Purification and characterization of tissue plasminogen activator secreted by human embryonic lung diploid fibroblasts, IMR-90 cells.
The anti-urokinase-IgG-resistant plasminogen activator secreted by human embryonic lung diploid fibroblasts, IMR-90 cells (ATCC, CCL186) was purified to homogeneity from serum-free conditioned medium by a four-step procedure. The fibroblast plasminogen activator was identified as tissue plasminogen activator (t-PA) by the N-terminal sequence of the purified material and the complete amino acid sequence deduced from its complementary DNA (cDNA). The apparent molecular weight was the range of 64,000 to 68,000 by SDS-PAGE and was in the range of 69,000 to 72,000 by gel filtration. The fibroblast t-PA showed a stricter substrate specificity than urokinase in enzymatic hydrolysis of various chromogenic substrates. Compared to urokinase, the fibrobrast t-PA was more stable by heating at 95 degrees C for five min and was stable from pH 5 to 10. The fibrorast t-PA had a higher affinity for fibrin than urokinase.
Research Institute of Life Science, Snow Brand Milk Products Co., Ltd., Tochigi, Japan.
Y
Y
Itagaki
H
H
Yasuda
T
T
Morinaga
S
S
Mitsuda
K
K
Higashio
D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004171:Diploidy; D004795:Enzyme Stability; D005347:Fibroblasts / Q000737:chemistry* / Q000502:physiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008168:Lung / Q000737:chemistry* / Q000166:cytology; D008969:Molecular Sequence Data; D013696:Temperature; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D004247:DNA; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator