Effect of cobra and viper venoms on alpha 2-macroglobulin activity in human, bovine, and goat sera

Incubation of human serum with cobra or viper venoms (10 micrograms/0.1 ml serum) caused negligible decrease in total protease inhibitory activity whereas alpha 2-macroglobulin activity was reduced by 67.0-82.0% in 16 hr. The action of venoms on MG activity was time dependent. Human alpha 2-macroglobulin activity was reduced to a much greater extent than goat or bovine factors by the venoms. While 25 micrograms venoms/0.1 ml serum caused 60-100% inhibition of human alpha 2-macroglobulin activity, the bovine factor was not affected under similar conditions. Goat alpha 2-macroglobulin was affected to the extent of 0-20%. Evidence is provided to show that venom proteases generate endogenous proteases in situ in human plasma or serum which in turn bind to alpha 2-macroglobulin. The venom-mediated action was abolished by prior dialysis of the serum or its dilution. Ethylenediaminetetraacetate at 10(-3) M concentration also blocked the reaction. While phenylmethylsulfonyl fluoride had no effect, pepstatin in the concentration range 10(-2) to 10(-3) M caused partial inhibition of the venom-mediated inhibition of alpha 2-macroglobulin activity in human serum.     

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.     

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)     

Proteolytic activity of viper venoms

Proteolytic activities of venoms of vipers kept in a serpentarium for three years or captured in various environmental regions were estimated. Gurza venom contained considerable amounts of protein (830-930 micrograms/mg venom) and displayed a high proteolytic activity by tyrosine (80-140 micrograms/min mg protein). The proteolytic activity did not depend on season, as well as age or physiological state of snakes in the reproductive period. The proteolytic activity of venom in gurza offspring was similar to that in parent specimens. Proteolytic activities (by tyrosine) of venoms produced by Radde's vipers and common vipers were 77-90 and 18-36 micrograms/min mg protein, respectively. The proteolytic activity of venom in common vipers native to the north European part of Russia was 20-30% higher than that in common vipers inhabiting southern European Russia. An inhibition assay found various ratios of metalloendopeptidase and serine endopeptidase activities in venoms of gurza, Radde's viper, and common viper.     

Proteolytic activity of viper venoms

Proteolytic activities of venoms of vipers kept in a serpentarium for three years or captured in various environmental regions were estimated. Gurza venom contained considerable amounts of protein (830-930 μg/mg venom) and displayed a high proteolytic activity by tyrosine (80-140 μg/m in mg protein). The proteolytic activity did not depend on season or the age or physiological state of snakes in the reproductive period. The proteolytic activity of venom in gurza offspring was similar to that in parent specimens. Proteolytic activities (by tyrosine) of venoms produced by Radde’s vipers and common vipers were 77–90 and 18–36 μg/m in mg protein, respectively. The proteolytic activity of venom in common vipers native to the north European part of Russia was 20–30% higher than that in common vipers inhabiting southern European Russia. An inhibition assay found various ratios of metalloendopeptidase and serine endopeptidase activities in venoms of gurza, Radde’s viper, and common viper.     

Functional inactivation and structural disruption of human alpha 2-macroglobulin by neutrophils and eosinophils

Human alpha 2-macroglobulin (alpha 2M) rapidly lost functional and structural integrity in the course of a short-term incubation with either triggered neutrophils or eosinophils. In contrast to native alpha 2M, the modified antiproteinase was unable to bind neutrophil elastase or pancreatic elastase in a manner that restricted the enzymes' access to high molecular weight substrates. In addition to the complete loss of its antiproteolytic potential, the conformation of the dysfunctional inhibitor was radically altered and susceptible to further modification by exogenous proteinases as assessed by polyacrylamide gel electrophoresis. Analysis of the mechanism by which alpha 2M was inactivated by neutrophils revealed that the process was dependent on the generation of hypochlorous acid, an oxidant generated by the hydrogen peroxide-myeloperoxidase-chloride system. In contrast to the neutrophil, maximal eosinophil-dependent inactivation required the presence of physiologic concentrations of bromide and appeared to involve the generation of hypobromous acid. The ability of either hypochlorous acid or hypobromous acid to directly disrupt alpha 2M function and structure was confirmed under cell-free conditions. These results demonstrate that alpha 2M, an antiproteinase heretofore considered to be resistant to physiologic inactivation, could be destroyed by two populations of human phagocytes via oxidative modifications mediated by hypophalous acids.     

Evolution of human alpha 2-macroglobulin

A papain-binding protein (PBP) resembling human alpha 2-macroglobulin (alpha 2M) but of Mr half that of alpha 2M was purified from plaice (Pleuronectes platessa L.) plasma. The plaice protein displayed most of the distinctive inhibitory properties of the human macroglobulin, and was therefore considered, despite its smaller molecular size, to be homologous with alpha 2M. Plaice PBP was shown to consist of four dissimilar subunits; two I chains (Mr 105 000) and two II chains (Mr 90 000). Each of the larger I chains contained a "bait region" sensitive to proteolytic attack by a variety of proteinases, and an autolytic site analogous to the autolytic site of alpha 2M. Subunit I, almost certainly at the autolytic site, formed SDS-stable, covalent links with methylamine or a proportion of the trapped proteinase molecules. A scheme is proposed for the evolution of human alpha 2M from the smaller fish protein, and the possibility of a shared evolutionary origin for alpha 2M and the complement components C3 and C4 is discussed.     

Antiproteinase activity of alpha 2-macroglobulin

Blood serum separation by the method of gel filtration on Sephadex G-200 with the subsequent immunochemical determination of the quantitative content of basic proteolysis inhibitors permitted isolating the alpha 2-macroglobulin fraction while alpha 1-antitrypsin and alpha 1-antichymotrypsin separation was a failure. The immunochemical analysis of the antienzymic activity of the isolated inhibitors showed that 32.3 +/- 3.5% of the introduced kallikrein, 18.7 +/- 0.6% of trypsin and 14.4 +/- 4.1% of chymotrypsin were bound in the zone of alpha 2-macroglobulin. The rest of antienzymic activity was localized in the zone of alpha 1-antitrypsin and alpha 1-antichymotrypsin. After a preliminary saturation of blood serum with trypsin in the amount equivalent to its antitryptic capacity (200 micrograms/ml) the ability of alpha 2-macroglobulin to bind kallikrein and chymotrypsin lowers considerably (by 69 and 72%, respectively). In the zone of alpha 1-antitrypsin and alpha 1-antichymotrypsin a decrease in the ability to bind kallikrein and chymotrypsin amounted to 44 and 12% respectively. Thus, alpha 2-macroglobulin being bound with trypsin looses considerably its ability to bind other enzymes.     

Computer averaging of single molecules of alpha 2-macroglobulin and the alpha 2-macroglobulin/trypsin complex

From electron micrographs single molecules of alpha 2-macroglobulin in the "closed" form, the "open" form and as the trypsin complex have been computer averaged. The molecular images are discussed. Molecules of the electrophoretically fast migrating "F-form" have the "closed" form. In the case of the alpha 2-macroglobulin/trypsin complex the two attached trypsin molecules are located very near to each other and in the central part of the alpha 2-macroglobulin molecule.     

Estimation of serum alpha 2-macroglobulin based on the esterolytic activity of bound alpha-chymotrypsin

A colorimetric method to estimate alpha 2-macroglobulin (MG) in human serum is described which is based on the capacity of MG:alpha-chymotrypsin complex to hydrolyze N-acetyl L-tyrosine ethyl ester in the presence of excess crude preparation of a trypsin/chymotrypsin inhibitor from redwood seed. Acetyltyrosine formed was measured using Folin's reagent (7). The method was found to be as reliable as, but at least five times more sensitive than the procedures described using trypsin and soybean trypsin inhibitor. MG level is expressed in terms of micrograms of bovine alpha-chymotrypsin bound. Serum from healthy males had a lower value (150.5 +/- 31.9 micrograms/ml, n = 20) than in females (196.8 +/- 40.4, n = 20). No significant difference between the levels in fasting and postprandial conditions was observed.     

Platelet alpha2-macroglobulin and alpha1-antitrypsin.

Subcellular membrane and granule fractions derived from human platelets contain immunologically identifiable alpha2-macroglobulin and alpha1-antitrypsin. These platelet-derived inhibitors show a reaction of immunologic identity when compared to alpha2-macroglobulin and alpha1-antitrypsin purified from human plasma. Further, the platelet protease inhibitors possessed a similar subunit polypeptide chain structure to their plasma counterparts as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. Studies of the binding of radiolabeled trypsin to the various solubilized platelet subcellular fractions suggest that the granule-associated alpha2-macroglobulin and alpha1-antitrypsin, as well as membrane-associated alpha2-macroglobulin were functionally active. Quantitatively, circulating platelets contain relatively small concentrations of these inhibitors as compared to platelet-associated fibrinogen and factor VIIIAGN. Platelet protease inhibitors may modulate the protease-mediated events involved in the formation of hemostatic plugs and thrombi.     

Radioimmunoassay of rat acute-phase alpha 2-macroglobulin

A double-antibody radioimmunoassay (RIA) to acute-phase alpha 2-macroglobulin was developed for the quantitation of this large macromolecule in physiological fluids. The primary receptor for the RIA was a monospecific antiserum to purified acute-phase alpha 2-macroglobulin which produced a high titre (7.5 . 10(6)) antibody with a strong affinity for rat acute-phase alpha 2-macroglobulin (Ka = 1.24 . 10(11)) as measured by Scatchard analysis. The validity of the assay was confirmed by specificity for rat alpha 2-macroglobulin measured in various physiological fluids as assessed by parallel dose-response curves; and accuracy, measured by the analytical recovery of alpha 2-macroglobulin by the RIA in serum (104 +/- 7%) and buffer (103 +/- 7%), and the correlation (R = 0.999) of measurements of acute-phase alpha 2-macroglobulin-containing samples measured in serum and buffer. Reference acute-phase serum measured by this RIA and by rocket immunoelectrophoresis were 98.6% in agreement. Radioimmunoassay sensitivity was estimated at less than 1.0 ng alpha 2-macroglobulin/ml, measured over a range of 0-160 ng. Precision was assessed by intraassay (2.99 +/- 0.97%) and interassay (8.76 +/- 2.64%) variation. Evaluation confirmed that quantitation of rat acute-phase alpha 2-macroglobulin by this RIA met the criteria of sensitivity, validity and precision.     

Cadmium binding to human alpha 2-macroglobulin

alpha 2-Macroglobulin (alpha 2M) is one of the major cadmium-binding proteins of human plasma. As determined with equilibrium dialysis, alpha 2M bound 4.6 (+/- 0.7) mol Cd2+ per mol protein with an apparent dissociation constant of (9.6 (+/- 5.0] X 10(-7) M. Methylamine-modified alpha 2M (alpha 2M-Me) had a similar affinity for Cd2+ (Kd,app = 5.3 X 10(-7) M), but fewer binding sites. Cadmium produced a small increase in the amidolytic activity of trypsin in the presence of alpha 2M and soybean trypsin inhibitor. Using the binding parameters determined from the equilibrium dialysis studies, the Cd2+ concentration which produced a half-maximal increase in amidolytic activity corresponded to saturation of all Cd2+-binding sites in one-half of the alpha 2M molecules. From these results, a model is proposed in which one Cd2+-binding site is present in each of the four polypeptide chains which compose alpha 2M.     

Effect of king cobra venom on α2-macroglobulin and proteases in human blood plasma

Normal human blood plasma showed hydrolytic activities on several synthetic substrates for proteases, the most effective being H-D-Ile-Pro-Arg-p-nitroanilide, H-D-Pro-Phe-Arg-p-nitroanilide and H-D-Val-Leu-Arg-p-nitroanilide. When plasma was preincubated for 12 h at 37°C, there was no significant alteration of the hydrolytic activities. On incubation for 12 h with king cobra venom (2 μg for 0.1 ml plasma), there was considerable decrease in the activities and complete abolition of the protease binding capacity of α₂-macroglobulin. On chromatography on Sephadex G-200, α₂-macroglobulin activity and bulk of the protease activity of normal plasma were eluted in the void volume region. A minor protease peak was eluted with aVe/Vo value of 2.5. With venom treated plasma, there was no decrease with this peak. The major protease peak and α₂-macroglobulin activity were drastically reduced. Chromatography on red Sepharose showed that all the α ₂ ⁻ acroglobulin activity and bulk of the protease activity in normal plasma were bound to the column. In venom treated plasma there was marked reduction in the bound fraction. The data suggest that cobra venom proteases directly or through proteases generated in plasmain situ causes limited cleavage of α₂-macroglobulin as well as α₂-macroglobulin bound proteases, inactivating them.     

Proteolytic activity of snake venoms of Costa Rica on casein

Experimental conditions for the comparative measurement of proteolytic activity of Costa Rican snake venoms on casein are established, and the activity of 13 different venoms is described. Venoms showed marked differences in activity not only between species, but between specimens of the same species captured in different geographic regions of Costa Rica.