Studies on DNA alpha-polymerase of mouse myeloma: partial purification and comparison of three molecular forms of the enzyme.
Activity of DNA alpha-polymerase in extracts from MOPC-104E was not associated with a single protein molecule, but with several molecular species that differed in isoelectric point. The three most abundant of these enzyme species were first separated from other DNA polymerases and then resolved from each other by repeated chromatography on diethylaminoethylcellulose columns. Next, with the use of glycerol gradient centrifugation and DNA-cellulose column chromatography, the three species were further purified to a state representing more than 5000-fold purification over the crude extract. These three highly purified enzyme species exhibited very similar catalytic properties, and the main activity of each species sedimented at the same rate (6-7S) in glycerol gradients containing 0.5 M KCl. Analysis of the polypeptide content of each species revealed that polypeptides of about 150 000 and 60 000 daltons cofractionated with the DNA polymerase activity. The multiple alpha-polymerase species did not appear to result from in vitro proteolytic cleavage, since multiple species were observed in extracts prepared under several different types of conditions, including the presence of the protease inhibitors, phenylmethanesulfonyl fluoride, or trasylol. The three species were recovered in about the same relative amounts from both the nuclear and cytoplasmic fractions of MOPC-104E, and it appeared that multiple species of alpha-polymerase were also present in extracts from fetal bovine liver.
D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology; D003593:Cytoplasm / Q000201:enzymology; D004257:DNA Polymerase II / Q000302:isolation & purification / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification; D007527:Isoenzymes / Q000302:isolation & purification; D051379:Mice; D008970:Molecular Weight; D009101:Multiple Myeloma / Q000201:enzymology*; D013698:Templates, Genetic
D007527:Isoenzymes; D004257:DNA Polymerase II; D004259:DNA-Directed DNA Polymerase