NO synthase (NOS) catalyzes the oxidation of L-arginine to L-citrulline and nitric oxide (NO) or a NO-releasing compound. At least three isoforms of NOS exist (types I-III). The activities of the type I isoform purified from brain and the type III isoform purified from endothelial cells are regulated by the intracellular free calcium concentration ([Ca2+]i) and the Ca(2+)-binding protein calmodulin. At resting [Ca2+]i, both isozymes are inactive; they become fully active at [Ca2+]i greater than or equal to 500 nM Ca2+. Longer lasting increases in [Ca2+]i may downregulate NO formation, for in vitro phosphorylation by Ca2+/calmodulin protein kinase II decreases the Vmax of NOS. Besides the conversion of L-arginine, type I NOS, Ca2+/calmodulin dependently, generates H2O2 and reduces cytochrome c/P450. Other redox activities, i.e. the reduction of nitroblue tetrazolium to diformazan (NADPH-diaphorase) or of quinoid-dihydrobiopterin to tetrahydrobiopterin, by NOS appear to be Ca2+/calmodulin-independent.

Amino Acid Oxidoreductases↗

PubMedDate not supplied

Localization of nitric oxide synthase, NADPH diaphorase and soluble guanylyl cyclase in adult rabbit retina.

Nitric oxide (NO) acts as a neuronal messenger which activates soluble guanylyl cyclase (SGC) in neighboring cells and produces a wide range of physiological effects in the central nervous system (CNS). Using immunocytochemical and histochemical stains, we have characterized the NO/SGC system in the rabbit retina and to a lesser extent, in monkey retina. Based on staining patterns observed with an antibody to nitric oxide synthase (NOS) type I and a histochemical marker for NADPH diaphorase, a metabolic intermediate required for NOS activity, three major classes of neurons appear to generate NO in the rabbit retina. These include two subclasses of sparsely distributed wide field amacrine cells, rod and cone photoreceptors, and a subpopulation of ganglion cells. Equivalent cell populations were labeled in monkey retina. An antibody to SGC (tested only in rabbit retina), labeled large arrays of cone photoreceptors in the outer nuclear layer, both amacrine and bipolar cells in the inner nuclear layer (INL), as well as populations of neurons in the ganglion cell layer. These data suggest that the ability to generate NO is restricted to relatively few neurons in the inner retina and to photoreceptor cells in the outer retina; while presumptive target cells, containing pools of SGC, are widespread and form contiguous fields across the inner and outer nuclear layers (ONL) as well as the ganglion cell layer.

Animals↗

PubMedDate not supplied

Epidermal proteinases in the European eel.

A fluorescent-sensitive assay demonstrated the exhaustive detection of proteinase activities in the dorsal skin of the European eel. Two distinct skin extracts were prepared from skin mucus and epidermal cell layers with no mutual contamination, so that the latter extract contained significant susceptibility of all tested substrates. Optimum hydrolysis pH's for susceptible substrates were found in acidic and neutral ranges, and optimum hydrolysis temperatures for the same substrates fell mainly in the 40 degrees-50 degrees C range. In addition, diverse inhibitory influences on these hydrolyses were prompted by several proteinase inhibitors and metal chlorides, and some other reagents specifically affected the individual hydrolysis; among them, the inhibitions of all activities by p-tosyl-L-phenylalanyl-chloromethylketone CdCl2, CuCl2, HgCl2, and ZnCl2 were remarkable. Antipain, iodoacetamide, and CoCl2 induced a severe inhibition of all except three activities, whereas N-ethylmaleimide markedly inhibited only these three activities. These findings suggest that epidermal cell layers of the European eel retain a party of proteolytic enzymes, and this party is judged from their exhibiting specificities to be composed of four distinct proteinases, presumably cathepsins L and B, a serine proteinase, and an aminopeptidase.

Animals↗