Identification, purification, and localization of tissue kallikrein in rat heart.
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.
Xiong
W
W
Department of Pharmacology, Medical University of South Carolina, Charleston 29425.
Chen
L M
LM
Woodley-Miller
C
C
Simson
J A
JA
Chao
J
J
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D003327:Coronary Disease / Q000209:etiology; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D007610:Kallikreins / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification*; D009206:Myocardium / Q000201:enzymology*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains
D012333:RNA, Messenger; D009320:Atrial Natriuretic Factor; D007610:Kallikreins