Kenji Akazawa

Multiple molecular forms of acid-stable plasmin inhibitor derived from human urinary trypsin inhibitor.

It was found that cyanogen bromide (BrCN) treatment of the highly purified human urinary trypsin inhibitors (H-UTI; specific activity 1,897 U/mg protein, and L-UTI; specific activity 1,850 U/mg protein) readily produced new plasmin inhibitors with almost no loss of UTI activity. Five multiple forms of chemically cleaved inhibitors (UTIB-I, UTIB-II, UTIB-III, UTIB-IV and UTIB-V) could be isolated from BrCN-treated L-UTI by isoelectric focusing and gel filtration. These inhibitors were very acid-stable and their isoelectric points (pI) were 4.5, 4.6, 4.9, 5.1 and 6.4, respectively. The molecular weights by SDS-polyacrylamide gel electrophoresis were almost the same at about 23,000 +/- 3,000. Although these inhibitors showed both anti-plasmin and anti-trypsin activities, much higher anti-plasmin/anti-trypsin activities were observed in the cleaved inhibitors than in the parent UTI. They competitively inhibited human plasmin with Ki values of 3.0-4.1 X 10(-8) mol/l (H-D-Val-Leu-Lys-pNA substrate).

Immunochemical studies of high and low molecular forms of urokinase.

The immunochemical properties of high and low molecular forms of urokinase (HMW-UK, MW 53,000, 124,000 IU/mg protein; LMW-UK, MW 32,000, 230,000 IU/mg protein) were studied with specific antisera against the functionally active heavy chain (H chain, MW 31,000, 201,000 IU/mg protein) and the light chain (L chain, MW 18,000) of HMW-UK. Using a double immunodiffusion technique, LMW-UK did not demonstrate L chain antigenicity in the molecule. Anti-L-chain serum exerted no effect on LMW-UK and the H chain, but anti-H-chain serum strongly inhibited the fibrinolytic activity of all the active enzymes (HMW-UK, LMW-UK, and H chain). Anti-L chain serum was found to exert an antifibrinolytic effect on HMW-UK.