A DNA polymerase from maize axes: its purification and possible role.
Three different DNA polymerase activities can be resolved by passing a protein extract from 24 h imbibed maize axes through DEAE-cellulose. These activities have been numbered 1, 2 and 3, according to their elution order. One of them, DNA polymerase 2, elutes at 100-120 mM phosphates. This enzyme was further purified by passing it through Heparin-Sepharose, Sephacryl S-300 and DNA cellulose. Purification was nearly 5000-fold. The enzyme needs Mg2+, is stimulated by K+, has an optimum pH of 7.0 and its optimum temperature is 30-37 degrees C. Specific inhibitors for different types of polymerases, such as aphidicolin, dideoxythymidine triphosphate and N-ethyl maleimide, gave intermediate values of inhibition, making impossible the definition of the type of enzyme purified by its inhibitory pattern. SDS-PAGE indicated the presence of several bands of molecular masses of 28-40, 56 and 15 kDa. Most of these bands could be visualized when proteins from crude extracts were analyzed by western blot, using an antibody against calf thymus DNA polymerase alpha. A high molecular mass (around 500 kDa) was calculated by western blot of native gels using the same antibody. Finally, specific activity of this enzyme increased 100-fold during maize germination whereas polymerase 3 virtually did not increase. Furthermore, immunoprecipitation experiments with the antipolymerase alpha-antibody showed a decrease in DNA polymerase activity by 70%. The possibility that polymerase 2 is a replicative enzyme is discussed.
Coello
P
P
Departamento de Bioquímica, Facultad de Química, UNAM, D.F.
Rodríquez
R
R
García
E
E
Vázquez-Ramos
J M
JM
D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000302:isolation & purification*; D007158:Immunologic Techniques; D008970:Molecular Weight; D019384:Nucleic Acid Synthesis Inhibitors; D010940:Plant Proteins / Q000302:isolation & purification; D003313:Zea mays / Q000201:enzymology* / Q000254:growth & development
D019384:Nucleic Acid Synthesis Inhibitors; D010940:Plant Proteins; D004259:DNA-Directed DNA Polymerase