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 10(4) (80K subunit), 5.0 x 10(4) (50K subunit), and 2.5 x 10(4) (25K subunit) (K. Ito, Y. Matsuura, and N. Minamiura (1994) Arch. Biochem. Biophys. 309, 160-167) was 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 enzyme had about 25% activity restored by removal of urea, and the native form of the enzyme was also reconstituted. The urea inactivation and the dissociation of subunits were almost completely prevented by Ca2+ but not by glycerol. The enzyme was also inactivated by ethylenediaminetetraacetic acid (EDTA). From this method of inactivation, the 50K and 25K subunits were still associated, but the complex showed no nuclease activity. About 80% of the activity of the EDTA-inactivated enzyme was 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. The enzyme was inactivated by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) at much higher concentrations compared with the inactivation by EDTA. On the other hand, dissociation of the subunits by EGTA proceeded in a manner similar to that of the inactivation of the enzyme by EDTA. The 50K and 25K subunits were still associated, and the complex showed nuclease activity. These results indicated that the enzyme contains two kinds of metal ions. One metal ion, represented by Ca2+, is thought to stabilize the quanternary structure of the enzyme, especially to connect the 80K subunit and the complex of the 50K and 25K subunits. Another metal ion, represented by Mg2+, is thought 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 results, possible molecular states of the enzyme under various conditions are proposed.
Ito
K
K
Department of Biology, Faculty of Science, Osaka City University, Japan.
Hara
C
C
Matsuura
Y
Y
Minamiura
N
N
D001230:Aspergillus / Q000201:enzymology*; D002118:Calcium / Q000737:chemistry; D002614:Chelating Agents / Q000494:pharmacology*; D003851:Deoxyribonucleases / Q000737:chemistry*; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D008274:Magnesium / Q000737:chemistry; D008670:Metals / Q000494:pharmacology; D017433:Protein Structure, Secondary; D012260:Ribonucleases / Q000737:chemistry*; D014508:Urea / Q000737:chemistry / Q000494:pharmacology*
D002614:Chelating Agents; D008670:Metals; D004533:Egtazic Acid; D014508:Urea; D004492:Edetic Acid; D003851:Deoxyribonucleases; D012260:Ribonucleases; D008274:Magnesium; D002118:Calcium