Characterization of nitric oxide synthase in the rat parotid gland.
Nitric oxide (NO) acts as an inter- and intracellular signalling molecule of various cells such as vascular endothelium, macrophages, and neurones. NO is produced by nitric oxide synthase (NOS) from L-arginine. Here the characteristics of NOS in the rat parotid gland were investigated. Approximately 74% of total activity of NOS was present in the cytosolic fraction. For full activation of the NOS in the cytosolic fraction, tetrahydroxybiopterin, NADPH, Ca(2+) and calmodulin were needed as cofactors, because the activity was clearly reduced in the absence of tetrahydroxybiopterin, NADPH, or Ca(2+), or in the absence of calmodulin and presence of trifluoperazine, a calmodulin antagonist, in the reaction mixture. The partially purified NOS activity was completely abolished in the absence of calmodulin or Ca(2+), and activated by them in a dose-dependent manner; EC(50) for calmodulin and Ca(2+) were 10 and 340 nM, respectively. The K(m) for L-arginine was 1.57 microM. Immunoblot analysis revealed that a 165-kDa protein band in the rat parotid gland cytosolic fraction cross-reacted with a rabbit polyclonal antibody against human brain NOS. These results suggest that NOS of the rat parotid gland is a neuronal isoform and that its activity is regulated by physiological concentrations of calmodulin and Ca(2+).
Mitsui
Y
Y
Department of Physiology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271-8587, Japan.
Furuyama
S
S
D000818:Animals; D002847:Chromatography, Agarose / Q000379:methods; D003600:Cytosol / Q000737:chemistry / Q000201:enzymology; D015151:Immunoblotting / Q000379:methods; D008297:Male; D019001:Nitric Oxide Synthase / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D010306:Parotid Gland / Q000737:chemistry / Q000201:enzymology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013053:Spectrophotometry / Q000379:methods
D019001:Nitric Oxide Synthase