Rapid purification of mammalian cardiac troponin T and its isoform switching in rat hearts during development.
A rapid purification of troponin T from adult hearts of various species has been developed. The purification procedure included 60 degrees C treatment of the high salt extract, ammonium sulfate fractionation, and DEAE-cellulose column chromatography. The troponin T purified from the bovine left ventricle contained two isoforms, which differed in both apparent molecular mass and isoelectric point. Both isoforms were able to bind to F-actin filaments only in the presence of tropomyosin. Monoclonal antibody JLT12 against rabbit skeletal troponin T cross-reacted with both isoforms of bovine cardiac troponin T. There was no detectable difference in the relative amount of these two isoforms among different portions (atria, right and left ventricles) of the bovine heart. The purified protein was used as an antigen to immunize mice, and a mouse antiserum with high titer and specificity to both isoforms was subsequently obtained. This antiserum also cross-reacted with cardiac troponin T from chicken, rabbit, and rat. The antibodies were further used to probe cardiac development in rats by Western blotting and immunoprecipitation. The results clearly showed that there was a switch of troponin T isoforms between hearts from 20-day-old rat embryos and hearts from 14-day-old rats. Immunoprecipitation of the in vitro translation products of poly(A)+ RNA isolated from day 5 rat hearts revealed the presence of two isoforms of troponin T, suggesting that two mRNAs coding for these two isoforms existed in the heart cells. It is of interest to not that some profound changes in the morphology and function of cardiac muscle have also been detected at this time of development. Troponin T isoform switching thus may well represent an important marker for cardiac development and function.
Jin
J P
JP
Department of Biology, University of Iowa, Iowa City 52242.
Lin
J J
JJ
D000199:Actins / Q000378:metabolism; D000375:Aging; D000818:Animals; D002417:Cattle; D002645:Chickens; D002848:Chromatography, DEAE-Cellulose; D004622:Embryo, Mammalian; D004625:Embryo, Nonmammalian; D004797:Enzyme-Linked Immunosorbent Assay; D006321:Heart / Q000196:embryology / Q000254:growth & development*; D008970:Molecular Weight; D014176:Protein Biosynthesis; D051381:Rats; D013045:Species Specificity; D014336:Troponin / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D020107:Troponin T
D000199:Actins; D014336:Troponin; D020107:Troponin T