Abstract
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.
Highlights
A monoclonal antibody against purified rat urinary roducts of tissue kallikrein have been long known to affect kallikrein was coupled to agarose and used to isolate ehavior (12),cardiovascular function (13),brain aminelevels kallikrein from rat brain
Cell-free protein synthesis in wheat germ and rabbit reticulocytelysatesystems. [35S]Methionine-labeled kallikrein was identified by fluorography of sodium dodecyl sulfate-polyacrylamide gels after the translation products were subject to immunoprecipitation withaffinity-purifiedkallikrein antibody
Totaland immunoprecipitated translation products were analyzed by SDS-polyacrylamide gel electrophoresis and radioactive protein bands were visualized by fluorography as described above
Summary
Tissue kallikrein (EC 3.4.21.8) is being found in sites not Kallikrein-like TosArgOMe esterase activity was determinedas previously considered to contain this activity, such as plasmade, scribed previously (22, 23). This article must be hereby marked "aduertisewas blotted free of blood, minced, and homogenized in 0.05 M Tris/ HC1, pH 7.5, using a motor-driven homogenizer, and thencentrifuged a t 600 X g for 15 min.Deoxycholate was added to the supernatant to a final concentration of 0.5% (w/v), incubated at room temperature for 30 min,andthen centrifuged at 10,000 X g for 20 min. This supernatant was centrifuged at 100,000 X g for 60 min and dialyzed overnight a t 4 "C against abuffer containing 0.1 M NaC1,0.01 M ment" in accordance with 18 U.S.C. Section 1734 solely to indicate.
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