The human melanoma cell line (Bowes) secretes tissue-type plasminogen activator (t-PA) into the culture medium. As reported previously, the secretion of t-PA was depressed and t-PA was accumulated in the intracellular compartment at alternative sodium and potassium ion concentrations and also in the presence of monensin, an ionophore for monovalent cations. In the present study, the secretion and intracellular distribution of t-PA were investigated by radioisotope labeling and fractionation of the cell organelles under normal and monensin-treated conditions. Cell homogenate was fractioneted by discontinuous sucrose density gradient ultracentrifugation. The plasminogen activator (PA) activity or t-PA antigenicity in these fractions was not uniformly distributed, but rather localized in those fractions where cell organelles were rich. This implied that t-PA was enclosed by the intracellular membranous system. Cells were incubated with 35s-methionine and/or 3H-mannose to produce labeled intracellular glycoproteins including t-PA and its premature intermediates, which were separated by immunological adsorption to rabbit anti t-PA IgG-protein A Sepharose. Pulse labeling with 35s-methionine (3 min) demonstrated that intracellular t-PA was transported from the heavier fractions (rER), via intermediate ones (Golgi complex), to lighter ones. The radioactivity of the intracellular t-PA reached a maximum in 30 min, while that of secreted t-PA was observed in 30 min and increased linearly at least for the following 20 min. Enzymography revealed the major intracellular PA activity at 72 kDa and minor activity at 50 kDa. Monensin treatment (10 μM, 6 hr) caused accumulation of the 72 kDa component which was immunologically homologous to secreted t-PA. After long term (3 hr) simultaneous 35s-methionine and 3H-mannose labeling, the radioactivity ratio (3H/35S) the intracellular t-PA was increased in the presence of monensin. These results suggest that some sugar chain(s) in the t-PA molecule are of high mannose type, which is interfered with by monensin in the course of the intracellular processing and transport of t-PA.
Purification of an active plasminogen activator inhibitor immunologically related to the endothelial type plasminogen activator inhibitor from the conditioned media of a human melanoma cell line.