Miki B L, Neelin J M
Can J Biochem. 1977 Dec;55(12):1213-9. doi: 10.1139/o77-181.
The erythrocyte histones of trout, carp, white sucker, and chicken are subject to very different levels of autolytic activity. Carp erythrocyte histones extracted from typical nuclear preparations suffer extensively from degradation; histones 1, 5, and 3 (H1, H5, and H3 respectively) are preferentially cleaved and characteristic peptides designated P1, P2, and P3 appear during the course of proteolysis. Generally, erythrocytes from different fish species yield highly disparate proportions of H1 and H5, but this is not a consequence of the variable levels of proteolytic activity in these species. Phenylmethylsulfonyl fluoride (PMSF) (1.0 mM) was found to be superior to 50 mM sodium bisulfite as a protease inhibitor and was well suited for use in media employed for cell washes and the isolation of nuclei. Nonetheless, in carp erythrocytes residual protease activity (qualitatively the same as the uninhibited activity) persists even in the presence of PMSF. It is activated during cell lysis and remains associated with the nuclear fraction of the lysate during subsequent washes. The isolation of intact nuclei is important for the ultimate extraction of undegraded histone, especially from sources in which the risks of autolysis are high or unknown.
鳟鱼、鲤鱼、白鲑和鸡的红细胞组蛋白具有非常不同程度的自溶活性。从典型的细胞核制剂中提取的鲤鱼红细胞组蛋白会大量降解；组蛋白1、5和3（分别为H1、H5和H3）优先被裂解，在蛋白水解过程中出现了名为P1、P2和P3的特征性肽段。一般来说，不同鱼类的红细胞产生的H1和H5比例差异很大，但这并不是这些物种中蛋白水解活性水平不同的结果。已发现苯甲基磺酰氟（PMSF）（1.0 mM）作为蛋白酶抑制剂比50 mM亚硫酸氢钠更有效，并且非常适合用于细胞洗涤和细胞核分离所用的培养基。然而，在鲤鱼红细胞中，即使存在PMSF，残余的蛋白酶活性（在质量上与未抑制的活性相同）仍然存在。它在细胞裂解过程中被激活，并在随后的洗涤过程中与裂解物的细胞核部分相关联。完整细胞核的分离对于最终提取未降解的组蛋白很重要，特别是从自溶风险高或未知的来源中提取。
