Expression of human inducible nitric oxide synthase in a tetrahydrobiopterin (H4B)-deficient cell line: H4B promotes assembly of enzyme subunits into an active dimer

Edith Tzeng, Timothy R. Billiar, Paul D. Robbins, Margaret Loftus, Dennis J. Stuehr

Research output: Contribution to journalArticlepeer-review

170 Scopus citations

Abstract

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 106 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 iNnS 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 iNnS 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 posttranslational mechanism by which intracellular H4B can regulate iNOS expression.

Original languageEnglish (US)
Pages (from-to)11771-11775
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume92
Issue number25
DOIs
StatePublished - Dec 5 1995

Fingerprint Dive into the research topics of 'Expression of human inducible nitric oxide synthase in a tetrahydrobiopterin (H<sub>4</sub>B)-deficient cell line: H<sub>4</sub>B promotes assembly of enzyme subunits into an active dimer'. Together they form a unique fingerprint.

Cite this