ACTIVE 2-DIMENSIONAL ELECTROPHORESIS OF RAT-LIVER DNA-POLYMERASE ALPHA

The approach of so-called active gel analysis was used to determine th e position and appearance of the catalytic subunit of rat liver DNA po lymerase a on a two-dimensional (2-D) electrophoretic map. In this cas e a polyacrylamide gel containing DNA was used for the second dimensio n. DNA presence does not change the 2-D protein pattern but makes it p ossible to conduct a polymerase reaction directly in the gel after sep aration. A crude extract of rat liver nuclei was used for analysis. Th e extract is quickly isolated and contains mainly DNA polymerase ct ac tivity. It was shown that this enzyme restores its activity after 2-D electrophoresis and sodium dodecyl sulfate (SDS) elution. After polyme rase reaction with labeled dNTPs and autoradiography, the catalytic po lypeptide or, rather, polypeptide cluster is revealed as chains of spo ts (possibly because of the presence of different hydrolyzed and phosp horylated forms). These spots are located on the 2-D electrophoretic m ap in the region corresponding to molecular masses of 160, 140, and 13 0 kDa and pi 5.5-6.2.