抄録
A human melanoma cell line (Bowes) secretes tissue-type plasminogen activator (t-PA). In order to elucidate the mechanism of t-PA secretion, we analyzed intracellular distribution of t-PA by fractionating cell organelles of melanoma cells with discontinuous sucrose density gradient ultracentrifugation method, and investigated the effect of monensin, a secretion depressing agent, on the intracellular distribution of t-PA. The t-PA was determined by the radioactivity adsorbed to anti-t-PA IgG-Protein A-Sepharose. The distribution of plasminogen activator (PA) activity was similar to that of cellular protein concentration when they were separated by the ultracentrifugation. Enzymography showed that the major intracellular PA activity was present at 72kDa and the minor, activity at 50kDa. When t-PA secretion was depressed by monensin, intracellular PA was accumulated, especially the 72kDa component predominantly. After longterm (3h) simultaneous labeling with 35S-methionine and 3H-mannose, the 3H/35S ratio of t-PA increased in the presence of monensin. Pulse labeling experiments showed that intracellular t-PA was transported from the high density fraction to the low density one and reached a maximum concentration at 30min. Labeled t-PA began to appear in the culture medium in about 30min. These results suggest that t-PA secretion mechanism is similar to that of other secretory glycoproteins and that about 30 minutes are necessary from the ingestion of methionine to the secretion of t-PA.
