Vesicle membrane association of nitric oxide synthase in primary mouse macrophages.
The isoform of nitric oxide synthase (NOS) whose activity is independent of elevated Ca2+ and exogenous calmodulin (iNOS; NOS type II) is inducible in a wide variety of cells and plays a major role in pathophysiology. The notion that iNOS is predominantly cytosolic is based on studies of a transformed cell line; almost nothing is known about the subcellular localization of iNOS in primary cells. Accordingly, we undertook a combined immunoelectron microscopic and biochemical analysis of iNOS in primary mouse macrophages. Approximately one-half of their iNOS activity and protein could be sedimented from 1 M KCl at 100,000 x g. The morphologic counterpart of particulate iNOS was a population of 50 to 80 nm vesicles that did not correspond to lysosomes nor peroxisomes. Vesicular iNOS arose from cytosolic iNOS by undergoing a post-translational modification that increased its apparent molecular mass by 4.5 kDa and promoted its salt-, detergent-, acid- and urea-resistant association with membranes, in the absence of detectable alternative splicing, myristoylation, palmitoylation, acetylation, glycosylation, or COOH-terminal truncation. Although primary macrophage iNOS underwent phosphorylation, ubiquitinylation, and binding of calmodulin tightly enough to resist boiling in SDS, these modifications did not allow us to distinguish between the cytosolic and particulate variants. The apparently novel iNOS-positive vesicles may translocate to phagosomes containing appropriately opsonized particles.
Vodovotz
Y
Y
Beatrice and Samuel A. Seaver Laboratory, Department of Medicine, Cornell University Medical College, New York, NY 10021.
Russell
D
D
Xie
Q W
QW
Bogdan
C
C
Nathan
C
C
D000594:Amino Acid Oxidoreductases / Q000032:analysis*; D000818:Animals; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000201:enzymology; D008264:Macrophages / Q000201:enzymology* / Q000648:ultrastructure; D051379:Mice; D008815:Mice, Inbred Strains; D016253:Microscopy, Immunoelectron; D019001:Nitric Oxide Synthase; D015388:Organelles / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests
D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases