Rapid purification of mammalian cardiac troponin T and its isoform switching in rat hearts during development.
Rapid purification of mammalian cardiac troponin T and its isoform switching in rat hearts during development.
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发表时间：
1988-05
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通讯作者：
J. Jin;J. Lin
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作者：
J. Jin;J. Lin
从不同物种的成年心脏中快速纯化肌钙蛋白T已经被开发出来。纯化过程包括60 ℃处理高盐提取物，硫酸铵分级分离和DEAE-纤维素柱层析。从牛左心室中纯化的肌钙蛋白T含有两种亚型，其表观分子量和等电点均不同。这两种亚型都能够结合到F-肌动蛋白丝只有在原肌球蛋白的存在下。抗兔骨骼肌肌钙蛋白T单克隆抗体JLT 12与牛心肌肌钙蛋白T的两种亚型均发生交叉反应。在牛心脏的不同部分（心房、右心室和左心室）中，这两种亚型的相对量没有可检测到的差异。纯化后的蛋白质作为抗原免疫小鼠，获得了高效价和特异性的抗血清。该抗血清与鸡、兔和大鼠的心肌肌钙蛋白T也有交叉反应。通过Western印迹和免疫沉淀法进一步将抗体用于探测大鼠心脏发育。结果清楚地表明，在20日龄大鼠胚胎心脏和14日龄大鼠心脏之间存在肌钙蛋白T亚型的转换。从第5天大鼠心脏中分离的poly（A）+ RNA的体外翻译产物的免疫沉淀揭示了肌钙蛋白T的两种亚型的存在，表明编码这两种亚型的两种mRNA存在于心脏细胞中。令人感兴趣的是，在发育的这个时期，还发现了心肌形态和功能的一些深刻变化。因此肌钙蛋白T亚型转换可能是心脏发育和功能的重要标志。
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.