Ionic Requirements of the DNA Polymerase Associated with Serum Hepatitis B Antigen

Abstract

Activity of the DNA polymerase associated with hepatitis B antigen (HB Ag) was enhanced by increasing concentrations of monovalent cations. There was no inhibition of enzyme activity even at salt concentrations of 0.8 M. Enhancement of DNA polymerase activity increased with atomic radius of the monovalent cations. Enzyme activity was higher with chloride than with sulfate anion. The DNA polymerase was activated by increasing concentrations of MgCl₂, with activity increasing even at concentrations of 80 mM. HB Ag-associated DNA polymerase was not activated by MnCl₂, CaCl₂, ZnCl₂, CdCl₂, FeCl₃, or CuCl₂. However, some activation of enzyme activity was observed with FeCl₂. All of these divalent cations exerted marked inhibitory effects on the DNA polymerase reaction in the presence of MgCl₂ HB Agassociated DNA polymerase was markedly inhibited by p-hydroxymercuribenzoate and N-ethylmaleimide in the absence of thiol compounds. Calcium elenolate also inhibited the enzyme. Studies of inhibitors indicated that sulfhydryl groups were required for enzyme activity. In contrast to results obtained with HB Ag-associated polymerase, Escherichia coli and bovine liver alpha DNA polymerases showed optimal concentrations of both MgCl₂ and KCl for activity, and the enzymes were inhibited by high concentrations of either salt. The activity of HB Ag-associated DNA polymerase at high concentrations of monovalent and divalent cations afforded a differential assay for this enzyme in the presence of the other two DNA polymerases. It is suggested that the ionic requirements of HB Ag-associated DNA polymerase could be used for differential assay in the presence of cellular DNA polymerases.