ASCORBIC-ACID REGENERATION BY BOVINE IRIS-CILIARY BODY

Ocular tissues appear to require high levels of ascorbic acid and the elucidation of the mechanisms by which those tissues maintain the vita min in its reduced state remains an important objective. The regenerat ion of ascorbate from its oxidative by-product, dehydroascorbic acid ( DHAA), was studied in bovine iris-ciliary body. his-ciliary body was r emoved by scalpel, weighed, minced, and homogenized in 20 mM MOPS, 62 mM sucrose, and 0.1 mM EDTA at pH 7.0. The homogenate was centrifuged and precipitated with ammonium sulfate such that maximal DHAA reducing activity was enhanced in a 50-75% ammonium sulfate fraction. This fra ction was employed for subsequent characterization of DHAA reduction b y iris-ciliary body. Results indicate that the iris-ciliary body enzym atically reduces DHAA to ascorbate at a rate significantly greater tha n can be accounted for by a nonenzymatic glutathione-dependent mechani sm. In addition, saturation kinetics are observed, and the enzymatic a ctivity is dependent on protein concentration, DHAA concentration, and reduced glutathione (GSH) concentration. The activity is sensitive to pH, to high temperature, and to digestion by trypsin and is greatest in the presence of both GSH and NADPH. The reducing activity is theref ore attributed to one or more proteins that are distinct from die know n ascorbate regenerating enzyme, GSH-dependent DHAA reductase (EC 1.8. 5.1).