%0 Journal Article
%T Human platelet nitric oxide synthase activity: an optimized method
%A Kawamato
%A Elisa Mitiko
%A Glezer
%A Isaias
%A Munhoz
%A Carolina Demarchi
%A Bernardes
%A Cristiane
%A Scavone
%A Cristoforo
%A Marcourakis
%A Tania
%J Revista Brasileira de Ci¨ºncias Farmac¨ºuticas
%D 2002
%I Scientific Electronic Library Online
%R 10.1590/S1516-93322002000300006
%X we investigated the kinetic analysis of human platelet nitric oxide synthase (nos) activity by the rate of conversion of [3h] arginine to [3h]-citrulline in unstimulated fresh platelets. nos activity was present in the membrane fraction and cytosol, and was ca2+- and calmodulin dependent which is a characteristic of endothelial nos. nos activity was also dependent of nadph since the omission of this cofactor induced an important decrease (85,2%) in the enzyme activity. the kinetic varied with protein and arginine concentration but optimum concentrations were found up to 60 minutes, and up to 80 ¦Ìg of protein at 120 nm of arginine and 0.5 ¦Ìci of 3h-arginine. nos activity in the absence of fad (flavin adenine dinucleotide), fmn (flavin mononucleotide) and bh4 (tetrahydrobiopterin) was only 2.8% of the activity measured in the presence of these three cofactors. the enzyme activity was completely inhibited by l-name (1 mm) (98.1 %) and egta (5 mm) (98.8 %). trifluoperazine (tfp) caused 73.2% inhibition of the enzyme activity at 200 ¦Ìm and 83.8 % at 500 ¦Ìm. under basal conditions, nos km for l-arginine was 0.84 ¡À 0.08 ¦Ìm and mean vmax values were 0.122 ¡À 0.025 pmol.mg-1.min-1. mean human nos platelet activity was 0.020 ¡À 0.010 pmol.mg-1.min-1. results indicate that the enos in human platelet can be evaluated by conversion of [3h]-arginine to [3h]citrulline in an optimized method, which provide reproducible and accurate results with good sensitivity to clinical experiments involving neurological and psychiatric diseases.
%K nitric oxide synthase
%K platelets
%K arginine
%K citrulline.
%U http://www.scielo.br/scielo.php?script=sci_abstract&pid=S1516-93322002000300006&lng=en&nrm=iso&tlng=en