On the lack of specificity of the cobalt-bicarbonate method for carbonic anhydrase.
Data are presented that support a nonenzymic mechanism for the staining obtained with the cobalt-bicarbonate method. The biochemically inactive apocarbonic anhydrase and Cu+2 apocarbonic anhydrase stain positively and this stain is inhibited by acetazolamide. The staining of the acetazolamide resistant carbonic anhydrase of male rat liver is inhibited by 10-6 M acetazolamide, at which concentration no biochemical inhibition is observed. There is no correlation between the biochemical and histochemical inhibitory potencies of a number of sulfonamides. The nonsulfonamide inhibitor, KCNO, does not inhibit staining. When incubations are performed in media exposed to atmospheres of increasing CO2 content, staining is not abolished until the atmospheric pCO2 approaches that generated by the medium itself. This finding renders the carbonic anhydrase catalyzed dehydration of HCO3- an improbable reaction for the staining. Studies with modified media show differences in staining patterns and in sensitivity to acetazolamide inhibition which question the specificity of the method for carbonic anhydrase.
D000086:Acetazolamide / Q000494:pharmacology; D000143:Acids; D001051:Apoenzymes / Q000378:metabolism; D001639:Bicarbonates; D002245:Carbon Dioxide / Q000494:pharmacology; D002257:Carbonic Anhydrase Inhibitors; D002256:Carbonic Anhydrases / Q000378:metabolism*; D003035:Cobalt; D003300:Copper; D003485:Cyanates / Q000494:pharmacology; D007672:Kidney Cortex / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013194:Staining and Labeling / Q000379:methods*; D013379:Substrate Specificity; D015032:Zinc
D000143:Acids; D001051:Apoenzymes; D001639:Bicarbonates; D002257:Carbonic Anhydrase Inhibitors; D003485:Cyanates; D002245:Carbon Dioxide; D012967:Sodium Dodecyl Sulfate; D003035:Cobalt; D003300:Copper; D002256:Carbonic Anhydrases; D015032:Zinc; D000086:Acetazolamide