An alpha(2)-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 anil ide substrates of p-guanidino-L-phenylalanine was purified from ascites and pleural effusion of patients with pulmonary, breast gastric, and ovarian c ancers by chromatographic techniques. When this protein was separated on SD S-PAGE on nonreducing conditions, two bands corresponding to 720 and 360 kD a were seen to have gelatin-digestive activity in zymography assay. Moreove r, when it separated by SDS-PAGE on reducing conditions, it migrated as sev eral bands up to 180 kDa. The N-terminal amino acid sequence and immunoreac tivity of anti-alpha(2)-macroglobulin polyclonal antibody revealed that the 180-kDa band was intact alpha(2)-macroglobulin. The hydrolytic activity of this complex was completely inhibited by diisopropyl fluorophosphate (DFP) and p-amidinophenylmethanesulfonyl fluoride. In addition, the 65-kDa prote in observed under reducing conditions bound H-3-labeled DFP. These results suggest that the purified protein is a complex of the plasma proteinase inh ibitor cll-macroglobulin and a serine proteinase. Several monoclonal antibo dies were obtained when the purified complex was used as an antigen. One of these antibodies, which was immunoreactive to this complex but not to alph a(2)-macroglobulin, gave a positive band corresponding to 65 kDa on SDS-PAG E under reducing conditions: Use of this antibody in immunohistochemical st udies revealed immunoreactivities in numerous neoplastic tissues with stron g activity in advanced gastric cancers (e.g., poorly differentiated adenoca rcinoma). In addition, strong cross-reactivity was detected in glandular ce lls of the fetus intestine.