Identification, purification, and localization of tissue kallikrein in rat heart

Abstract

A tissue kallikrein has been isolated from rat heart extracts by DEAE-Sepharose and aprotinin-affinity column chromatography. The purified cardiac enzyme has both N-tosyl-L-arginine methyl ester esterolytic and kinin-releasing activities, and displays parallelism with standard curves in a kallikrein radioimmunoassay, indicating it to have immunological identity with tissue kallikrein. The enzyme is inhibited by aprotinin, antipain, leupeptin and by high concentrations of soybean trypsin inhibitor, but stimulated by lima-bean or ovomucoid trypsin inhibitor and low concentrations of soybean trypsin inhibitor. By using a specific monoclonal antibody to tissue kallikrein in Western blot as well as active-site labelling with [14C]di-isopropyl fluorophosphate, the cardiac enzyme was identified as a protein of 38 kDa, a molecular mass identical with that of tissue kallikrein. Immunocytochemistry at the electron-microscopic level localized this enzyme to the sarcoplasmic reticulum and granules of rat atrial myocytes. Two cardiac kallikrein precursors, (38 and 40 kDa) were identified from the translation in vitro of heart mRNA by immunoprecipitation and electrophoresis of [35S]methionine-labelled cell-free translation products. Kallikrein mRNA in the rat heart was also demonstrated by dotblot analysis using a tissue kallikrein cDNA probe. These results indicate that the tissue kallikrein gene is expressed in the rat heart and that the purified enzyme is indistinguishable from tissue kallikrein with respect to enzymic and immunological characteristics.

Increased tissue kallikrein levels in type 2 diabetes

“…Moreover, the failure of diabetes to influence blood kallidin and kininogen levels, despite increased tissue kallikrein levels, was consistent with the predominant tissue localisation of tissue kallikrein, where it may participate in local formation of kallidin peptides. Our finding that tissue kallikrein was produced by atrial myocytes was in agreement with the previous identification of tissue kallikrein mRNA in rat heart and the immunolocalisation of tissue kallikrein to rat atrial myocytes [23].…”

A local kallikrein-kinin system is present in rat hearts.

“…Thus, the presence of both the enzyme and its mRNA substantiate the existence of a kallikrein-kinin pathway in the heart. This extends our previous observations" and confirms the report of Xiong et al 12 When epicardial slices were incubated in vitro, kallikrein was released into the medium in concentrations too high to be the result of nonspecific release due to damage, as indicated also by the lack of association between kallikrein and LDH activity. Kallikrein release was significantly decreased by pretreatment with the protein synthesis inhibitor puromycin.…”

Cardiac Angiotensin II Type 2 Receptor Activates the Kinin/NO System and Inhibits Fibrosis

“…15 Components of the kallikrein-kinin system are identified in the heart. 24 Samples from heart homogenates were incubated with bovine kininogen (2000 ng, Seikagaku Kogyo) for 30 minutes at 37°C. The amount of kinins generated during the incubation was measured by radioimmunoassay.…”