An alpha2-macroglobulin-serine proteinase complex from human carcinomatous ascites and pleural effusion: isolation, monoclonal antibody preparation, and immunohistochemical study.

A protein with the apparent molecular mass of 720 kDa which hydrolyzes anilide substrates of p-guanidino-L-phenylalanine was purified from ascites and pleural effusion of patients with pulmonary, breast, gastric, and ovarian cancers by chromatographic techniques. When this protein was separated on SDS-PAGE on nonreducing conditions, two bands corresponding to 720 and 360 kDa were seen to have gelatin-digestive activity in zymography assay. Moreover, when it separated by SDS-PAGE on reducing conditions, it migrated as several bands up to 180 kDa. The N-terminal amino acid sequence and immunoreactivity of anti-alpha2-macroglobulin polyclonal antibody revealed that the 180-kDa band was intact alpha2-macroglobulin. The hydrolytic activity of this complex was completely inhibited by diisopropyl fluorophosphate (DFP) and p-amidinophenylmethanesulfonyl fluoride. In addition, the 65-kDa protein observed under reducing conditions bound 3H-labeled DFP. These results suggest that the purified protein is a complex of the plasma proteinase inhibitor alpha2-macroglobulin and a serine proteinase. Several monoclonal antibodies were obtained when the purified complex was used as an antigen. One of these antibodies, which was immunoreactive to this complex but not to alpha2-macroglobulin, gave a positive band corresponding to 65 kDa on SDS-PAGE under reducing conditions. Use of this antibody in immunohistochemical studies revealed immunoreactivities in numerous neoplastic tissues with strong activity in advanced gastric cancers (e.g., poorly differentiated adenocarcinoma). In addition, strong cross-reactivity was detected in glandular cells of the fetus intestine.

[The evolutionary problems of the family of human and animal macroglobulins]. / Problemy évoliutsii semeistva makroglobulinov cheloveka i zhivotnykh.

Studies have been made on physicochemical and immunochemical properties of macroglobulins, as well as of associated with pregnancy glycoproteins, from human subjects, mammals, birds, fishes and invertebrates. It was shown that these proteins exhibit similar composition, structure and capacity to bind proteinases inhibiting the latter. Using immunochemical methods, reactions of antigenic identity of these proteins were investigated. A hypothesis of evolutionary formation of macroglobulin family is discussed.

Isolation of nine human plasma proteinase inhibitors by sequential affinity chromatography.

Purification of nine plasma proteinase inhibitors and one zymogen from a single batch of human plasma, using affinity chromatography has been accomplished. Those isolated were plasminogen (lysine-Sepharose), alpha-2-antiplasmin (plasminogen-Sepharose), high and low molecular weight kininogens (CM-papain-Sepharose), alpha-2-macroglobulin (Zn++ chelate-Sepharose), alpha-1-proteinase inhibitor, alpha-1-antichymotrypsin, Cl-inhibitor, inter-alpha-trypsin inhibitor (Blue-Sepharose) and antithrombin III (heparin-Sepharose). Alpha-2-macroglobulin and alpha-1-proteinase inhibitor required gel filtration as additional purification steps. Each protein was recovered in both high yield and purity.

Physical and chemical characterization of macroglobulin from marine turtle eggwhite.

Macroglobulin was purified from the eggwhite of the marine turtle (Caretta caretta Linn.) by gel filtration through Sephadex G-200 and Sepharose 4B. It was characterized by physical techniques including sedimentation velocity, diffusion and viscosity. Its molecular weight (Mr) was determined as 724,000 with four subunits of equal molecular weight. A large amount of water was hydrodynamically associated with the macroglobulin. It inhibited the activities of trypsin and papain and did not cross-react with human alpha 2-macroglobulin. Its amino acid composition was similar to that of human alpha 2-macroglobulin. Results suggest that turtle eggwhite macroglobulin is a homologous but distinct protein from human alpha 2-macroglobulin.

Purification and characterization of alpha-macroglobulin and ovomacroglobulin of the green turtle (Chelonia mydas japonica).

The plasma alpha-macroglobulin and egg white ovomacroglobulin were purified from the sea turtle, Chelonia mydas japonica, and their structural and functional properties were studied with the aim of clarifying the degree of evolutional divergence of two homologous proteins specific to different tissues of the same animal. The concentration of alpha-macroglobulin in green turtle plasma was about 4 mg/ml. The protein was purified from the plasma by precipitation with polyethylene glycol 6000, followed by zinc chelate chromatography and gel chromatography on Sepharose CL-6B. The concentration of ovomacroglobulin in green turtle egg white was about 0.4 mg/ml. Ovomacroglobulin was purified by gel chromatography on Sepharose CL-6B. The two proteins had similar molecular weights and amino acid compositions, and both inhibited proteinases such as trypsin, chymotrypsin, papain, and thermolysin. The amino terminal sequences of the two proteins were homologous to each other but higher homologies were found between the ovomacroglobulin of turtle and chicken, and between the serum macroglobulins of the same animals. The functional difference between turtle alpha-macroglobulin and ovomacroglobulin became clear when they were treated with methylamine, which is known to destroy the inhibitory activity of human alpha 2-macroglobulin by splitting internal thiolester bonds. The inhibitory activity of the turtle plasma protein was completely destroyed by methylamine but that of ovomacroglobulin was only partially affected. The number of sulfhydryl groups as titrated with 5,5'-dithiobis(2-nitrobenzoate) before and after treatment with proteinases or methylamine was different for the two proteins. The amount of radioactive methylamine that was incorporated was also different between the two proteins. The two proteins purified in this study had no immunological cross-reactivity.

Venom resistance in the hedgehog, Erinaceus europaeus: purification and identification of macroglobulin inhibitors as plasma antihemorrhagic factors.

Hedgehog (Erinaceus europaeus) plasma contains factor(s) which neutralize the hemorrhagic activity of European viper (Vipera berus) venom. These antihemorrhagic factor(s) were purified by gel filtration on Sephacryl S-200 and chromatography on Cibacron Blue Sepharose. The macroglobulin fraction obtained was characterized for proteinase inhibiting activity, molecular weight and purity by polyacrylamide gel electrophoresis and immunological cross-reactivity and was found to contain the three macroglobulin proteinase inhibitors--alpha 2-macroglobulin, alpha 2-beta-macroglobulin and beta-macroglobulin. The purified macroglobulins were totally able to neutralize Vipera berus venom hemorrhagic activity, but no distinction between them in this respect was possible.

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.

Tumor-induced growth-inhibitory activity isolated from the Yoshida ascites fluid by extraction with methanol.

Growth-inhibitory activity was isolated from the Yoshida ascites fluid by sequential precipitation with polyethylene glycol, extraction with methanol, LH 20 Sephadex chromatography and preparative agarose gel electrophoresis. The cell non-specific activity was tumor-related as far as analogous fractions prepared from normal sera were inactive. Flow cytometric analysis indicates that the inhibition of cell growth was caused by blockage of the G1-S and possibly the G2-M phase transitions. The active component migrates electrophoretically associated to an unidentified alpha 1-antigen. In aqueous solution the molecular size of the inhibitor is ill defined, due to a tendency to autoaggregation and hydrophobic interaction with the chromatographic media. The molecular weight of the inhibitor as estimated by LH 20 Sephadex chromatography in methanol is approximately 350 daltons. This chromatographic fraction contains prostaglandin-E2 cross-reactive material in amounts suggesting the participation of a prostaglandin derivative in the observed growth-inhibitory activity.

Human macromolecular insoluble cold globulin (MICG). I. T-cell origin of T-MICG and null cell origin of N-MICG.

Although surface immunoglobulin characterizes B cells in man, there are few surface markers that distinguish T cells. We have described a new protein synthesized in human T cells, termed T-MICG. This protein is a macromolecule of 225,000 daltons, is insoluble in the cold, and migrates as a beta-globulin on electrophoresis. Separation of human peripheral blood lymphocytes into T and B-cell populations by rosette sedimentation and anti-human-Fab columns clearly demonstrated the T-cell origin of the 225,000 dalton component. Furthermore, null cells were shown to synthesize a protein of 185,000 daltons, termed N-MICG, with physical properties similar to T-MICG, T-MICG and N-MICG were shown to be antigenically dissimilar, employing antiserum to each of these proteins. The present studies demonstrate two novel cell surface markers, T-MICG and N-MICG, which characterize T cells and null cells, respectively.

Towards characterization of epidermal cell migration promotion activity in serum.

The effect of various fractions of serum on active epidermal cell migration in vitro has been studied by incubating small explants of normal human skin in the presence of the fractions and determining the extent of migration after 48 h incubation. Serum was fractionated by combining ammonium sulphate precipitation and molecular sieve chromatography using Sephadex G-200. The components of serum responsible for most, if not all, of the migration promotion activity were localized in the G-200 third peak (albumin fraction) of the supernatant after precipitation of the serum with ammonium sulphate at 50% saturation.

[Antithrombin III deficiency and tendency to thrombosis (author's transl)]. / Antithrombin-III-Mangel und Thromboseneigung

Antithrombin III (AT III) was determined in 290 patients with deep venous thrombosis and/or pulmonary embolism by immunological methods (radial immunodiffusion, Laurell technique) and by biological activity (heparin cofactor activity and anti-Xa activity). Patients with venous thrombosis had a significantly lower AT III concentration, as determined by the immunological methods or biological method (heparin cofactor activity), than normal persons without any history of venous thrombosis. A decreased level of AT III was found in 27 patients. In these patients the immunoreactive antithrombin III was decreased to the same degree as biological activity (heparin cofactor activity or anti-Xa activity). Thirteen out of these 27 patients belonged to 9 families and, hence, congenital AT III deficiency can be assumed in these cases. The aetiology was unknown in the other half. Patients with AT III deficiency are prone to spontaneous and/or recurrent venous thrombosis. A high incidence of pulmonary embolism and particularly, of fatal pulmonary embolism is remarkable. In more than half of the patients the first thrombotic event occurred before the age of 35. The treatment of choice in such patients is with oral anticoagulants of the coumarin group.

The effect of human alpha 2-macroglobulin on the restoration of humoral responsiveness in x-irradiated mice.

The effect of human alpha 2-macroglobulin on the recovery of the humoral immune response in X-irradiated mice has been assessed by measurement of antibody-forming cells in the spleen 5 days after challenge with sheep erythrocytes. The capacity of this protein to promote the recovery of immune responsiveness was variable, and appeared to be influenced by the strain and sex of the mice, and the dose of alpha2M given.