Studies on DNA α-polymerase of mouse myeloma: partial purification and comparison of three molecular forms of the enzyme

Abstract

Activity of DNA .alpha.-polymerase in extracts from MOPC-104E [mouse plasmacytoma] was associated with several molecular species that differed in isoelectric point. The 3 most abundant of these enzyme species were 1st separated from other DNA polymerases and then resolved from each other by repeated chromatography on DEAE cellulose columns. Next, with the use of glycerol gradient centrifugation and DNA-cellulose column chromatography, the 3 species were further purified more than 5000-fold over the crude extract. These 3 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 3 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.