Partial Purification and Characterization of Epidermal Plasminogen Activator and Their Inhibitor

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Plasminogen activator (PA) and PA inhibitor were partially purified from 2-d-old rat epidermis and characterized in order to elucidate the enzyme-inhibitor interaction in epidermis. PA extracted with buffer containing KSCN was first purified by Blue-Sepharose affinity chromatography. Separation of two PAs, with relative molecular mass (M_r) of 66000 and 44000,was accomplished by Con A-Sepharose column chromatography. The M_r 66000 enzyme had the properties of tissue-type PA (t-PA), while the M_r 44000 enzyme showed those of urokinase-type PA (u-PA) as determined by immunological and fibrin-binding studies. PA inhibitor was extracted in 1,4-piperazinediethanesulphonic acid buffer and purified by (NH_4)_2SO_4 precipitation, gel filtration followed by a Mono Q column in an FPLC system. This inhibitor showed M_r 60000 and inhibited human u-PA activity in a dose- and time-dependent manner, but did not inhibit the activity of t-PA from human and murine melanoma cells or plasmin. It inhibited epidermal PA, M_r 44000,more effectively than it did the M_r 66000 epidermal PA. It was stable at 60℃ for 60 min or between pH 5 and 11. This study indicates that both u-PA and t-PA function in normal rat epidermis. On the other hand, an inhibitor which preferentially acts against u-PA exists, but inhibitor to t-PA does not appear to operate under normal epidermal functions.
社団法人日本薬学会の論文
1989-07-25