Document Detail

Expression of human inducible nitric oxide synthase in a tetrahydrobiopterin (H4B)-deficient cell line: H4B promotes assembly of enzyme subunits into an active dimer.
MedLine Citation:
PMID:  8524846     Owner:  NLM     Status:  MEDLINE    
Murine inducible nitric oxide (NO) synthase (iNOS) is catalytically active only in dimeric form. Assembly of its purified subunits into a dimer requires H4B. To understand the structure-activity relationships of human iNOS, we constitutively expressed recombinant human iNOS in NIH 3T3 cells by using a retroviral vector. These cells are deficient in de novo H4B biosynthesis and the role of H4B in the expression and assembly of active iNOS in an intact cell system could be studied. In the absence of added H4B, NO synthesis by the cells was minimal, whereas cells grown with supplemental H4B or the H4B precursor sepiapterin generated NO (74.1 and 63.3 nmol of nitrite per 10(6) cells per 24 h, respectively). NO synthesis correlated with an increase in intracellular H4B but no increase in iNOS protein. Instead, an increased percentage of dimeric iNOS was observed, rising from 20% in cytosols from unsupplemented cells to 66% in H4B-supplemented cell cytosols. In all cases, only dimeric iNOS displayed catalytic activity. Cytosols prepared from H4B-deficient cells exhibited little iNOS activity but acquired activity during a 60- to 120-min incubation with H4B, reaching final activities of 60-72 pmol of citrulline per mg of protein per min. Reconstitution of cytosolic NO synthesis activity was associated with conversion of monomers into dimeric iNOS during the incubation. Thus, human iNOS subunits dimerize to form an active enzyme, and H4B plays a critical role in promoting dimerization in intact cells. This reveals a post-translational mechanism by which intracellular H4B can regulate iNOS expression.
E Tzeng; T R Billiar; P D Robbins; M Loftus; D J Stuehr
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Proceedings of the National Academy of Sciences of the United States of America     Volume:  92     ISSN:  0027-8424     ISO Abbreviation:  Proc. Natl. Acad. Sci. U.S.A.     Publication Date:  1995 Dec 
Date Detail:
Created Date:  1996-01-24     Completed Date:  1996-01-24     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  7505876     Medline TA:  Proc Natl Acad Sci U S A     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  11771-5     Citation Subset:  IM    
Department of Surgery, University of Pittsburgh, PA 15261, USA.
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MeSH Terms
3T3 Cells
Biopterin / analogs & derivatives*,  deficiency,  metabolism
Blotting, Northern
Blotting, Western
Cytosol / enzymology
Dose-Response Relationship, Drug
Enzyme Induction
Genetic Engineering
Genetic Vectors
Nitric Oxide Synthase / biosynthesis*,  genetics
Nitrites / analysis
Protein Conformation
Pteridines / metabolism
Recombinant Proteins / biosynthesis
Retroviridae / genetics
Grant Support
Reg. No./Substance:
0/Nitrites; 0/Pteridines; 0/Pterins; 0/Recombinant Proteins; 17094-01-8/sepiapterin; 17528-72-2/5,6,7,8-tetrahydrobiopterin; 22150-76-1/Biopterin; EC Oxide Synthase

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