Subcellular localization and characterization of neuronal nitric oxide synthase.
In contrast to the predominantly particulate, Ca2+/calmodulin-dependent nitric oxide (NO) synthase in endothelial cells, the corresponding neuronal isoenzyme is considered to be mainly soluble, presumably owing to the lack of a posttranslational myristoylation. However, preliminary findings from this and other laboratories suggest that a substantial portion of the neuronal NO synthase activity may in fact be membrane bound. We have therefore investigated the distribution of this enzyme among subcellular fractions of the rat and rabbit cerebellum in more detail. Up to 60% of the total NO synthase activity was found in the particulate fraction and, according to density gradient ultracentrifugation, associated mainly with the endoplasmic reticulum fraction. There was no apparent difference between the soluble and particulate enzymes with respect to their specific activity, Ca2+ and pH dependency, inhibitor sensitivity, or immunoreactivity, suggesting that both rat and rabbit cerebella contain a single Ca2+/calmodulin-dependent NO synthase. The inhibition by the cytochrome P450 inhibitor SKF-525A of the NO synthase activity in these subcellular fractions (IC50 = 90 microM) and the fact that mammalian cytochrome P450 enzymes are endoplasmic reticulum-bound proteins support the notion that the cerebellar NO synthase is a cytochrome P450-type hemoprotein. Moreover, the aforementioned findings suggest that posttranslational myristoylation may not be the only factor determining the intracellular localization of NO synthase.
Hecker
M
M
Centre of Physiology, Johann-Wolfgang-Goethe-University Clinic, Frankfurt/Main, F.R.G.
Mülsch
A
A
Busse
R
R
D000594:Amino Acid Oxidoreductases / Q000032:analysis* / Q000037:antagonists & inhibitors; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002499:Centrifugation, Density Gradient; D002531:Cerebellum / Q000201:enzymology* / Q000648:ultrastructure; D065607:Cytochrome P-450 Enzyme Inhibitors; D003600:Cytosol / Q000201:enzymology; D004721:Endoplasmic Reticulum / Q000201:enzymology; D007425:Intracellular Membranes / Q000201:enzymology; D007527:Isoenzymes / Q000032:analysis*; D009474:Neurons / Q000201:enzymology* / Q000648:ultrastructure; D019001:Nitric Oxide Synthase; D019335:Nitroarginine; D011335:Proadifen / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology*
D002147:Calmodulin; D065607:Cytochrome P-450 Enzyme Inhibitors; D007527:Isoenzymes; D019335:Nitroarginine; D001120:Arginine; D011335:Proadifen; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D002118:Calcium