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.     

Effect of methylamine and plasmin on the conformation of human alpha 2-macroglobulin as revealed by differential scanning calorimetric analysis.

Differential scanning calorimetric analysis was used as a probe of the conformational alteration in human alpha 2-macroglobulin (AM) upon its complex formation with methylamine and with the protease, human plasmin. The slow electrophoretic form of AM displayed a single thermal transition, characterized by a temperature midpoint (Tm) of 65.8 +/- 0.3 degrees, a calorimetric enthalpy (delta Hc) of 2,550 +/- 150 kcal/mol and a van't Hoff enthalpy (delta Hvh) of 140 kcal/mol. In the presence of sufficient methylamine to irreversibly disrupt the four thiol ester bonds in AM, a single thermal transition was obtained, characterized by a Tm of 62.8 +/- 0.3 degrees, a delta Hc of 1,700 +/- 100 kcal/mol, and a delta Hvh of 169 kcal/mol. These data suggest that a major conformational alteration is produced in AM upon complex formation with methylamine. When plasmin interacts with AM, the resulting thermogram displays Tm values for AM of 68-69 degrees and 77 degrees, also suggestive of a large conformational alteration in AM. However, this latter alteration appears dissimilar to the change induced by methylamine.     

Physical properties of human alpha 2-macroglobulin following reaction with methylamine and trypsin

Circular dichroism spectroscopy, sedimentation velocity and ultraviolet difference spectroscopy were used to compare alpha 2-macroglobulin, alpha 2-macroglobulin-trypsin complex and alpha 2-macroglobulin-methylamine complex. The circular dichroic spectrum of native alpha 2-macroglobulin is significantly changed in shape and magnitude following reaction with either trypsin or methylamine. The spectra of alpha 2-macroglobulin-trypsin and alpha 2-macroglobulin-methylamine are, however, indistinguishable. The ultraviolet difference spectrum between alpha 2-macroglobulin-methylamine and native alpha 2-macroglobulin displays a tyrosine blue shift consistent with the exposure of several tyrosine residues to solvent. The conformational change which occurs in alpha 2-macroglobulin during reaction with methylamine follows pseudo-first-order kinetics. T 1/2 was 10.5 min for the reaction with 200 mM methylamine at pH 8.0 and 45 min for the reaction with 50 mM methylamine, also at pH 8.0. Reaction of methylamine with alpha 2-macroglobulin results in loss of trypsin-binding activity which appears to be a direct consequence of the conformational change induced by methylamine. A sedimentation coefficient (S0(20),W) of 20.5 was determined for alpha 2-macroglobulin-methylamine compared to a value of 18.5 for unreacted alpha 2-macroglobulin. This increase in sedimentation velocity is attributed to a 10% decrease in alpha 2-macroglobulin Stokes radius. alpha 2-Macroglobulin-trypsin complex prepared by reaction of the protease at a 2-fold molar excess with the inhibitor was a S0(20),W of 20.3. Although this sedimentation coefficient does reflect compacting of the alpha 2-macroglobulin structure compared to native alpha 2-macroglobulin, it is not large enough to rule out significant protrusion of the proteases from pockets in the alpha 2-macroglobulin structure.     

Effect of methylamine on the reaction of alpha 2-macroglobulin with enzymes.

The kinetics of reaction of alpha 2-macroglobulin (alpha 2M) with thrombin and with trypsin were studied in the presence and absence of methylamine. The rate of enzyme-induced thiol release was found to be the same whether or not amine was present. The result suggests that covalent bond formation and enzyme-catalyzed amine incorporation proceed via a common (enzyme-dependent) rate-determining step. The reaction of lysyl-modified enzymes (which show poor covalent binding with alpha 2M) was similarly unaffected by amine, indicating that enzyme-catalyzed steps were also rate determining for hydrolysis of the thiol ester. The products of the reactions were analyzed by native and denaturing gel electrophoresis. Methylamine did not affect the total binding of enzyme to alpha 2M but did cause a substantial decrease in covalent binding. Surprisingly, not all covalent complexes were affected by the presence of amine: complexes in which enzyme was covalently bound to one half-molecule increased compared to the reaction with no amine; complexes in which two half-molecules are cross-linked by two bonds to a single enzyme were substantially reduced, however. The results are consistent with a mechanism of reaction in which an enzyme-dependent step is rate determining. This step is accompanied by activation of two thiol esters. One of these reacts immediately with the bound enzyme (or may be hydrolyzed if the enzyme amine groups are blocked). The other activated center is capable of reaction with external nucleophiles such as methylamine.     

Mechanism of hypochlorite-mediated inactivation of proteinase inhibition by alpha 2-macroglobulin.

The proteinase-proteinase inhibitor balance plays an important role in mediating inflammation-associated tissue destruction. alpha 2-Macroglobulin (alpha 2M) is a high-affinity, broad-spectrum proteinase inhibitor found abundantly in plasma and interstitial fluids. Increased levels of alpha 2M and proteinase-alpha 2M complexes can be demonstrated in patients with sepsis, emphysema, peridontitis, rheumatoid arthritis, and other inflammatory diseases. Despite these increased levels, proteolysis remains a significant problem. We hypothesized that a mechanism for inactivating alpha 2M-mediated proteinase inhibition must exist and recently demonstrated that alpha 2M isolated from human rheumatoid arthritis synovial fluid is oxidized and has decreased functional activity. The oxidant responsible for alpha 2M inactivation and the mechanism of such destruction were not studied. We now report that while hypochlorite and hydroxyl radical both modify amino acid residues on alpha 2M, only hypochlorite can abolish the ability of alpha 2M to inhibit proteinases. Hydrogen peroxide, on the other hand, has no effect on alpha 2M structure or function. Protein unfolding with increased susceptibility to proteolytic cleavage appears to be involved in alpha 2M inactivation by oxidation. The in vivo relevance of this mechanism is supported by the presence of multiple cleavage fragments of alpha 2M in synovial fluid from patients with rheumatoid arthritis, where significant tissue destruction occurs, but not in patients with osteoarthritis. These results provide strong evidence that hypochlorite oxidation contributes to enhanced tissue destruction during inflammation by inactivating alpha 2M.     

Inhibition of inflammatory proteinase, medullasin, by alpha 2-macroglobulin and ovomacroglobulin

The in vitro activity of inflammatory proteinase, medullasin, was stoichiometrically inhibited by a serum proteinase inhibitor, alpha 2-macroglobulin, and its homolog, chicken ovomacroglobulin. The two inhibitors were cleaved by medullasin only in the bait region. The effectiveness of alpha 2-macroglobulin to inhibit medullasin in competition with alpha -1-proteinase inhibitor was measured under a simulated in vivo condition and an estimation was made that about 60-70% medullasin is inhibited by alpha-1-inhibitor and 30-40% by alpha 2-macroglobulin.     

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

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

Reaction of methylamine with human alpha 2-macroglobulin. Mechanism of inactivation

The human protease inhibitor alpha 2-macroglobulin (alpha 2 M) is inactivated by reaction with methylamine. The site of reaction is a protein functional group having the properties of a thiol ester. To ascertain the relationship between thiol ester cleavage and protein inactivation, the rates of methylamine incorporation and thiol release were measured. As expected for a concerted reaction of a nucleophile with a thiol ester, the rates were identical. Furthermore, both rates were first order with respect to methylamine and second order overall. The methylamine inactivation of alpha 2M was determined by measuring the loss of total protease-binding capacity. This rate was slower than the thiol ester cleavage and had a substantial initial lag. However, the inactivation followed the same time course as a conformational change in alpha 2M that was measured by fluorescent dye binding, ultraviolet difference spectroscopy, and limited proteolysis. Thus, the methylamine inactivation of alpha 2M is a sequential two-step process where thiol ester cleavage is followed by a protein conformational change. It is the latter that results in the loss of total protease-binding capacity. A second assay was used to monitor the effect of methylamine on alpha 2M. The assay measures the fraction of alpha 2M-bound protease (less than 50%) that is resistant to inactivation by 100 microM soybean trypsin inhibitor. In contrast to the total protease-binding capacity, this subclass disappeared with a rate coincident with methylamine cleavage of the thiol ester. alpha 2M-bound protease that is resistant to a high soybean trypsin inhibitor concentration may reflect the fraction of the protease randomly cross-linked to alpha 2M. Both the thiol ester cleavage and the protein conformational change rates were dependent on methylamine concentration. However, the thiol ester cleavage depended on methylamine acting as a nucleophile, while the conformational change was accelerated by the ionic strength of methylamine. Other salts and buffers that do not cleave the thiol ester increased the rate of the conformational change. A detailed kinetic analysis and model of the methylamine reaction with alpha 2M is presented. The methylamine reaction was exploited to study the mechanism of protease binding by alpha 2M. At low ionic strength, the protein conformational change was considerably slower than thiol ester cleavage by methylamine. Thus, at some time points, a substantial fraction of the alpha 2M had all four thiol esters cleaved, yet had not undergone the conformational change. This fraction (approximately 50%) retained full protease-binding capacity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Selective cleavage of glycyl bonds by papaya proteinase IV

The specificity of papaya proteinase IV (PPIV) has been examined with small substrates and a protein. With both classes of substrate, the enzyme shows a marked selectivity for cleaving glycyl bonds. Boc-Ala-Ala-Gly-NHPhNO₂ is a convenient substrate for routine assays that discriminate well against chymopapain, the most common contaminant of PPIV. Sixteen cleavage points in β-trypsin were identified, of which 13 are glycyl bonds. Tentative suggestions are made as to the reasons for lack of cleavage of some other glycyl bonds. The structure of PPIV has been modelled on that of papain, and we suggest that the replacement of the highly conserved residues Gly-65 and Gly-23 by arginine and glutamic acid, respectively, can account for the specificity of PPIV.     

Electron microscopical localization of the alpha 2-macroglobulin thiol ester sites

The active thiol ester groups of alpha 2-macroglobulin (alpha 2M) were reacted with a biotin derivative and the sites labelled with avidin-ferritin complexes. Electron micrographs show a strong preference of attachment of the ferritins to the ends of the rods of the H-shaped molecules. A mutual "cross-labelling" was observed in an alpha 2M preparation which yielded dimers of the molecules which must have been formed during purification. The molecules were mostly attached to each other at the ends of the rods of the H-shaped molecules. It is concluded that the thiol esters responsible for the covalent attachment of the proteinases (and other molecules) may be located more in the distal parts of the alpha 2M molecules, while the proteinase molecules are finally trapped near to the centre of the alpha 2M molecules.     

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.     

The effect of alpha2-macroglobulin from bovine serum on bovine alpha-chymotrypsin.

Bovine alpha2-globulin contains a protein which increases the activity of bovine alpha-chymotrypsin against synthetic substrates. The active protein fraction migrates slowly on polyacrylamide gel electrophoresis, so it was named slow alpha2-globulin (Salpha2). The fraction was isolated from bovine serum and purified. Its sedimentation constant S20 was 18.5 S. It was thus identified with the alpha2-macroglobulin (alpha2M). By kinetic studies, the dissociation constant of the alpha-chymotrypsin-alpha2 M complex was calculated to be of the order of 10(-7) l/mol. The purified alpha2 M was shown to bind alpha-chymotrypsin at a definite rate. If the binding ratio was assumed to be 1:2, the molecular weight was calculated to be about 8 X 10(5).     

Spin-label and fluorescence labeling studies of the thioester bonds in human alpha 2-macroglobulin

Upon cleavage of the reactive thioester bonds (Cys-949-Glx-952) of tetrameric human alpha 2-macroglobulin (alpha 2M) by methylamine, one sulfhydryl group per alpha 2M subunit is exposed. These identical sulfhydryl group sites were labeled with the thiol-specific nitroxide spin-labels (1-oxy-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methyl methanethiosulfonate and (1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)methyl methanethiosulfonate, a homologous series of maleimide spin-labels, and the thiol-specific fluorescent probe 2-[(4-maleimidophenyl)amino]naphthalene-6-sulfonic acid sodium salt (MANS). The ESR and fluorescence results showed that these sulfhydryl group sites were at the base of a narrow crevice that is greater than or equal to 8 A deep. Although the bound MANS fluorophore was slightly blue shifted with an enhanced quantum yield vs the free label in water, the environment of the sulfhydryl site appeared to be of a polar nature when compared with the emission maxima in several solvents of varying polarity. The Glx residue participating in the thioester linkage in the intact protein was labeled with 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl. The distance between the Glx and Cys moieties was estimated at greater than or equal to 10-25 A from double spin-labeling experiments.