An improved method for the quantitative isolation of rat liver nuclear RNA polymerases.
Rat liver nuclear RNA polymerases exist in two functional states, one of which is active towards the endogenous chromatin template (engaged enzyme), while the other is inactive (free enzyme) (Yu, F.L. (1974) Nature 251, 344-346). This paper reports the direct separation of these two populations of RNA polymerases from isolated rat liver nuclei by a simple extraction procedure. It is estimated that as much as 50% of the total nuclear RNA polymerase activity in normal rat liver may exist in the form of the free enzyme. Evidence is also presented to indicate that the free enzyme activity is easily lost when the nuclear isolation procedure involves the use of an isotonic buffer medium, or when the isolated nuclei are subjected to sonication as is required for the solubilization of the nuclear RNA polymerases by the conventional method. Based on these new findings, it is proposed that nuclei be isolated directly in hypertonic sucrose and that the free enzyme be extracted before the nuclei are subjected to sonication to solubilize the engaged enzyme. This method circumvents the loss of the free RNA polymerase population and, as a result, the total yield of the nuclear RNA polymerases is greatly increased. The possible functional role of the free RNA polymerase in gene expression is discussed.
D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology*; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008297:Male; D008722:Methods; D009994:Osmolar Concentration; D051381:Rats; D012995:Solubility; D013698:Templates, Genetic; D014461:Ultracentrifugation; D014500:Uracil Nucleotides / Q000378:metabolism
D014500:Uracil Nucleotides; D006160:Guanosine Triphosphate; D012321:DNA-Directed RNA Polymerases