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.
Arima
K
K
Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Iizuka, Japan.
Nakamura
Y
Y
Takami
N
N
Mizusaki
K
K
Yoshida
N
N
Okamoto
K
K
D000230:Adenocarcinoma / Q000473:pathology; D000911:Antibodies, Monoclonal; D001201:Ascites; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007150:Immunohistochemistry; D007422:Intestines / Q000033:anatomy & histology; D008259:Macroglobulins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D010996:Pleural Effusion / Q000737:chemistry*; D012697:Serine Endopeptidases / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D008259:Macroglobulins; D012697:Serine Endopeptidases