MOLECULAR-STATES OF FUNGAL NUCLEASE COMPOSED OF HETEROGENEOUS SUBUNITS AS ESTIMATED FROM THE EFFECTS OF UREA AND CHELATING-AGENTS

Fungal nuclease composed of subunits with molecular weights of 8.0 X 1 0(4) (80K subunit), 5.0 X 10(4) (50K subunit), and 2.5 X 10(4) (25K su bunit) (K. Ito, Y. Matsuura, and N. Minamiura (1994) Arch. Biochem. Bi ophys. 309, 160-167) wars inactivated by urea and dissociated into its subunits. The urea inactivation depended on the concentration of urea , the incubation period and the temperature. The urea-inactivated enzy me had about 25% activity restored by removal of urea, and the native form of the enzyme was also reconstituted. The urea inactivation and t he dissociation of subunits were almost completely prevented by Ca2+ b ut not by glycerol. The enzyme was also inactivated by ethylenediamine tetraacetic acid (EDTA). From this method of inactivation, the 50K and 25K subunits were still associated, but the complex showed no nucleas e activity. About 80% of the activity of the EDTA-inactivated enzyme w as restored by the addition of Ca2+ or Sr2+ and 20-40% by Mn2+, Ba2+, Mg2+, or Co2+. The reactivation of the enzyme by these metal ions was accompanied by the reconstitution of the native form of the enzyme. Th e enzyme was inactivated by ethylene glycol bis(beta-aminoethyl ether) -N,N'-tetraacetic acid (EGTA) at much higher concentrations compared w ith the inactivation by EDTA. On the other hand, dissociation of the s ubunits by EGTA proceeded in a manner similar to that of the inactivat ion of the enzyme by EDTA. The 50K and 25K subunits were still associa ted, and the complex showed nuclease activity. These results indicated that the enzyme contains two kinds of metal ions. One metal ion, repr esented by Ca2+, is thought to stabilize the quaternary structure of t he enzyme, especially to connect the 80K subunit and the complex of th e 50K and 25K subunits. Another metal ion, represented by Mg2+, is tho ught to be bound to the complex of the 50K and 25K subunits and to be required for activity appearance of the complex. Along with these resu lts, possible molecular states of the enzyme under various conditions are proposed. (C) 1995 Academic Press, Inc.