A comparison of methods for extracting ribonucleic acid polymerases from rat liver nuclei
- 1 October 1979
- journal article
- research article
- Published by Portland Press Ltd. in Biochemical Journal
- Vol. 183 (1) , 43-54
- https://doi.org/10.1042/bj1830043
Abstract
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 × 10(4) molecules of RNA polymerases I and III (altogether) and 1 × 10(4) molecules of form-II enzymes per original nucleus, whereas subsequent sonication released 2 × 10(4)-2.5 × 10(4) form-I and -III enzyme molecules (altogether) and a further 7 × 10(3)-8 × 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.This publication has 35 references indexed in Scilit:
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