Abstract
A fibrinolysis inhibitor was extracted and partially purified from human cornified cells. The inhibitor activity was quantitatively measured for plasmin and urokinase in amydolytic reactions of chromogenic tri-peptide substrates, H-D-Val-Leu-Lys-pNA and PyroGlu-Gly-Arg-pNA. Human cornified cells scraped from heels were homogenized in 0.1M Tris-HCl+0.14M NaCl, pH 8.0, and supernatant was separated by centrifugation at 24, 000g. The extract did not show inhibition for plasmin. One mg of protein of the extract, whereas, inactivated urokinase that hydrolyzes 3.0 nmole of the substrate per minute (3.0mIU/mg protein). The inhibitory effect was found to increase with time for 30 minutes in a enzyme-inhibitor mixture, illustrating “time dependent inhibition”. Sephacryl S-200 gel chromatography and subsequent DEAE Sepharose ion exchange chromatography yielded the urokinase inhibitor fraction that exhibited 254mIU/mg protein in specific activity. SDS-polyacrylamide 10% gel electrophoresis showed seven protein bands in this fraction. Molecular weight of the urokinase inhibitor was estimated to be approximately 35, 000 in the gel chromatography.
A proteinase inhibitor that progressively inactivates urokinase was demonstrated in human cornified cells. We suggest that a regulatory function of the urokinase inhibitor possibly participates in physiological and pathological proteolytic mechanisms in the epidermis.
