Hexokinase inactivation induced by ascorbic acid/Fe(II) in rabbit erythrocytes is independent of glutathione-reductive processes and appears to be mediated by dehydroascorbic acid.
Recent studies performed in our laboratory demonstrated that rabbit red blood cell hexokinase was remarkably inhibited by the cocktail ascorbic acid/Fe(II) (Stocchi et al., 1994, Arch. Biochem. Biophys. 311, 160-167) and that the formation of dehydroascorbic acid was a key event in this process (Fiorani et al., 1996, Arch. Biochem. Biophys, 334, 357-361). The present study was undertaken to determine the final hexokinase-inactivating species using cell-free extract as a model. Our results demonstrate superimposable kinetics of hexokinase decay promoted by either ascorbic acid/Fe(II) or dehydroascorbic acid in erythrocyte lysates in which the reduced glutathione (GSH) levels were variously manipulated. In particular, neither removal nor addition of this tripeptide was able to significantly alter the rate or extent of hexokinase inhibition. Thus, GSH-reductive processes are dispensable events in the process of hexokinase inhibition promoted by ascorbic acid/Fe(II) in red blood cells. As a consequence, dehydroascorbic acid appears to be the species which directly inhibits hexokinase. This inference is further supported by the observation that addition of dehydroascorbic acid to the purified enzyme leads to a remarkable inhibition in its activity.
Fiorani
M
M
Instituto di Chimica Biologica Giorgio Fornaini, Università degli Studi di Urbino, Italy.
De Sanctis
R
R
Saltarelli
R
R
Stocchi
V
V
D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology*; D003683:Dehydroascorbic Acid / Q000378:metabolism / Q000494:pharmacology*; D004912:Erythrocytes / Q000187:drug effects / Q000201:enzymology*; D005296:Ferrous Compounds / Q000494:pharmacology*; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism*; D019803:Glutathione Disulfide; D006593:Hexokinase / Q000037:antagonists & inhibitors* / Q000097:blood; D006861:Hydrogen Peroxide / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D011817:Rabbits
D005296:Ferrous Compounds; D006861:Hydrogen Peroxide; D006593:Hexokinase; D005978:Glutathione; D001205:Ascorbic Acid; D010100:Oxygen; D019803:Glutathione Disulfide; D003683:Dehydroascorbic Acid