| Co-Investigator(Kenkyū-buntansha) |
TAKADA Yumiko Hamamatsu Univ., Sch. Med. Dept. Physiol., Assistant, 医学部, 助手 (90092981)
URANO Tetsumei Hamamatsu Univ., Sch. Med. Dept. Physiol., Assoc. Prof., 医学部, 助教授 (50193967)
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| Research Abstract |
We transfected CHO cells or E. Coli with CDNA of plasminogen activator inhibitor-1(PAI-1), and obtained eukaryotic PAI-1(rePAI-1)and prokaryotic PAI-1(rpPAI-1), and studied their interactions with tissue plasminogen activator(t-PA). PAI-1 treated with 0.01 % SDS could not form a stable complex with t-PA or u-PA. On the other hand, PAI-1 treated with 0.1 % SDS did not form a complex with t-PA, and a new band appeared on SDS-PAGE which was smaller in molecular weight about 5 KD, suggesting a C terminal peptide split at the reaction site Arg-Met. PAI-1 treated with 0.01 % SDS showed a weaker inhibition of t-PA, and in the presence of 0.1 % SDS, PAI-1 did not show any inhibition of the activity of t-PA. The measurement using circular dichroism showed that PAI-1 had little extent of a helix, but PAI-1 treated with SDS had higher extent of a helix, suggesting that PAI-1 changed its secondary conformation after the treatment with SDS. These results show that changes in the conformation converted PAI-1 from inhibitor to substrate, providing a good model for the analysis of enzymesubstrate interaction. In order to study the role of PAI-1 in the presence of PAI-1, we used euglobulin clot lysis time(ECLT). These was a significantly positive correlation between ECLT and the concentration of PAI-1, suggesting that the fibrinolytic potential of the blood is primarily determined by tile concentration of PAI-1. The high concentration of PAI-1 inactivates t-PA, and low concentration of PAI-1 keeps enough amounts of t-PA to cause fibrinolysis by activating plasminogen.
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