotein isolated from the plasma of the soft tick Ornithodoros moubata.

We report the identification of the first representative of the alpha-2-macroglobulin family identified in terrestrial invertebrates. An abundant acidic glycoprotein was isolated from the plasma of the soft tick Ornithodoros moubata. Its molecular mass is about 420 kDa in the native state, whereas in SDS/PAGE it migrates as one band of 190 kDa under nonreducing conditions and a band of 92 kDa when reduced. Chemical deglycosylation reveals that it is composed of two different subunits, designated A and B. The N-terminal amino-acid sequence of subunit A is similar to the N-terminus of invertebrate alpha-2-macroglobulin. Sequence analysis of several internal peptides confirms that the tick protein belongs to the alpha-2-macroglobulin family, and the protein is therefore referred to as tick alpha-macroglobulin (TAM). Functional analyses strengthen this assignment. TAM inhibits trypsin and thermolysin cleavage of the high-molecular-weight substrate azocoll in a manner similar to that of bovine alpha-2-macroglobulin. This effect is abolished by pre-treatment of TAM with methylamine. In the presence of TAM, trypsin is protected against active-site inhibition by soybean trypsin inhibitor. We cloned and sequenced a PCR product containing sequences from both subunits and spanning the N-terminus of subunit B and the putative 'bait region' (a segment of alpha-2-macroglobulin which serves as target for various proteases). This indicates that the two subunits are generated from a precursor polypeptide by post-translational processing.     

Clostridiopeptidase B inhibition by plasma marcroglobulins and microbial antiproteases.

Clostridiopeptidase B (EC 3.4.22.8) was not inhibited by stoichiometric amounts of lima bean trypsin inhibitor, ovomucoid trypsin inhibitor, Kuntiz bovine trypsin inhibotor, Kunitz soybean trypsin inhibitor or ovoinhibitor. Activity was diminished at relatively high concentrations of the three latter inhibitors. Human plasma alpha 2-macroglobulin inhibited both the amidase and protease activity of the enzyme. Rat and dog plasmas contained high molecular weight inhibitors, presumably macroglobulins as well. Inhibition by this component was greater in rat plasma than in dog plasma, which may be related to the observation that clostridiopeptidase B-induced generation of kinin activity is indirect in the former plasma, but direct in the later. Leupeptin (N-acetyl-L-leucyl-L-leucyl-L-argininal) and antipain ([S)-1-carboxy-2-phenylethyl] carbamoyl-L-arginyl-L-valyl-L-argininal) inhibited clostridiopeptidase B (Ki of 2 . 10(-8) and 3 . 10(-8) M, respectively). They were potent inhibitors of clostridiopeptidase B-induced kinin release in dog plasma.     

Three high molecular weight protease inhibitors of rat plasma. Reactions with trypsin

We have compared the reactions of trypsin with human alpha 2-macroglobulin (alpha 2M), and three rat plasma protease inhibitors, alpha 1-macroglobulin (alpha 1M), alpha 1-inhibitor III (alpha 1I3), and alpha 2M. All four of these proteins appear to contain reactive thiol esters. The electrophoretic mobility in agarose gels of human and rat alpha 2M is increased by 1 mol of trypsin, while the mobility of alpha 1M and alpha 1I3 is decreased. Treatment with methylamine causes similar mobility changes, except in the case of rat alpha 2M. Titration of human and rat macroglobulins by repeated small additions of trypsin and by assay of liberated SH groups or enhanced ligand fluorescence revealed a stoichiometry of about 1 mol of trypsin/mol of inhibitor. In contrast, addition of macroglobulin to a fixed amount of trypsin and detection of residual amidase or protease activity revealed a stoichiometry of about 2 mol of trypsin for 1 mol of human alpha 2M, about 1.4 mol for rat alpha 1M, and about 1 mol for rat alpha 2M. One mol of trypsin reacted with 2 or more mol of alpha 1I3 by the criteria of SH groups liberated or protease inhibition. Methylamine-treated rat alpha 2M binds a significant amount of trypsin releasing about 2 mol of SH. Radioactive beta-trypsin was covalently bound to subunits of the purified plasma inhibitors. The Mr of the labeled products with rat and human alpha 2M had molecular weights which suggested trypsin was bound to intact as well as cleaved subunit chains and also to multiple chains via cross-linking. Rat alpha 1M also produced a product which may be an intact subunit alpha chain plus trypsin. Greater than 80% of the trypsin was bound covalently to these inhibitors at low molar ratios.     

Binding of zinc to alpha-2-macroglobulin and its role in enzyme binding activity.

Physicochemical studies performed on alpha-2-macroglobulin were correlated with the biological activities of this protein. Equilibrium dialysis of the binding of 65Zn by alpha-2-macroglobulin at pH 7.9 showed heterogeneous binding which could be attributed to two classes of binding sites. The site of greatest affinity for zinc had an apparent stoichiometry (n1 in gatoms/mol of alpha-2-macroglobulin monomer) of 12 and an apparent association constant (K1) of 3.06.10(7). The second binding site had an n2 of 60 and K2 of 1.32.10(5). The trypsin binding activity of alpha-2-macroglobulin did not depend on the presence of zinc in this protein since all but traces of this metal could be removed by EDTA without loss of trypsin binding activity. Saturation of site 1 with zinc did not affect the trypsin binding activity of alpha-2-macroglobulin, but binding of the metal by site 2 progressively decreased the trypsin binding activity by causing an irreversable association of the alpha-2-macroglobulin molecules. Removal of excess zinc from alpha-2-macroglobulin did not restore its trypsin binding activity. Our results also indicate that the high zinc content of alpha-2-macroglobulin (320--770 microgram/g protein) reported in the literature is an artifact and that native alpha-2-macroglobulin contains approximately 150--180 microgram Zn/g protein.     

Circular dichroic spectroscopy of non-human alpha-macroglobulins

Bovine, chicken and frog alpha-macroglobulins and ovomacroglobulin were studied by circular dichroic spectroscopy over the region 205-250 nm. All four spectra exhibited negative ellipticity with minima at about 215 nm similar to that reported for human alpha 2-macroglobulin. On reaction of the alpha-macroglobulins with trypsin, the spectrum of each of the four changed similarly. However, these proteins exhibited different conformational changes when treated with methylamine. These differences were exploited to determine which characteristics of alpha-macroglobulins correlate with changes in circular dichroic spectroscopy.     

Isolation and partial characterization of rat urinary esterase A2

An enzyme, esterase A2, which hydrolyzes tosyl-arginine methyl ester was isolated from the urine of female, inbred, Dahl-salt-resistant rats using DEAE-Sephadex ion-exchange, aprotinin-agarose affinity and molecular sieve column chromatography. The purest preparation obtained showed four closely migrating bands on polyacrylamide gel electrophoresis. All four bands of the esterase A2 preparation had enzyme activity since all were stainable on zymograms using N-acetyl-L-methionine alpha-naphthyl ester as substrate. Three of these four bands showed decreased electrophoretic mobility following treatment with neuraminidase, indicating that variable sialic acid content accounts for part of the microheterogeneity. The preparation of esterase A2 used was free of rat urinary kallikrein as shown by radioimmunoassay, electrophoretic and isoelectric focusing experiments. The relative kinin-generating ability of rat urinary kallikrein and esterase A2 was highly dependent on the assay used. Using canine plasma as a source of kininogen and the rat uterus to bioassay kinins, esterase A2 was 47% as active as kallikrein; using pure bovine low-molecular-weight kininogen and a radioimmunoassay to measure generated kinins, esterase A2 was only 6% as active as kallikrein. Esterase activity of A2 was activated non-specifically by proteins and detergents. Esterase A2 was 50% inhibited by an 8-fold molar excess of aprotinin and by a 26.5-fold molar excess of soybean trypsin inhibitor, but ovomucoid inhibitor was not inhibitory.     

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

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

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.     

alpha-1-Anti-trypsin, an inhibitor of renin.

A naturally occurring competitive inhibitor of pig kidney renin has been identified in human plasma. The inhibitor was shown to be alpha-1 anti-trypsin and the effect in vitro on the renin activity was examined. The slope in the Hill plot is compatible with the assumption of one-site competitive inhibition. Other proteinase inhibitors, such as alpha-2-macroglobulin and C1 inactivator, however, have no inhibitory effect on the renin-angiotensinogen reaction.     

Trypanosoma cruzi: inhibition of host cell uptake of infective bloodstream forms by alpha-2-macroglobulin

The infection of murine macrophages and fibroblasts by recently isolated infective bloodstream trypomastigotes of Trypanosoma cruzi is inhibited by the addition of human plasma protease inhibitor alpha-2-macroglobulin (alpha 2M) or of soybean trypsin inhibitor. The ingestion of the non-infective epimastigotes by macrophages is not affected by the physiological protease inhibitor. Incubation of bloodstream trypomastigotes for 20 h in a serum-free axenic medium enhances their ability to infect macrophages in a process influenced by the temperature and sensitive to alpha 2M. After this period the infectivity of the parasites to cells was not sensitive to alpha 2M. These observations suggest that proteases located on the surface and/or secreted by the bloodstream trypomastigote form of T. cruzi may modulate its ability to infect host cells.     

Inactivation of alpha- and beta-thrombin by antithrombin-III, alpha 2-macroglobulin and alpha 1-proteinase inhibitor.

Inactivation of alpha- and beta-thrombin by alpha 2-macroglobulin, by alpha 1-proteinase inhibitor and by antithrombin-III and heparin was studied. The amount of alpha- and beta-thrombin inactivated by antithrombin-III was proportional to the concentration of the inhibitor, but the inactivation rates of the two forms of thrombin were different. Heparin facilitated complex-formation between alpha-thrombin and antithrombin-III, whereas inactivation of beta-thrombin by antithrombin was only slightly influenced, even at a heparin concentration two orders of magnitude higher. alpha 2-Macroglobulin inhibited both alpha- and beta-thrombin activity similarly, i.e. the amount of alpha- and beta-thrombin inactivated as well as the rates of their inhibition were the same. alpha 1-Proteinase inhibitor also formed a complex with alpha- and beta-thrombin, similarly to antithrombin-III, although the inactivation of the enzyme needed high inhibitor concentration and long incubation time. These results suggest that the inactivation of beta-thrombin, if it occurs in the plasma, is also controlled by plasma inhibitors.     

Differential interactions of rabbit plasma alpha-1-antiproteinases S and F with porcine trypsin

Two major forms of rabbit plasma alpha-1-antiproteinase, S and F, were separated by affinity chromatography on Red Sepharose, and their modes of interaction with porcine trypsin were studied. The S form interacted with trypsin much more slowly than the F form, and the resulting complex partially retained the amidolytic and proteolytic activities towards benzoyl-L-arginine p-nitroanilide and remazol brilliant blue hide powder, respectively. This S form-trypsin complex also prevented the inactivation of bound trypsin by soybean trypsin inhibitor. In marked contrast, an equimolar complex of trypsin and the F form retained neither amidolytic nor proteolytic activity. These results suggest that the F form blocks the active site of trypsin while the S form does not bind directly to the active site, thereby preserving the catalytic potential of trypsin. No similar interaction was observed, however, between the S form and either bovine chymotrypsin or porcine pancreatic elastase. Both the S and F forms inactivated these proteinases in a stoichiometric manner with differing inhibitor/proteinase binding ratios. The S form showed about twofold greater capacity to inhibit elastase than the F form, whereas the reverse was the case for chymotrypsin.     

alpha2-Macroglobulin from hemolymph of the freshwater crayfish Astacus astacus.

1. A high mol. wt proteinase inhibitor has been purified from the haemolymph of the freshwater crayfish Astacus astacus. 2. The protein is a disulphide-bonded dimer (Mr 390,000) of two identical polypeptide chains (Mr 185,000). 3. The inhibitor displays a broad specificity and protects trypsin from inhibition by soybean trypsin inhibitor and thus is similar to vertebrate alpha 2-macroglobulin. 4. The alpha 2-macroglobulin-like inhibitor from Astacus interacts with bovine trypsin in an equimolar stoichiometry thereby decreasing tryptic hydrolysis of N-benzoyl-L-arginine-ethylester to 50% residual activity. In contrast, the activity of Astacus protease, a digestive zinc proteinase from crayfish toward succinyl-alanyl-alanyl-alanyl-4-nitroanilide is inhibited almost completely. 5. Sensitivity of the inhibitor to methylamine and autolytic cleavage suggests the presence of an internal thioester bond. 6. The N-terminal amino acid sequence of Astacus alpha 2-macroglobulin is strongly related to the alpha 2-macroglobulins from Pacifastacus leniusculus (91% identity) and from the lobster Homarus americanus (72% identity). In contrast, only 25% of the residues are identical with the alpha 2-macroglobulin from the horseshoe crab Limulus polyphemus. There is also a faint similarity to human complement protein C3 and human alpha 2-macroglobulin.