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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 dot-blot 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.

SUBMITTER: Xiong W

PROVIDER: S-EPMC1131345 | biostudies-other | 1990 May

REPOSITORIES: biostudies-other

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Project description:We have identified a tissue-kallikrein-binding protein in human serum and in the serum-free culture media from human lung fibroblasts (WI-38) and rodent neuroblastoma X glioma hybrid cells (NG108-15). Purified and 125I-labelled tissue kallikrein and human serum form an approximately 92,000-Mr SDS-stable complex. The relative quantity of this complex-formation is measured by densitometric scanning of autoradiograms. Complex-formation between tissue kallikrein and the serum binding protein was time-dependent and detectable after 5 min incubation at 37 degrees C, with half-maximal binding at 28 min. Binding of 125I-kallikrein to kallikrein-binding protein is temperature-dependent and can be inhibited by heparin or excess unlabelled tissue kallikrein but not by plasma kallikrein, collagenase, thrombin, urokinase, alpha 1-antitrypsin or kininogens. The kallikrein-binding protein is acid- and heat-labile, as pretreatment of sera at pH 3.0 or at 60 degrees C for 30 min diminishes complex-formation. However, the formed complexes are stable to acid or 1 M-hydroxylamine treatment and can only be partially dissociated with 10 mM-NaOH. When kallikrein was inhibited by the active-site-labelling reagents phenylmethanesulphonyl fluoride or D-Phe-D-Phe-L-Arg-CH2Cl no complex-formation was observed. An endogenous approximately 92,000-Mr kallikrein-kallikrein-binding protein complex was isolated from normal human serum by using a human tissue kallikrein-agarose affinity column. These complexes were recognized by anti-(human tissue kallikrein) antibodies, but not by anti-alpha 1-antitrypsin serum, in Western-blot analyses. The results show that the kallikrein-binding protein is distinct from alpha 1-antitrypsin and is not identifiable with any of the well-characterized plasma proteinase inhibitors such as alpha 2-macroglobulin, inter-alpha-trypsin inhibitor, C1-inactivator or antithrombin III. The functional role of this kallikrein-binding protein and its impact on kallikrein activity or metabolism in vivo remain to be investigated.

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

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