PURIFICATION AND CHARACTERIZATION OF TISSUE PLASMINOGEN ACTIVATOR PRODUCED BY IMR-90 CELLS

Tissue plasminogen activator ( t-PA ) produced by IMR-90 ( human embryonic fibroblast ) cells cultured in the serum-free medium ( DMEM ) containing 1% proteos^e peptone and 1.6 - 3.6mM CaCl₂ was purified by the procedure consisted of ultrafiltration, immunoadsorpt ion chromatography, HPLC and lysine-Sepharose chromatography. The yield of t-PA from the culture broth was approximately 47%. The purified t-PA migrated as a single band on SDS-polyacrylamide gels. The molecular weight of the t-PA was estimated to be 66,000 by SDS-polyacrylamide gel electrophoresis and 69,000 by gel filtration method. Purified t-PA had a specific activity of 36 × 10⁴ IU/mg protein by fiblin plate method or 54 - 56 X 10⁴ IU/mg protein by clot lysis method using t-PA obtained from WHO as a standard. The amino acid composition of fibroblast t-PA was very similar to those of melanoma t-PA and uterine t-PA. Isoelectric point of fibroblast t-PA ranged from 5-7 to 8.2. The t-PA had twice as much affinity for fiblin as did high molecular weight urokinase ( UK ). Both t-PA and UK had optimum temperature at 41°C and optimum pH between 8.0 - 9.0. The polyclonal and monoclonal antibodies raised against t-PA quenched t-PA activity but had no effect on UK activity. The inhibitors of serine proteases, difluorophos-phate and gabexate mesilate, strongly inhibited the activities of fibroblast t-PA and UK. The nucleotide sequence analysis of the t-PA cDNA isolated from the cDNA library prepared from IMR-90 mRNA revealed the nucleotide changes at two positions in the coding region as compared to that of melanoma t-PA cDNA. Neither of the changes replaced the coded amino acid. The N-terminal amino acid of fibroblast t-PA was determined to be valine, indicatig the structural similarity of fibroblast t-PA to uterine t-PA.