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| NITRIC-OXIDE SYNTHASE REGULATORY SITES - PHOSPHORYLATION BY CYCLIC AMP-DEPENDENT PROTEIN-KINASE, PROTEIN-KINASE-C, AND CALCIUM CALMODULIN PROTEIN-KINASE - IDENTIFICATION OF FLAVIN AND CALMODULIN BINDING-SITES |
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| Author(s): BREDT DS, FERRIS CD, SNYDER SH |
| Source: JOURNAL OF BIOLOGICAL CHEMISTRY Volume: 267 Issue: 16 Pages: 10976-10981 Published: JUN 5 1992 |
| Times Cited: 507 References: 44 |
| Abstract: Nitric oxide (NO) is an important molecular messenger accounting for endothelial-derived relaxing activity in blood vessels, mediating cytotoxic actions of macrophages, and functioning as a neurotransmitter in the brain and periphery. NO synthase (NOS) from brain has been purified to homogeneity and molecularly cloned. We now report that NOS is stoichiometrically phosphorylated by cAMP dependent protein kinase, protein kinase C, and calcium/calmodulin-dependent protein kinase, with each kinase phosphorylating a different serine site on NOS. Activation of PKC in transfected cells reduces NOS enzyme activity by almost-equal-to 77% in intact cells and by 50% in protein homogenates from these cells. Utilizing fluorescence spectroscopy we find that purified monomer NOS contains 1 molar equivalent of both FMN and FAD. This stoichiometry is supported by enzymatic digestion of the flavins with phosphodiesterase, and titration of the FMN with a specific FMN binding protein. We demonstrate that purified NOS is labeled by a photoaffinity derivative of calmodulin. These recognition sites on NOS provide multiple means for regulation of NO levels and "cross-talk" between second messenger systems. |
| Document Type: Article |
| Language: English |
Addresses:
1. JOHNS HOPKINS UNIV, SCH MED, DEPT NEUROSCI, BALTIMORE, MD 21205 USA
2. JOHNS HOPKINS UNIV, SCH MED, DEPT PHARMACOL & MOLEC SCI, BALTIMORE, MD 21205 USA
3. JOHNS HOPKINS UNIV, SCH MED, DEPT PSYCHIAT & BEHAV SCI, BALTIMORE, MD 21205 USA |
| Publisher: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 9650 ROCKVILLE PIKE, BETHESDA, MD 20814 |
| Subject Category: Biochemistry & Molecular Biology |
| IDS Number: HX169 |
| ISSN: 0021-9258 |
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