Abstract
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.
Highlights
A rapid purificationof troponin T from adulhtearts major contractile and regulatory proteins in striated muscle, of various species has been developed
With high titer and specificity to both isoforms was have recently reported that these two isoforms have different subsequently obtained. This antiserum cross-re- influences on the overall response of the reconstituted thin acted with cardiac troponin T from chicken, rabbit, filaments to Ca2+, there is no detectable difference and rat
The results clearlyshowed that expression of these two isoforms may modulate cardiac functhere was a switch of troponin T isoforms between tion
Summary
(30 mM NaCl, 10 mM imidazole HCI, pH 7.0, 15 mM 2-mercaptoethanol, 0.5 mM EGTA, and 6 M urea), the sample was loaded onto a DEAE-cellulose column (2.5 X 10cm) equilibrated in the column buffer. The column was washed with 150ml of the column buffer and Purifkation and Characterization of Bovine Cardiac Troponin T-The TnT purification method described here consists of 1 M KC1 extraction, 60 "C heat treatment,ammonium eluted with a linear gradient of 0-300 mM NaCl in the column sulfate fractionation, and DEAE-cellulose columnchromatogbuffer (400ml total).The fractionscontainingtroponin T were raphy. The treatment very effectively removedmost contaminants from purification procedure described here was readily suitable for isolating cardiac troponin T from other species, such as chicken, rabbit, and rat. 4200k of rabbit skeletal muscle as described previously (Lin et al.,1984b)
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