Identification of tissue kallikrein in brain and in the cell-free translation product encoded by brain mRNA.
A monoclonal antibody against purified rat urinary kallikrein was coupled to agarose and used to isolate kallikrein from rat brain. The purified enzyme has N alpha-tosyl-L-arginine methyl esterase activity with a pH optimum at 9.0, kinin-releasing activity from a purified low molecular weight kininogen, and a parallelism with standard curves of rat urinary kallikrein in a direct radioimmunoassay. Brain kallikrein is inhibited by a series of tissue kallikrein inhibitors with IC50 values similar to those for urinary kallikrein. The purified brain enzyme was labeled with [14C]diisopropylphosphorofluoridate and visualized by fluorography on a sodium dodecyl sulfate-polyacrylamide gel. Electrophoretic mobility of the enzyme was closely similar to that of urinary kallikrein with estimated Mr of approximately 38,000. With Western blot analyses using a rabbit anti-kallikrein antibody, both brain and urinary kallikrein were visualized at identical positions by immunoperoxidase staining and by autoradiography with 125I-protein A binding. Brain mRNA was used to direct cell-free protein synthesis in wheat germ and rabbit reticulocyte lysate systems. [35S]Methionine-labeled kallikrein was identified by fluorography of sodium dodecyl sulfate-polyacrylamide gels after the translation products were subject to immunoprecipitation with affinity-purified kallikrein antibody. Collectively, the data show that tissue kallikrein exists in brain and can be synthesized by brain mRNA.
D000818:Animals; D000911:Antibodies, Monoclonal; D001923:Brain Chemistry; D002474:Cell-Free System; D002846:Chromatography, Affinity; D007610:Kallikreins / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D011817:Rabbits; D051381:Rats
D000911:Antibodies, Monoclonal; D012333:RNA, Messenger; D007610:Kallikreins