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.