Immunological analysis of rat pancreatic prokallikrein activation.
The present study shows that tissue kallikrein is present in rat pancreas as a proenzyme that can be converted by autolysis to a 38 000 Da active enzyme. The activation of pancreatic prokallikrein was examined by direct radioimmunoassay, enzymatic assays, active-site labeling with immunoprecipitation, and Western blot analyses. A monoclonal antibody (V1C3), which binds only active kallikrein, was used in a direct radioimmunoassay to monitor the appearance of the active enzyme. During a 22-h autolysis of pancreatic extract, a time-dependent increase in active kallikrein concentration paralleled the increase of kallikrein activities measured by both TosArgOMe esterase and kininogenase assays. The activation process was further analyzed by labeling the pancreatic extract with [14C]diisopropylphosphorofluoridate [( 14C]DFP) followed by immunoprecipitation with sheep anti-kallikrein antiserum. Pancreatic prokallikrein was not labeled by [14C]DFP; however, upon autolysis, a 38 000 Da active kallikrein can be labeled with [14C]DFP and increase in quantity with time. Western blot analysis, using a monoclonal antibody (V4D11) which recognizes both latent and active tissue kallikreins, identified a 39 000 Da pancreatic prokallikrein prior to autolysis and a 38 000 Da active kallikrein after 7 h of autolysis. The results indicate that the pancreatic prokallikrein exists as a 39 000 Da protein which may be converted to a 38 000 Da active kallikrein, indistinguishable from purified urinary, brain, spleen or submandibular gland kallikrein.
D000818:Animals; D000911:Antibodies, Monoclonal; D004789:Enzyme Activation; D007531:Isoflurophate / Q000378:metabolism; D007610:Kallikreins / Q000276:immunology / Q000378:metabolism*; D008297:Male; D008970:Molecular Weight; D010179:Pancreas / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000378:metabolism; D011288:Prekallikrein / Q000378:metabolism*; D011863:Radioimmunoassay; D051381:Rats; D011919:Rats, Inbred Strains; D012756:Sheep
D000911:Antibodies, Monoclonal; D007531:Isoflurophate; D011288:Prekallikrein; D010447:Peptide Hydrolases; C023073:tosylarginine methyl ester hydrolase; D007610:Kallikreins