Novikoff hepatoma deoxyribonucleic acid polymerase. Identification of a stimulatory protein bound to the beta-polymerase.
The Novikoff hepatoma DNA polymerase-beta sediments as a 7.3S form in crude extracts but during purification sediments as a 4.1S form (after diethylaminoethyl-Sephadex chromatography) or as a 3.3S form (after DNA-cellulose chromatography). If 0.25 M ammonium sulfate or 0.5 M NaCl is included in the sucrose gradients, the 7.3S form sediments at 3.3 S; after removal of the salt, it sediments again at 7.3 S, indicating the reversibility of the aggregation phenomenon. By careful adjustment of ionic strength in the gradient, four distinct and reproducible forms of the enzyme sedimenting at 7.3, 5.8, 4.1, and 3.3 S can be generated. The isoelectric point of the DNA polymerase also changes during purification; the 7.3S form has a pI of 7.5, while the 4.1S form isoelectrically focuses at a pH of 8.5. During DNA-cellulose chromatography, the Novikoff beta-polymerase is separated from a stimulatory factor designated as Novikoff factor IV. Factor IV is a protein as shown by its sensitivity to protease and resistance to nucleases. It is responsible for converting the 3.3S enzyme to the 4.1S form since the 3.3S homogeneous DNA polymerase-beta sediments at 4.1 S in the presence of factor IV. Factor IV confers stability to the polymerase in low ionic strength buffers as well as stability to heat denaturation. Factor IV has the ability to increase the activity of the 3.3S homogeneous polymerase by about fourfold.
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000201:enzymology*; D004257:DNA Polymerase II / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004789:Enzyme Activation; D007700:Kinetics; D008113:Liver Neoplasms; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000302:isolation & purification / Q000502:physiology*; D009374:Neoplasms, Experimental / Q000201:enzymology; D051381:Rats
D009363:Neoplasm Proteins; D004257:DNA Polymerase II; D004259:DNA-Directed DNA Polymerase