Wright W E, Catala F, Farmer K
Department of Cell Biology and Neuroscience, U.T. Southwestern Medical Center, Dallas 75235.
Symp Soc Exp Biol. 1992;46:79-87.
Sucrose gradients and molecular sieve chromatography were used to determine the native molecular weight of the basic HLH proteins myogenin, MyoD and E12. The muscle bHLH proteins not only formed dimers but also associated in a variety of higher order complexes. Although homodimers bind to DNA sequences such as the MEF-1 site in the creatine kinase enhancer, homotetramers and larger forms do not recognize this DNA sequence. Little evidence for complexes larger than dimers was found for the ubiquitous bHLH protein E12. Most of the myogenin remains in large complexes when myogenin and E12 are mixed. The same result was obtained in nuclear extracts from differentiated myotubes, in which most of the myogenin was found to be present in large complexes that do not bind to the creatine kinase enhancer. A fusion protein that contains only the myogenin HLH region fused to glutathione-S-transferase also forms large homomeric complexes. A model to explain these results is that each helix of the HLH motif can associate with a different subunit to form chains or ring structures. The presence of myogenin in nuclear extracts as both dimers that recognize known DNA sequences as well as higher order complexes that do not raises significant issues concerning the regulation of skeletal muscle bHLH protein activity during myogenesis.
采用蔗糖梯度离心和分子筛层析法测定碱性HLH蛋白肌细胞生成素、MyoD和E12的天然分子量。肌肉bHLH蛋白不仅能形成二聚体，还能形成多种高阶复合物。尽管同型二聚体可结合DNA序列，如肌酸激酶增强子中的MEF-1位点，但同型四聚体及更大的形式无法识别该DNA序列。对于普遍存在的bHLH蛋白E12，未发现大于二聚体的复合物的证据。当肌细胞生成素和E12混合时，大多数肌细胞生成素仍存在于大复合物中。在分化的肌管的核提取物中也得到了相同的结果，其中发现大多数肌细胞生成素存在于不与肌酸激酶增强子结合的大复合物中。一种仅包含与谷胱甘肽-S-转移酶融合的肌细胞生成素HLH区域的融合蛋白也能形成大的同源复合物。一个解释这些结果的模型是，HLH基序的每个螺旋都可以与不同的亚基结合，形成链状或环状结构。核提取物中肌细胞生成素以既能识别已知DNA序列的二聚体形式存在，也以不能识别的高阶复合物形式存在，这一现象引发了关于成肌过程中骨骼肌bHLH蛋白活性调控的重大问题。
