Reconstitution of the Deinococcus radiodurans aposuperoxide dismutase.
Deinococcus radiodurans, a radiation-resistant aerobe, synthesized a 43,000 Mr dimeric superoxide dismutase. The holoenzyme, sp act 3300 U/mg, contained 1.5 g-atoms Mn, 0.6 g-atom Fe, and 0.1 g-atom Zn per mole dimer. Apoprotein, prepared by dialysis of the holoenzyme in denaturant plus chelator and then renatured in chelex-treated Tris chloride buffer, rapidly regained superoxide dismuting activity upon incubation in 1 mM MnCl2. Reconstitution was dependent on Mn concentration and pH. The Mn-reconstituted protein, sp act 3560 U/mg, contained 1.7 g-atoms Mn per mole dimer. The holoenzyme and Mn-reconstituted apoprotein migrated with the same patterns in 10% acrylamide gels and focused to the same pattern upon isoelectric focusing. Fluorescence emission maxima of the holoenzyme, Mn-reconstituted apoprotein, and the renaturated apoprotein were 329 +/- 1 nm but differed from the denatured apoprotein (352 nm). Apoprotein bound 1.7 g-atoms Zn and from 3-7 g-atoms Fe per mole dimer on incubation with 1 mM ZnSO4 and Fe(NH4)2(SO4)2, respectively. Although neither Zn nor Fe restored superoxide dismuting activity, the ferrous and the zinc salt inhibited reconstitution of the apoprotein with manganese. Metal addition to renatured aposuperoxide dismutase offers a novel approach to reconstitution of procaryote superoxide dismutases.
Juan
J Y
JY
Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg 24060.
Keeney
S N
SN
Gregory
E M
EM
D000596:Amino Acids / Q000032:analysis; D000645:Ammonium Sulfate; D001051:Apoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D001420:Bacteria, Aerobic / Q000201:enzymology*; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006094:Gram-Positive Bacteria / Q000201:enzymology*; D007501:Iron / Q000032:analysis; D007700:Kinetics; D046911:Macromolecular Substances; D008345:Manganese / Q000032:analysis; D008970:Molecular Weight; D013482:Superoxide Dismutase / Q000302:isolation & purification* / Q000378:metabolism; D015032:Zinc / Q000032:analysis
D000596:Amino Acids; D001051:Apoenzymes; D046911:Macromolecular Substances; D008345:Manganese; D007501:Iron; D013482:Superoxide Dismutase; D015032:Zinc; D000645:Ammonium Sulfate