Characterization of Drosophila nitric oxide synthase: a biochemical study

Sengupta, R., Sahoo, R., Mukherjee, S., Regulski, M., Tully, T., Stuehr, D. J., Ghosh, S. (June 2003) Characterization of Drosophila nitric oxide synthase: a biochemical study. Biochemical and Biophysical Research Communications, 306 (2). pp. 590-597. ISSN 0006-291X

URL: http://www.ncbi.nlm.nih.gov/pubmed/12804606
DOI: 10.1016/S0006-291X(03)01003-9,

Abstract

The heme and flavin-binding domains of Drosophila nitric oxide synthase (DNOS) were expressed in Escherichia coli using the expression vector pCW. The denatured molecular mass of the expressed protein was 152 kDa along with a proteolytically cleaved product of 121 kDa. The DNOS heme protein exhibited very low Ca2+/calmodulin-dependent NO synthase activity. The trypsin digestion patterns were different from nNOS. The full-length DNOS protein had high degree of stability against trypsin. The activity assay of trypsin-digested protein confirmed the same result. Urea dissociation profile of DNOS full-length protein showed that the reductase domain activity was much more susceptible towards urea than the oxygenase domain activity. Urea gradient gel of DNOS full-length protein established distinct transition of dissociation and unfolding in the range 3-4 M urea. Reductase domain activity of full-length DNOS protein against external electron acceptors like cytochrome c indicated slow electron transfer from FMN. The bacterial expression of DNOS full-length protein represents an important development in structure-function studies of this enzyme and comparison with other mammalian NOS enzymes which is evolutionary significant. (C) 2003 Elsevier Science (USA). All rights reserved.

Item Type: Paper
Uncontrolled Keywords: Drosophila nitric oxide synthase arginine tetrahydrobiopterin calmodulin trypsin hydrolysis urea gradient gel Griess assay MACROPHAGE NO SYNTHASE ELECTRON-TRANSFER BIDOMAIN STRUCTURE REDUCTASE DOMAIN ACTIVE-SITE L-ARGININE CALMODULIN TETRAHYDROBIOPTERIN EXPRESSION IDENTIFICATION
Subjects: organism description > animal > insect > Drosophila
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > enzymes
bioinformatics > genomics and proteomics > small molecules > nitric oxide
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > enzymes > nitric oxide synthase
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types
CSHL Authors:
Communities: CSHL labs > Tully lab
Depositing User: Matt Covey
Date: June 2003
Date Deposited: 11 Jun 2013 20:50
Last Modified: 11 Jun 2013 20:50
Related URLs:
URI: http://repository.cshl.edu/id/eprint/27942

Actions (login required)

Administrator's edit/view item Administrator's edit/view item
CSHL HomeAbout CSHLResearchEducationNews & FeaturesCampus & Public EventsCareersGiving