Purification and properties of the nuclease inhibitor of Aspergillus oryzae and kinetics of its interaction with crystalline nuclease O.
A nuclease inhibitor found in the mycelia of Aspergillus oryzae has been purified 158,000-fold by ammonium sulfate precipitation, chromatography on Sephadex G-75, DEAE-Sephadex A-50 and Bio-Gel p-60 columns, preparative disc electrophoresis on acrylamide gel, and electrofocusing in ampholite. The purified inhibitor is nearly homogeneous as judged by disc electrophoresis. It shows a typical ultraviolet absorption curve for protein, and the inhibitory activity is inactivated by chymotrypsin. The inhibitor and nuclease O (EC 3.1.4.9, a crystalline enzyme from the mycelia of the same organism) form a stable enzyme inhibitor complex. The molecular weights of nuclease O, the inhibitor and the enzyme inhibitor complex are estimated to be 46,000, 22,000 and 73,000 respectively, by Sephadex G-100 gel filtration. The isoelectric points of the enzyme and the inhibitor are 10.0 and 4.09, respectively, as determined by electrofocusing in ampholite. The inhibition is noncompetitive, and the inhibitor constant (K1) is 3.2 X 10(-12) M, whereas the Michaelis constant (Km) for DNA is 2.2 X 10(-8) M. The inactive enzyme-inhibitor complex is reactivated by chymotrypsin through inactivation of the inhibitor. The reactivated enzyme can be inactivated again by the inhibitor, which shows that desensitization of the enzyme does not occur by the action of chymotrypsin.
D001230:Aspergillus; D001236:Aspergillus oryzae; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D004589:Electrophoresis, Disc; D004706:Endodeoxyribonucleases; D004720:Endonucleases / Q000037:antagonists & inhibitors*; D004722:Endoribonucleases; D007525:Isoelectric Focusing; D007700:Kinetics
D001426:Bacterial Proteins; D004706:Endodeoxyribonucleases; D004720:Endonucleases; D004722:Endoribonucleases; C021835:Serratia marcescens nuclease