Purification and characterization of RNA polymerase II from Artemia salina.

摘要

Total RNA polymerase activity was extracted from the nuclei isolated from developing Artemia salina. The crude enzyme extract was stable for two weeks at -20°C and up to two months at -80°C. DEAE BioGel A and DEAE Affigel Blue chromatography were used for the purification of the enzyme. SDS-PAGE analysis of RNA polymerase II from the 12 hr embryos showed 14 subunits, the 24 hr enzyme had 17 subunits and the 36 hr enzyme showed 16 subunits with molecular weights ranging from 258 to 15.0 kDa. Lectin blotting analysis of the enzyme preparations with Sambucus nigra agglutinin detected the presence of terminal sialic acid on the enzyme isolated from 24 embryos and Datura stramonium agglutinin detected the presence of Galbeta-(1-4)-N-acetylglucosamine on the enzymes isolated from the 12 and 36 hr embryos. Staining with ProQ Diamond phospho gel stain showed several phosphorylated subunits.