An Inactive Form of Kallikrein in Human Urine
An inactive form of human urinary kallikrein (inactive HUK) was highly purified from fresh urine collected from healthy men. Inactive HUK was separated from the active kallikrein (HUK) initially presents in the urine by affinity chromatography on a column of aprotinin immobilized on Sepharose 4B and further purified by gel filtration, ion-exchange chromatography and immunoaffinity chromatography on an anti-HUK antibody immobilized Sepharose 4B column.
Inactive HUK was rapidly actigvted by a trace amount of trypsin. While, plasmin, urokinase, thrombin and chymotrypsin caused no activation of inactive HUK.
The molecular weights of inactive HUK and HUK were estimated to be 4.8 × 104 and 4.5 × 104, respectively. The molecular weight of active HUK generated from inactive HUK by the action of trypsin (HUK″) was almost the same as that of HUK.
The mobility of inactive HUK was slightly slower than that of HUK on both Immunoelectrophoresis and polyacrylamide gel disc electrophoresis. On the other hand, the electrophoretic mobility of HUKK″ was almost the same as that of HUK. These two types of active HUK had no significant difference in the Km values for H-Pro-Phe-Arg-MCA hydrolysis and inhibition profiles by various protease inhibitors and anti-HUK antibody.
Inactive HUK was unable to be measured by the direct radioimmunoassay (RIA) but HUK″ generated by the action of trypsin could be measured by the RIA.
KeywordsHuman Urine Immunoaffinity Chromatography Approximate Molecular Weight Fresh Urine Urinary Kallikrein
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