Abstract
Nitroprusside appears to inhibit the known types of superoxide dismutases irrespective of their metal prosthetic group and regardless of the source from which the enzymes were isolated. Thus the copper-zinc enzyme from bovine erythrocyte or Neurospora crassa behaved identically as did the manganese enzymes from Escherichia coli or red alga and the iron enzyme from E. coli and a blue-green alga. The inhibition was dose dependent with a Ki = 2.5 X 10(-5) for nitroprusside. Nitroprusside does not bind to the copper moiety of copper-zinc enzyme and seems to compete with O2- for superoxide dismutase. These inhibitions by nitroprusside, which were elicited not only in purified enzymes but also in crude soluble extracts of biological samples, were rapidly reversible. Nitroprusside was found to react with O2- to form a paramagnetic species with three absorption lines of equal width with a separation AN = 15.0 G and a g value of 2.028. The spin adduct appears to be a nitroxide radical and was stable for several minutes.
Highlights
Superoxide dismutases, which appearto be anessential defense againstthe toxicity of oxygen,havebeenisolated from a wide range of organisms
We have investigated the effects of nitroprusside on superoxide dismutase and have observed that nitroprusside inhibits all superoxide dismutasestothesame degree,irrespective of theirmetal prosthetic group and regardless of the organism from which the enzyme was isolated
The copper-zinc-superoxide dismutases were prepared from bovine erythrocytes [33] and Neurosporacrassa [34]
Summary
The copper-zinc-superoxide dismutases were prepared from bovine erythrocytes [33] and Neurosporacrassa [34]. The Mn-containing superoxide dismutases were isolated from Escherichia coli [35] and Porphyridium cruentum [36]. The Fe-containing superoxide dismutases were purified from E. coli [37] and Plectonema boryanurn [38]. These enzymes were routinely assayed either in terms of their ability to prevent the reduction of cytochrome3+c by 0, [33] or to augment riboflavin-sensitized photooxidation of dianisidine [39]. Cytochrome3+c (type III), and dianisidine were purchased from Sigma, while riboflavin was obtained from Eastman. The rate of cytochrome3+c reduction was calculataesdsuming that between the oxidized and reduced states is 2.1 X lo4M” cm” [41].EPR spectra were obtained with a Varian model E-109 X-band instrument Spectrophotometric assayswere performed in a Gilford model 2000 absorbance recorder at 25 “C. The rate of cytochrome3+c reduction was calculataesdsuming that between the oxidized and reduced states is 2.1 X lo4M” cm” [41].EPR spectra were obtained with a Varian model E-109 X-band instrument
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