ACTIVE-SITE MODIFICATION OF ALDOSE REDUCTASE BY NITRIC-OXIDE DONORS

Nitric oxide (NO) donors sodium nitrosoprusside (SNP), S-nitroso-N-ace tylpenicillamine (SNAP), and 3-morpholinosydnonemine (SIN-1) caused a time-and concentration-dependent loss of catalytic activity of recombi nant human placental aldose reductase. Modification of the enzyme was prevented by NADPH and NADP and reversed partially by dithiothreitol ( DTT) and sodium borohydride. The protection by NADPH was lost in the p resence of both substrates (NADPH and glyceraldehyde), indicating that the enzyme becomes sensitive to inhibition by SNP during catalysis. S ite-directed mutant form of the enzyme, in which active site cys-298 w as substituted with serine (C298S) was not inactivated by NO donors, w hereas, ARC80S and ARC303 were as sensitive as the wild type enzyme, i ndicating that inactivation of aldose reductase is due to modification of the active site at cys298, These results suggest that NO may be an endogenous regulator of aldose reductase, and consequently the polyol pathway of glucose metabolism; which has been implicated in the patho genesis of secondary diabetic complications. (C) 1997 Elsevier Science B.V.