Involvement of sulfhydryl residues in aldose reductase-inhibitor interaction.
Purification and storage of aldose reductase isolated from human placenta or kidney in beta-mercaptoethanol-containing buffers causes a time-dependent decrease in its catalytic activity, as well as in its sensitivity to inhibition by aldose reductase inhibitors such as sorbinil. Dithiothreitol (DTT) slowly regenerated the enzyme activity, as well as reversed the alterations in the sensitivity of the enzyme to sorbinil. In contrast to sorbinil, the inhibition of aldose reductase by tolrestat was less affected by purification and/or storage in beta-mercaptoethanol-containing buffers. Kinetic analysis of the rate of increase in sensitivity of the enzyme to sorbinil on incubation with DTT reveals that the reaction follows two kinetically distinct rate constants. Also, sorbinil protected the enzyme from inactivation with sulfhydryl-modifying reagents 5,5'-dithiobis(2-nitrobenzoic acid) and glutathione disulfide. The enzyme stored in beta-mercaptoethanol migrates as two distinct bands, one corresponding to molecular weight 36,000 and the other to molecular weight 33,000, on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas under reducing conditions the protein migrates as a single discrete band corresponding to molecular weight 36,000. Moreover, the molecular weight 33,000 form of the enzyme could be converted to the molecular weight 36,000 form on reduction with DTT, indicating that the molecular weight 33,000 form of the enzyme is due to intramolecular disulfide bond(s) formed, which presumably cause the protein to assume a more folded conformation and migrate faster through the gel, and not due to proteolysis. These studies indicate that oxidation of sulfhydryl residues, including disulfide bond formation, during purification and storage in beta-mercaptoethanol-containing buffers alters the sensitivity of the enzyme to some inhibitors.
Bhatnagar
A
A
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77550.
Liu
S
S
Das
B
B
Srivastava
S K
SK
D000449:Aldehyde Reductase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D004229:Dithiothreitol / Q000494:pharmacology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D048289:Imidazolidines; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D008970:Molecular Weight; D009249:NADP / Q000494:pharmacology; D013401:Sugar Alcohol Dehydrogenases / Q000037:antagonists & inhibitors*; D013438:Sulfhydryl Compounds / Q000032:analysis*
D007093:Imidazoles; D048289:Imidazolidines; D013438:Sulfhydryl Compounds; D009249:NADP; D008623:Mercaptoethanol; D013401:Sugar Alcohol Dehydrogenases; D000449:Aldehyde Reductase; C026411:sorbinil; D004229:Dithiothreitol