Neary J T, Armstrong A, Davidson B, Maloof F, Soodak M
Biochim Biophys Acta. 1975 Jan 30;379(1):262-70. doi: 10.1016/0005-2795(75)90029-x.
The molecular size of microsomal membrane proteins from frozen porcine thyroids before and after solubilization by proteolytic and non-proteolytic techniques has been investigated by means of polyacrylamide-gel electrophoresis in the presence of 1% sodium dodecylsulfate. When thyroid microsomal membrane proteins are solubilized by non-proteolytic methods such as high pH, n-butanol, or deoxycholate, no major change in the electrophoretic pattern compared to untreated microsomes has been observed, thereby suggesting that these non-proteolytic methods are capable of extracting membrane proteins from thyroid microsomes without altering their molecular size. However, treatment of microsomes with protein-solubilizing levels of trypsin (1-5 mug trypsin per mg thyroid protein) results in degradation of all major proteins with a molecular weight greater than 30 000. The high-molecular-weight proteins are particularly susceptible to attack by trypsin. Thus, these experiments indicate that the use of trypsin to solubilize thyroid microsomal membrane proteins, particularly thyroid peroxidase, will result in fragmented proteins and should be avoided if intact membrane proteins are desired.
利用在1%十二烷基硫酸钠存在下的聚丙烯酰胺凝胶电泳，研究了经蛋白水解和非蛋白水解技术溶解前后的冷冻猪甲状腺微粒体膜蛋白的分子大小。当甲状腺微粒体膜蛋白通过非蛋白水解方法（如高pH值、正丁醇或脱氧胆酸盐）溶解时，与未处理的微粒体相比，电泳图谱没有观察到主要变化，这表明这些非蛋白水解方法能够从甲状腺微粒体中提取膜蛋白而不改变其分子大小。然而，用胰蛋白酶的蛋白溶解水平（每毫克甲状腺蛋白1 - 5微克胰蛋白酶）处理微粒体会导致所有分子量大于30000的主要蛋白质降解。高分子量蛋白质尤其容易受到胰蛋白酶的攻击。因此，这些实验表明，使用胰蛋白酶溶解甲状腺微粒体膜蛋白，特别是甲状腺过氧化物酶，会导致蛋白质片段化，如果需要完整的膜蛋白，应避免使用。
