Härtner K T, Kirschbaum B J, Pette D
Fakultät für Biologie, Universität Konstanz, Federal Republic of Germany.
Eur J Biochem. 1989 Jan 15;179(1):31-8. doi: 10.1111/j.1432-1033.1989.tb14517.x.
Polyclonal antibodies were raised in guinea pigs against troponin-T (TnT) isoforms purified from fast- and slow-twitch rabbit muscles. With the use of these antibodies and immunoblots of one- and two-dimensional electrophoreses, the distribution of fast and slow TnT isoforms was investigated in normal and chronically stimulated hindlimb muscles of the rabbit. According to differences in their apparent molecular masses, six fast TnT isoforms (TnTcf, TnT1f, TnT2f, TnT3f, TnT4f, TnT5f) were distinguished in normal tibialis anterior and extensor digitorum longus muscles. These muscles also contained low amounts of TnT1s and TnT2s which were the predominant TnT isoforms in slow-twitch soleus muscle. Fast and slow TnT isoforms were found to exist in several charge variants, i.e. one for TnTcf, three different charge forms for TnT1f, seven for TnT2f, four for TnT3f, three for TnT4f, one for TnT5f, four for TnT1s, and three for TnT2s. Some charge variants were phosphorylated isoforms because treatment with alkaline phosphatase reduced the number of the 19 fast and 7 slow variants to 12 and 3, respectively. The stimulation-induced fast-to-slow transition caused progressive decreases in fast and increases in slow isoforms. The decrease and the disappearance of the major fast isoforms followed a sequence of TnT2f, TnTcf, TnT4f, TnT1f, and TnT3f. This decrease in fast isoforms fits well with the reduction of fast TnT mRNAs assessed by Northern blot analysis. Prolonged stimulation ultimately created a TnT isoform pattern similar to that found in normal slow-twitch muscle. Stimulation also induced changes in the tropomyosin subunit pattern with a decrease in the fast and an increase in the slow alpha-tropomyosin subunit without altering the alpha/beta-tropomyosin subunit ratio. Similar to slow-twitch soleus muscle, long-term stimulated muscles contained appreciable amounts of the fast alpha-tropomyosin subunit, but only traces of fast TnT isoforms. This combination indicated that the predominant slow TnT isoforms may be capable of interacting with fast tropomyosin in these muscles.
用从快肌和慢肌兔肌肉中纯化的肌钙蛋白 - T（TnT）同工型在豚鼠体内制备多克隆抗体。利用这些抗体以及一维和二维电泳的免疫印迹，研究了兔正常和慢性刺激后肢肌肉中快、慢TnT同工型的分布。根据其表观分子量的差异，在正常胫骨前肌和趾长伸肌中区分出六种快TnT同工型（TnTcf、TnT1f、TnT2f、TnT3f、TnT4f、TnT5f）。这些肌肉中还含有少量的TnT1s和TnT2s，它们是慢肌比目鱼肌中的主要TnT同工型。发现快、慢TnT同工型存在多种电荷变体，即TnTcf有1种，TnT1f有3种不同电荷形式，TnT2f有7种，TnT3f有4种，TnT4f有3种，TnT5f有1种，TnT1s有4种，TnT2s有3种。一些电荷变体是磷酸化同工型，因为用碱性磷酸酶处理后，19种快变体和7种慢变体的数量分别减少到12种和3种。刺激诱导的快 - 慢转变导致快同工型逐渐减少，慢同工型逐渐增加。主要快同工型的减少和消失顺序为TnT2f、TnTcf、TnT4f、TnT1f和TnT3f。快同工型的这种减少与通过Northern印迹分析评估的快TnT mRNA的减少非常吻合。长时间刺激最终产生了一种与正常慢肌中发现的TnT同工型模式相似的模式。刺激还诱导了原肌球蛋白亚基模式的变化，快α - 原肌球蛋白亚基减少，慢α - 原肌球蛋白亚基增加，而α/β - 原肌球蛋白亚基比例不变。与慢肌比目鱼肌类似，长期刺激的肌肉含有相当数量的快α - 原肌球蛋白亚基，但只有微量的快TnT同工型。这种组合表明，在这些肌肉中，主要的慢TnT同工型可能能够与快原肌球蛋白相互作用。
