Characterization of Drosophila nitric oxide synthase: a biochemical study.
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 152kDa along with a proteolytically cleaved product of 121kDa. The DNOS heme protein exhibited very low Ca(2+)/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 3-4M 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.
Sengupta
Rajib
R
Department of Biochemistry, University College of Sciences, Calcutta University, 35 Ballygunge Circular Road, Kolkata, West Bengal 700 019, India.
Sahoo
Rupam
R
Mukherjee
Shaeri
S
Regulski
Michael
M
Tully
Tim
T
Stuehr
Dennis J
DJ
Ghosh
Sanjay
S
D000818:Animals; D001120:Arginine / Q000737:chemistry; D001708:Biopterins / Q000031:analogs & derivatives* / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004330:Drosophila; D004583:Electrons; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006418:Heme / Q000737:chemistry; D006861:Hydrogen Peroxide; D006868:Hydrolysis; D009249:NADP / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000737:chemistry*; D009573:Nitrites / Q000378:metabolism; D016366:Open Reading Frames; D017434:Protein Structure, Tertiary; D013053:Spectrophotometry; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D014466:Ultraviolet Rays; D014508:Urea / Q000494:pharmacology
D009566:Nitrates; D009573:Nitrites; D001708:Biopterins; D009569:Nitric Oxide; D006418:Heme; D009249:NADP; D014508:Urea; D001120:Arginine; D006861:Hydrogen Peroxide; D019001:Nitric Oxide Synthase; D014357:Trypsin; C003402:sapropterin