Differentiation and characterization of the cytoplasmic and nuclear deoxyribonucleic acid polymerases from baby hamster kidney cells.
Distinct DNA polymerase activities have been found in the cytoplasmic and nuclear fractions of a baby hamster kidney cell line. They were separated by chromatography on DEAE-cellulose and partially purified by ammonium sulfate fractionation, DNA - cellulose and linear sucrose gradients. The cytoplasmic DNA polymerase exhibited an S-coefficient of 6.95 S in 0.15 M NaCl and its activity was highly sensitive to inhibition by N-ethylmaleimide and elevated temperatures, regardless of the presence of DNA template or other cofactors. It was stimulated by monovalent salts in the order of NH4 Cl greater than KCl greater than NaCl greater than CsCl greater than LiCl (inhibitory). The DNA polymerase extracted from nuclei sedimented with an S-value of 3.47 S, was resistant to inactivation by N-ethylmaleimide, and maximally stimulated by NaCl, while also being inhibited by LiCl. For optimal activity, both DNA polymerase activities required a divalent cation, with MgCl2 being more effective than MnCl2. Although the optimal pH values for the two enzyme activities differed slightly, glycine - NaOH buffer induced an alkaline shift of 1.5 pH units in the optimum of both enzymes. This was accompanied by an increase in the effectiveness of MnCl2 relative to MgCl2 for the cytoplasmic DNA polymerase.
D000818:Animals; D002414:Cations, Monovalent; D002458:Cell Fractionation; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology*; D006224:Cricetinae; D003593:Cytoplasm / Q000201:enzymology; D004254:DNA Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D004355:Drug Stability; D005033:Ethylmaleimide / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007668:Kidney; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D009994:Osmolar Concentration
D002414:Cations, Monovalent; D008345:Manganese; D004254:DNA Nucleotidyltransferases; D008274:Magnesium; D005033:Ethylmaleimide