Biochemical characterization of NADPH diaphorase in the chick embryo retina: stimulation by calcium ions and inhibition by arginine analogs
Braz. j. med. biol. res
; 28(2): 252-5, Feb. 1995. graf
Article
Dans En
| LILACS
| ID: lil-154273
Responsable en Bibliothèque :
BR1.1
ABSTRACT
Nitric oxide is an important intercellular messenger in the central nervous system. NADPH-diaphorase, reported to be identical to nitric oxide synthase, is prsent in specific groups of cells in several neural tissues, including the retina. We determined NADPH-diaphorase activity in homogenates of the chick embryo retina. The enzyme activity was measured spectophotometrically at 585 nm after incubating retinal total homogenates (100-150 µg protein) with 1mMNADPH and 0.5 mM nitroblue tetrazolium in 50 mMTris buffer, pH8.1, at 37ºC. NADPH-diaphorse was detected in 14-day old retinas and 53-65 per cent of the enzyme activity was inhibited by 3 mM NG-nitro-L-arginine (NARG), the arginine analog. One mM L-N5-(1-iminoethyl)ornithine (NIO) was the most potent inhibitor (63 per cent inhibition) while 3 mM NG-nitro-L-arginine methyl ester (NAME) (33 per cent inhibition) and I mMNG-monomethyl-L-arginine acetate (NMMA) (14 per cent inhbition) were less effective. Enzyme activity was increased by 48 per cent by 2 mM calcium chloride, and effect reversed by 1 mMEGTA or EDTA. Basal enzyme levels were also partially inhibited by the chelators, indicating the presence of calcium-dependent and -independent isoforms of nitric oxide synthase in the retina. The results show that the NADPH-diaphorase assay is sample and sensitive and that the different isoforms of nitric oxide synthase expressed in chick retinal cells during development can be demonstrated
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Indice:
LILACS
Sujet Principal:
Rétine
/
Amino-acid oxidoreductases
/
Dihydrolipoamide dehydrogenase
Type d'étude:
Diagnostic_studies
langue:
En
Texte intégral:
Braz. j. med. biol. res
Thème du journal:
BIOLOGIA
/
MEDICINA
Année:
1995
Type:
Article