Serum concentration of alpha-2-macroglobulin, alpha-1-antitrypsin and alpha-2-antichymotrypsin in patients with lung carcinoma

Serum concentration of alpha-2-macroglobulin, alpha-1-antitrypsin and alpha-2-antichymotrypsin was evaluated in 26 patients with lung carcinoma. We observed an evident decrease in alpha-2-M and alpha-1-antitrypsin level and no differences between tested and control groups in alpha-1-antichymotrypsin concentration. The deficiency of protease inhibitors may be due to the increased level of protease activity in malignant cells. Infiltration of granulocytes near tumor and released enzymes from them may exhaust proteolytic inhibitory capacity, too. Increased protease activity is associated with transformation and uncontrolled proliferation, therefore antiproteases may be accepted as anticancerogenic factors. Further investigations are needed to bring us closer to understanding this question.     

Activity of selected proteinase inhibitors in patients with disorders of lipid metabolism]

The study was aimed at verification of previously found, in animals with experimentally induced atherosclerosis, the disturbances of serum proteinases inhibitors: alpha-2-antiplasmin, alpha-1-antitrypsin and alpha-2-macroglobulin. In humans with hypercholesterolemia the decrease of serum activity of alpha-2-antiplasmin was observed. In humans with hypercholesterolemia and increased serum concentration of triacylglycerols no significant changes in activity of alpha-1-antitrypsin and alpha-2-macroglobulin were found--in comparison with control subjects.     

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.     

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.     

Isolation and characterization of alpha-1-antitrypsin from rhesus-monkey serum.

A simple, relatively gentle, procedure for isolation of rhesus-monkey alpha-1-antitrypsis from serum is described. The method consists of chromatographic separation of the fraction precipitated by 50-75%-satd. (NH4)2SO4 from pooled monkey serum on DEAE-cellulose followed by affinity chromatography on Sepharose-bound concanavalin A. Approx. 30% of the trypsin-inhibitory activity present in the original serum was recovered when alpha-1-antitrypsin was reconstituted with physiological saline (0.85% NaCl). Pure alpha-1-antitrypsin exhibitied a single band on sodium docecyl sulphate/polyacrylamide-gel electrophoresis, with an estimated mol.wt. of 60000 and four bands in acid/starch-gel electrophoresis. The acid/starch-gel-electrophoretic pattern and mobility of isolated material were identical with those of the alpha-1-antitrypsin bands in the original serum sample. The most rapdily migrating bands resembled the pattern and mobility for the normal human phenotype PiM in 28 monkeys. A starch strip from the acid/starch-gel-electrophoresis as the origin for antigen-antibody electrophoresis was used to examine alpha-1-antitrypsin for microheterogeneity; no evidence for microheterogeneity was observed in samples from 18 monkeys. In addition, isolated alpha-1-antitrypsin exhibited a single arc when subjected to immunoelectrophoresis. Amino acid and carbohydrate compositions of isolated monkey alpha-1-antitrypsin were similar to those of human alpha-1-antitrypsin.     

The identification of distinctive forms of human alpha 2-macroglobulin by using the numerical relationship between trypsin binding in alpha- and beta-modes.

Interactions between the serine proteinase trypsin and the protein proteinase inhibitors in human blood were expressed in terms of a coupled set of non-linear differential equations, which has been solved for each of 110 samples of serum obtained from colleagues and from a variety of hospital sources. Optimization of nine unknown theoretical parameters and 21 experimental rate measurements of the hydrolytic activity of trypsin in free and bound states after admixture with various amounts of a given serum was achieved by an iterative procedure using initial estimates of the parameters derived from the "four-straight-line" model described in the preceding paper [Topping & Seilman (1979) Biochem. J. 177, 493--499.] Such a procedure yielded the following information for each sample of serum examined: (a) the concentrations of alpha 1-antitrypsin and alpha 2-macroglobulin; (b) the unequivocal assignment of alpha 2-macroglobulin into one of seven categories on the basis of trypsin binding in two kinetically differentiated modes (alpha and beta); (c) the hydrolytic activities of trypsin (versus Bz-Arg-OEt) when bound to alpha 1-antitrypsin, and to alpha 2-macroglobulin in the alpha- and beta-modes. Molecular interpretations of the binding of trypsin to alpha 2-macroglobulin are discussed and the potential clinical value of recognizing the nature of such binding is reported.     

The evolution of alpha-1 and alpha-2-macroglobulin levels in the serum of rabbit fetus and newborn

The study of the evolution of alpha-1 and alpha-2-macroglobulin levels in the rabbit fetus and newborn reveals an analogy between alpha-1-macroglobulin in the rabbit and alpha-2-macroglobulin in humans.     

Preparation of human alpha-2-macroglobulin by chromatography on Cibacron Blue Sepharose in the presence of pregnancy- associated alpha-2-glycoprotein

The simple and efficient procedure for the isolation of alpha-2-macroglobulin (alpha-2-M) from human sera (hp-type 1-1) by means of affinity chromatography on Cibacron Blue Sepharose is not convenient to separate it from pregnancy-associated alpha-2-glycoprotein (alpha-2-PAG) which is present in high amounts in sera of estrogen-treated women, at pregnancy, and under other conditions. With this method both proteins are eluted in the same fractions; gel filtration of these fractions does not lead to their separation. Therefore, the use of male sera (tesed by monospecific antisera to alpha-2-PAG) with low alpha-2-PAG, content (hp-type 1-1) is recommended for alpha-2-M preparation.     

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.     

The serum protein content of human follicular fluid and its correlation with the maturity of oocytes

The authors determined, by an immunochemical method, the alpha-1-antitrypsin, Gc globulin, prealbumin, albumin, haemopexin, transferrin, haptoglobin, IgA, IgG, ceruloplasmin, alpha-2-macroglobulin contents of 98 follicular fluids, 10 peritoneal fluids and 24 blood sera. Out of these proteins analysed, change in the concentration of alpha-1-antitrypsin showed correlation with the maturity and fertilisation of the oocyte. The alpha-1-antitrypsin content was 1.6 +/- 0.26 g/l in the case of mature oocytes, and 3.1 +/- 1.12 g/l in the case of immature ones. Fertilisation was also concomitant of low alpha-1-antitrypsin levels.     

Tissue alpha-globulins in keloid formation.

The deposition of alpha-1-antitrypsin and alpha-2-macroglobulin, both known to be inhibitors of human skin collagenase, is significantly increased in keloids and in hypertrophic scars (as compared to normal skin). However, following intralesional triamcinolone treatment, a marked resorption of these abnormal scars occurs along with a significant reduction of the alpha-1-antitrypsin deposits. These findings suggest that alpha-globulins are involved in abnormal scar formation, and that triamcinolone may remove collagenase and/or protease inhibitors--thereby allowing activation of the collagenase with subsequent breakdown and resorption of the excessive collagen.     

Modifications of serum glycoproteins the days following a prolonged physical exercise and the influence of physical training.

Eight male subjects (mean age 24.1 +/- 2.6 years) performed at intervals of 2 weeks successively a 3 h and two 2 h runs of different running speed. The days following the running there were moderate elevations of C-reactive protein, haptoglobin, alpha-1-acid glycoprotein, coeruloplasmin, transferrin, alpha-1-antitrypsin and plasminogen. There were small or no changes of albumin, alpha-2-macroglobulin and hemopexin. The elevations of the "acute phase reactants" were examined in three male subjects following a 2 h run before and after an endurance training period of 9 weeks. This demonstrated a decreased acute phase response after training as illustrated by the changes of C-reactive protein, haptoglobin and alpha-1-acid glycoprotein in spite of higher posttraining running speeds. Well-trained athletes have elevated levels of the serum protease inhibitors alpha-1-antitrypsin, alpha-2-macroglobulin and C1-inhibitor. These antiproteolytic glycoproteins might limit exercise-induced inflammatory reactions.     

Purification and N-terminal characterization of Chinchilla villidera alpha-1-antitrypsin

1. Chinchilla, Chinchilla villidera, alpha-1-antitrypsin has been purified to homogeneity and partially characterized according to mol. wt, amino acid and carbohydrate composition and N-terminal amino acid sequence (30 residues). 2. The mol. wt is between 52,000 and 55,000 as determined by PAGE or sedimentation equilibrium. 3. The best alignment between chinchilla, human and baboon alpha-1-antitrypsin amino acid sequences offsets the chinchilla sequence 6 positions vs the primate structures. 4. This alignment suggests potential importance of the sequence His-Glu-Gln-Glu-His at positions 11-15. 5. Additionally, the segment Leu-Ala-Glu-Phe-Ala, positions 25-29, is strictly conserved. 6. Shorter N-terminal sequences available for rat and rabbit alpha-1-antitrypsin appear to follow the offset alignment vs the primate structures.     

Inactivation of human alpha-1-antitrypsin by a tissue-destructive protease of Legionella pneumophila

Three extracellular proteases produced by Legionella pneumophila during growth in liquid medium were examined for their effects on human alpha-1-antitrypsin (alpha-1-AT). One of these proteases, tissue-destructive protease (TDP) destroyed completely the trypsin-inhibitory capacity of alpha-1-AT at protease: inhibitor molar ratios down to 0.002:1. After inactivation by TDP, the Mr of alpha-1-AT was reduced by 5000 in SDS-PAGE. This suggested that inactivation entailed only limited cleavage.     

Interaction between human pancreatic elastase and plasma protease inhibitors

1 ml of human serum inhibits about 0.9 mg of purified human pancreatic elastase owing to complexation with alpha 1-antitrypsin and alpha 2-macroglobulin. On addition to serum, elastase is preferentially bound by alpha 2-macroglobulin. The complexes between elastase and alpha 1-antitrypsin and alpha 2-macroglobulin, respectively, migrate as alpha 2-globulin on agarose gel electrophoresis. Elastase bound by alpha 1-antitrypsin is precipitated by antibodies against enzyme as well as inhibitor, while the alpha 2-macroglobulin-bound elastase is only precipitated by antibodies against the inhibitor. The molar combining ratio for elastase/alpha 1-antitrypsin is 1:1 and for elastase/alpha 2-macroglobulin 2:1. The elastase bound by alpha 2-macroglobulin retains its activity against low molecular weight substrates, while that bound by alpha 1-antitrypsin is enzymologically inactive.     

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.     

Detection of inhibitory inactive fraction of alpha-1-antitrypsin in human serum

Pseudomonas aeruginosa elastase is not inhibited by human serum alpha-1-antitrypsin, it damages inhibitory activity of alpha-1-antitrypsin activity instead. A simple method, based on electrophoresis in urea containing polyacrylamide gel, is described for the separation of active part of the inhibitor from that inactivated by the bacterial enzyme.     

Rat plasma murinoglobulin: isolation, characterization, and comparison with rat alpha-1- and alpha-2-macroglobulins

Murinoglobulin, a newly identified mouse plasma protein with trypsin-protein esterase activity (Saito, A. & Sinohara, H. (1985) J. Biol. Chem. 260, 775-781), was also found in rat plasma and purified to apparent homogeneity. The serum level of rat murinoglobulin was 14.1 mg/ml, amounting to 1/3 of the total serum globulin fraction. Rat murinoglobulin was a monomeric glycoprotein (Mr = 210,000) containing 12% carbohydrate. Rat plasma contained two isoforms of murinoglobulin, termed I and II, which showed complete immunological identity on double diffusion analysis using rabbit antiserum raised against isoform I or II. These antisera also showed partial cross-reactivity towards mouse murinoglobulin and rat alpha-1-macroglobulin but not towards rat or human alpha-2-macroglobulin. The chemical compositions, peptide mapping patterns and electrophoretic mobilities of the two isoforms resembled each other but clearly differed from those of rat alpha-1- or alpha-2-macroglobulin. Rat murinoglobulin inhibited the proteolytic activity of trypsin towards casein and remazol brilliant blue hide powder. The inhibition as to the latter substrate was greater than that as to the former. When molar ratios of inhibitor to trypsin were low, murinoglobulin and the two alpha-macroglobulins stimulated the amidolytic activity of trypsin towards a synthetic substrate. At higher ratios, however, murinoglobulin, but not the alpha-macroglobulins, inhibited the same activity. The trypsin-protein esterase activity of murinoglobulin and the two alpha-macroglobulins was impaired by a molar excess of soybean trypsin inhibitor. Murinoglobulin and the two alpha-macroglobulins were inactivated by methylamine with a concomitant unmasking of the thiol group. Murinoglobulin was much more sensitive to soybean trypsin inhibitor and methylamine than the two alpha-macroglobulins.     

Low pH stability of alpha-1-antititrypsin.

According to the previous findings of others, the trypsin inhibitory capacity of alpha-1-antitrypsin is irreversibly lost in acidic solutions below pH 5.0. In contrast, experiments reported herein show that considerable inhibitory activity can be regenerated as a time-dependent phenomena following titration to basic media. The rate of recovery of activity is accompanied by a decreasing amplitude in the fluorescent emission spectrum at 335 nm of acidified alpha-1-antitrypsin solutions following adjustment to pH 8.0. Acidic media also results in the slow, progressive formation of protein aggregates as measured using Sephadex gel filtration. This latter process is more prominent at pH 4.0, near the isoelectric point of alpha-1-antitrypsin than at pH 3 or 2. Both monomer and polymeric forms of alpha-1-antititrypsin were isolated before or after adjustment to basic media. Isolated monomeric material shows a high recovery of biological and immunological activity; aggregate forms, however, are immunologically cross-reactive but show little enzyme inhibitory activity.