Human urinary kallikrein (HUK): large-scale purification and direct solid-phase radioimmunoassay (RIA).

HUK was purified from 1,000 liters of fresh urine by the following procedures: silica gel adsorption, gel filtration on Sephadex G-75, DEAE-Sephadex chromatography, bentonite treatment, affinity chromatography on aprotinin-Sepharose 4B, and rapid gel filtration on a TSK Gel G-3000 SWG column. Seventeen mg of HUK being found to be pure by means of various analyses was obtained. The pI values of the heterogeneous components of HUK were 3.5, 3.8, and 4.1, while the corresponding molecular weights of these components were 5.4 X 10(4), 4.9 X 10(4), and 4.4 X 10(4), respectively. The antigens (125I- and non-labeled HUK) were incubated for 4 hrs at 37 degrees C in polystyrene test tubes to which anti-HUK rabbit IgG had been immobilized. The quantitative range of the standard curve was 1-128 ng. This Ria principally recognized active form of HUK. Therefore, total and inactive HUK also could be determined by the combination of the RIA with trypsin treatment of the urine sample. The RIA correlated closely with both S-2266 amidolytic assay and kininogenase assay.

Some components of the kinin system in Cohn's unheated human plasma proteins.

The presence of a kininase and a kininogenase was demonstrated in unheated human plasma proteins. The kininase was found to inactivate synthetic bradykinin and a kinin-like substance released from a kininogen-like material in unheated plasma proteins by human urinary kallikrein. It was heat-labile and inhibited by EDTA, HgCl2 and 1,10-phenanthroline. This enzyme was estimated to be kininase I from its inhibition pattern. Inactivation of the kininase revealed the presence of a kininogenase. Five per cent heat-treated human plasma protein solution has been demonstrated to have no kininase activity but to contain the kinin-like substance. A mechanism for the formation of the kinin-like substance in 5 per cent heat-treated plasma protein solution was discussed in terms of these kinin forming components present in the unheated plasma proteins.