Large scale isolation of RNA polymerase I of Tetrahymena pyriformis and the identification of polymerase-associated polypeptides

A procedure has been developed for a large scale and rapid isolation of RNA polymerase I (EC 2.7.7.6) of Tetrahymena pyriformis. The enzyme is precipitated from the cell homogenate by Polymin P, extracted from the sediment and separated from RNA polymerase II by a treatment with phosphocellulose. The further purification procedure involves sedimentation in glycerol gradients and chromatography on heparin-Sepharose and DEAE-Sephadex. The last step achieved the separation of RNA polymerase I from RNA polymerase III. On the basis of different criteria RNA polymerase I is assumed to consist of two large subunits of 180 and 118 kDa and nine subunits smaller than 50 kDa. Additional polypeptides have been identified which are associated with RNA polymerase I but are not found in integral stoichiometric amounts. Except for certain minor differences RNA polymerase I purified from the cell homogenate shows the same structure as the enzyme obtained from isolated macronuclei (Mueller et al., 1985).     

Isolation and purification of protein kinase C from human leukemia ML-1 cells phosphorylation of human leukemia RNA polymerase II in vitro

Protein kinase C (PKC) was purified to near homogeneity from human leukemia ML-1 cells. The purified enzyme showed a single polypeptide band of 80 kDa on SDS-polyacrylamide gel after electrophoresis, and was totally dependent on Ca2+/phospholipid for activity. Diacylglycerol and the tumor-promoting on Ca2/phospholipid for activity. Diacylglycerol and the tumor-promoting phorbol esters stimulated the enzyme activity. Autophosphorylation of PKC purified from phenyl-Sepharose column showed both 80- and 37 kDa polypeptides. Further fractionation of PKC on a hydroxyapatite column revealed two peaks of enzyme activity, indicating that there may be two different forms of protein kinase C present in human leukemia cells. The purified PKC was used to phosphorylate RNA polymerase II of human leukemia cells in vitro and the autoradiogram showed that RNA polymerase II large subunits (240, 220 and 150 kDa) were phosphorylated in a time-dependent manner.