Nitric oxide (NO) is known to be synthesized in several tissues and to increase the formation of cyclic GMP through the activation of soluble guanylate cyclases. Since cyclic GMP plays an important role in visual transduction, we investigated the presence of nitric oxide synthesizing activity in retinal rod outer segments. Bovine rod outer segments were isolated intact and separated into membrane and cytosolic fractions. Nitric oxide synthase activity was assayed by measuring the conversion of L-arginine to L-citrulline. Both membrane and cytosolic fractions were active in the presence of calcium and calmodulin. The activity in both fractions was stimulated by the nitric oxide synthase cofactors FAD, FMN, and tetrahydrobiopterin and inhibited by the L-arginine analog, L-monomethyl arginine. The Km for L-arginine was similar, about 5 microM for the enzyme in both fractions. However, the two fractions differed in their calcium/calmodulin dependence: the membrane fraction exhibited basal activity even in the absence of added calcium and calmodulin while the cytosolic fraction was inactive. But the activity increased in both fractions when supplemented with calcium/calmodulin: in membranes from about 40 to 110 fmol/min/mg of protein and in the cytosol from near zero to about 350 fmol/min/mg of protein in assays carried out at 0.3 microM L-arginine. The two enzymes also responded differently to detergent: the activity of the membrane enzyme was doubled by Triton X-100 while that of the cytosolic enzyme was unaffected. These results show that NO is produced by cytosolic and membrane-associated enzymes with distinguishable properties.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗

PubMedDate not supplied

Interactions of glutathione disulfide with lens crystallins.

The study was designed to investigate the formation of mixed disulfides (PSSG) of protein and glutathione (GSH) in the lens. The possibility that oxidized GSH reacts with lens protein sulfhydryls was examined by incubating tritiated glutathione disulfide ([3H] GSSG) with a solution of dialyzed water-soluble bovine lens crystallins. Gel chromatography on a Sephacryl S-200 column showed that all crystallins have become labeled. The ratios of radioactivity incorporated into each crystallin relative to the protein concentration (cpm/0.D. ratios) were 0.5, 6, 12 and 11 for alpha, beta H, beta L and gamma-crystallins, respectively. When [3H] GSSG was covalently linked to and immobilized on derivatized agarose beads (Affi-Gel 10) and then treated with dialyzed lens crystallins part of the radioactivity was released from the gel. Most of the released radioactivity was associated with the protein and was not dialyzable but susceptible to removal by dithiothreitol or glutathione, indicating that it is linked to the protein as PSSG. Dialysis of [3H] PSSG against increasing concentrations of unlabeled GSSG or GSH showed that only GSH could effectively cause the release of radioactivity into the dialysis medium due to the formation of GSSG. It can be concluded that GSSG rather than GSH is involved in the formation of protein-glutathione disulfide and that such a reaction may retard or prevent protein aggregation due to the protein-protein disulfide bond formation that leads to lens opacification.

Animals↗