Progressive inhibition of human glandular (urinary) kallikrein by human serum and identification of the progressive antikallikrein as alpha 1-antitrypsin (alpha 1-protease inhibitor).

Human urinary kallikrein was inhibited by human alpha 1-antitrypsin (alpha 1-protease inhibitor) in a similar way as by equivalent amounts of human serum. The inhibitor present in the kallikrein-inhibitor complex formed was identified as alpha 1-antitrypsin by two-dimensional immunoelectrophoresis. Under the experimental conditions applied, 90 mIU (= 185 microgram) alpha 1-antitrypsin inhibits about 9 microgram of human urinary kallikrein in 24 h at 37 degrees C, 1 ml of human serum, containing 2-4 mg alpha 1-antitrypsin, inhibits about 70 microgram kallikrein. At an incubation temperature of 25 degrees C, the rate of inactivation is significantly lower than at 37 degrees C. No inhibition was observed at 0 degrees C or when alpha 1-antitrypsin was presaturated with trypsin.

Biochemistry and functional aspects of human glandular kallikreins.

Human urinary kallikrein was purified by gel filtration on Sephacryl S-200 and affinity chromatography on aprotinin-Sepharose, followed by ion exchange chromatography on DEAE-Sepharose. In dodecylsulfate gel electrophoresis two protein bands with molecular weights of 41,000 and 34,000 were separated. The amino acid composition and the carbohydrate content of the kallikrein preparation were determined; isoleucine was identified as the only aminoterminal amino acid. The bimolecular velocity constant for the inhibition by diisopropyl fluorophosphate was determined as 9 +/- 2 1 mol-1 min-1. The hydrolysis of a number of substrates was investigated and AcPheArgOEt was found to be the most sensitive substrate for human urinary kallikrein. Using this substrate an assay method for kallikrein in human urine was developed. It was shown by radioimmunoassay that pig pancreatic kallikrein can be absorbed in the rat intestinal tract. Furthermore, in dogs the renal excretion of glandular kallikrein from blood was demonstrated by radioimmunological methods.

Isolation and characterization of human urinary kallikrein.

Human urinary kallikrein was purified by gel filtration on Sephacryl S-200 and affinity chromatography on aprotinin-Sepharose, followed by ion exchange chromatography on DEAE-Sepharose. Thus an enzyme preparation with a specific activity (using AcPheArgOEt as substrate) of 1 100 U/mg protein was obtained. A specific bioligical activity of 2 300 KE/mg was measured in the dog blood pressure assay and of 0.742 HMW kininogen-U/mg, corresponding to the liberation of 787 micrograms bradykinin per mg enzyme per min from HMW-kininogen, in the rat uterus assay. In dodecyl sulfate electrophoresis two protein bands with apparent molecular weights of 41 000 and 34 000 were separated. The amino acid composition was determined and isoleucine was identified as the only aminoterminal amino acid. On isoelectric focusing six protein bands with isoelectric points of 3.75, 3.80, 3.90, 4.00, 4.05 and 4.25 were separated. The kinetic constants for the kallikrein-catalyzed hyrdolysis of AcPheArgOEt and D ValLeuArgNHNp were determined. The bimolecular velocity constant for the inhibition by diisopropyl fluorophosphate was determined as 9 +/- 2l x mol-1 x min-1. Immunological studies showed that a close relationship exists between the urinary enzyme and other human glandular kallikreins. Deoxycholate, lysolecithin and other amphiphiles activated human urinary kallikrein.