title: Biochemical characterization and histochemical localization of nitric oxide synthase in the nervous system of the snail, Helix pomatia.
abstract: Nitric oxide synthase (NOS) in the snail Helix pomatia was characterized by biochemical and molecular biological techniques and localized by histochemical methods. Central ganglia contained particulate paraformaldehyde-sensitive and cytosolic paraformaldehyde-insensitive NADPH-diaphorase. The cytosolic NADPH-diaphorase activity coeluted with NOS activity. The activity of NOS was dependent on Ca2+ and NADPH and was inhibited by N(G)-nitro-L-arginine (L-NNA). Proteins purified by 2',5'-ADP affinity chromatography were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and migrated at 150, 60, 40, and 30 kDa. An antibody to mammalian NOS exclusively labeled the 60-kDa protein. Characterization of the cDNA of the corresponding 60-kDa NOS-immunoreactive protein revealed no sequence homology with any known NOS isoform. The recombinant protein exhibited Ca2+- and NADPH-dependent NOS activity, which was partially inhibited by EGTA and L-NNA. Histochemistry showed NADPH-diaphorase activity in discrete regions of the central and peripheral nervous system. About 60% of the NADPH-diaphorase-positive neurons colocalize with immunoreactive material detected by antibodies to mammalian NOS. Comparison of organs showed the highest NADPH-diaphorase activity in the nervous system, whereas moderate activity was present in muscle tissue, digestive tract, and gonads. Our study suggests the presence of NOS and a putative NOS-associated/regulating protein in mollusk nervous tissue.
authors.affiliation: Department of Animal Physiology, Institute of Zoology, University of Salzburg, Austria.
authors.forename: S
authors.initials: S
authors.lastname: Huang
authors.forename: H H
authors.initials: HH
authors.lastname: Kerschbaum
authors.forename: E
authors.initials: E
authors.lastname: Engel
authors.forename: A
authors.initials: A
authors.lastname: Hermann
mesh_terms: D000818:Animals; D001483:Base Sequence; D002490:Central Nervous System / Q000201:enzymology; D018076:DNA, Complementary / Q000235:genetics; D006372:Helix, Snails / Q000201:enzymology*; D006651:Histocytochemistry; D015151:Immunoblotting; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009252:NADPH Dehydrogenase / Q000378:metabolism; D009420:Nervous System / Q000166:cytology / Q000201:enzymology*; D009474:Neurons / Q000201:enzymology; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010525:Peripheral Nerves / Q000201:enzymology; D014018:Tissue Distribution
chemical_list: D018076:DNA, Complementary; D019001:Nitric Oxide Synthase; D009252:NADPH Dehydrogenase