Beebee T J
Biochem J. 1979 Oct 1;183(1):43-54. doi: 10.1042/bj1830043.
Nuclei were prepared from rat liver after homogenization of the tissue in hyperosmotic sucrose and RNA polymerases (EC 2.7.7.6) extracted by two methods applied sequentially. Optimal conditions for washing loosely bound enzymes out of nuclei were determined first, and involved short (10 min) incubations at 0 degrees C in the presence of 5 mM-Mg2+ and 60 mM-(NH4)2SO4. Subsequent sonication of the residual nuclear pellet after resuspension and lysis at high ionic strength resulted in further release of RNA polymerases. The primary wash yielded about 2 x 10(4) molecules of RNA polymerases I and III (altogether) and 1 x 10(4) molecules of form-II enzymes per original nucleus, whereas subsequent sonication released 2 x 10(4)-2.5 x 10(4) form-I and -III enzyme molecules (altogether) and a further 7 x 10(3)-8 x 10(3) form-II enzyme molecules, as measured by end-labelling of nascent RNA. RNA polymerase II was partially purified from both types of extracts and shown to initiate very poorly on high-molecular-weight homologous DNA irrespective of the source of the enzyme.
将大鼠肝脏组织在高渗蔗糖中匀浆后制备细胞核，并通过两种方法依次提取RNA聚合酶（EC 2.7.7.6）。首先确定了从细胞核中洗去松散结合酶的最佳条件，即在5 mM - Mg2+和60 mM - (NH4)2SO4存在下于0℃短时间（10分钟）孵育。在高离子强度下重悬并裂解后，对剩余核沉淀进行后续超声处理，导致RNA聚合酶进一步释放。初次洗涤每原代细胞核产生约2×10⁴个RNA聚合酶I和III分子（总计）以及1×10⁴个II型酶分子，而后续超声处理释放2×10⁴ - 2.5×10⁴个I型和III型酶分子（总计）以及另外7×10³ - 8×10³个II型酶分子，这是通过新生RNA的末端标记测量的。RNA聚合酶II从两种提取物中部分纯化，并显示无论酶的来源如何，在高分子量同源DNA上起始能力都很差。
