Biochemical Characterization and Histochemical Localization of Nitric Oxide Synthase in the Nervous System of the Snail, Helix pomatia |
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Authors: | Shile Huang Hubert H Kerschbaum Edwin Engel Anton Hermann |
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Institution: | Department of Animal Physiology, Institute of Zoology, and; Institute of Genetics and General Biology, University of Salzburg, Salzburg, Austria |
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Abstract: | 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. |
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Keywords: | Nitric oxide NADPH-diaphorase Nitric oxide synthase Neuron Mollusk Helix pomatia |
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