The applicant proposed to characterize and study inactive glandular kallikrein in urine. It is known that approximately 50% of immunoreactive kallikrein in urine exists in an inactive form. It is not known whether this inactive moiety represents a pro-enzyme, kallikrein bound to an inhibitor or a combination of these two species. A defect in activation of pro-kallikrein or the presence of excess inhibitory activity may be responsible for the low kallikrein activity noted in human urine in certain disease states such as essential hypertension. We plan to isolate prokallikrein using a combination of Trasylol-sepharose affinity chromatography followed by immuno-affinity chromatography with specific anti-kallikrein antiserum. We anticipate that these steps will yield a relatively pure preparation of prokallikrein. We will then characterize prokallikrein and study its activation in vitro. Kallikrein inhibitor in urine will be isolated using insolubilised kallikrein (bound to sepharose or agarose) to compete with endogenous kallikrein for any bound inhibitor. The inhibitor wll then be eluted from the kallikrein-sepharose gel. One we have characterized both prokallikrein and kallikrein inhibitor in urine, we plan to study these moieties in disease states associated with decreased kallikrein activity in urine, e.g. essential hypertension and chronic renal disease.
NIH ExPorter
NIH ExPorter