Subcellular localization and characterization of neuronal nitric oxide synthase

Area of Science:

• Neuroscience
• Biochemistry
• Cell Biology

Background:

• Endothelial nitric oxide synthase (eNOS) is particulate, while neuronal NOS (nNOS) is considered mainly soluble.
• Posttranslational myristoylation is thought to dictate nNOS solubility, but preliminary data suggest membrane association.

Purpose of the Study:

• To investigate the subcellular distribution of neuronal nitric oxide synthase (nNOS) in rat and rabbit cerebellum.
• To determine if nNOS activity is predominantly soluble or particulate in cerebellar tissue.

Main Methods:

• Subcellular fractionation of rat and rabbit cerebella.
• Density gradient ultracentrifugation to isolate cellular components.
• Assay of nitric oxide synthase activity and characterization of enzyme properties.

Main Results:

• Up to 60% of total nitric oxide synthase activity was found in the particulate fraction of cerebellar homogenates.
• Density gradient ultracentrifugation localized the particulate nNOS primarily to the endoplasmic reticulum.
• Soluble and particulate nNOS exhibited similar specific activity, Ca2+/calmodulin dependency, pH dependency, inhibitor sensitivity, and immunoreactivity.
• Inhibition by the cytochrome P450 inhibitor SKF-525A (IC50 = 90 μM) suggests nNOS is a cytochrome P450-type hemoprotein.

Conclusions:

• Cerebellar nitric oxide synthase is predominantly particulate, associated with the endoplasmic reticulum.
• Rat and rabbit cerebella contain a single type of Ca2+/calmodulin-dependent NOS, which appears to be a cytochrome P450-type hemoprotein.
• Posttranslational myristoylation may not be the sole factor controlling the intracellular localization of neuronal nitric oxide synthase.