Skip to main content
PMC home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1992 Aug 1;176(2):599–604. doi: 10.1084/jem.176.2.599

Calmodulin is a subunit of nitric oxide synthase from macrophages

PMCID: PMC2119310  PMID: 1380065

Abstract

A central issue in nitric oxide (NO) research is to understand how NO can act in some settings as a servoregulator and in others as a cytotoxin. To answer this, we have sought a molecular basis for the differential regulation of the two known types of NO synthase (NOS). Constitutive NOS's in endothelium and neurons are activated by agonist- induced elevation of Ca2+ and resultant binding of calmodulin (CaM). In contrast, NOS in macrophages does not require added Ca2+ or CaM, but is regulated instead by transcription. We show here that macrophage NOS contains, as a tightly bound subunit, a molecule with the immunologic reactivity, high performance liquid chromatography retention time, tryptic map, partial amino acid sequence, and exact molecular mass of CaM. In contrast to most CaM-dependent enzymes, macrophage NOS binds CaM tightly without a requirement for elevated Ca2+. This may explain why NOS that is independent of Ca2+ and elevated CaM appears to be activated simply by being synthesized.

Full Text

The Full Text of this article is available as a PDF (758.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alexander K. A., Cimler B. M., Meier K. E., Storm D. R. Regulation of calmodulin binding to P-57. A neurospecific calmodulin binding protein. J Biol Chem. 1987 May 5;262(13):6108–6113. [PubMed] [Google Scholar]
  2. Bredt D. S., Hwang P. M., Glatt C. E., Lowenstein C., Reed R. R., Snyder S. H. Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase. Nature. 1991 Jun 27;351(6329):714–718. doi: 10.1038/351714a0. [DOI] [PubMed] [Google Scholar]
  3. Bredt D. S., Snyder S. H. Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc Natl Acad Sci U S A. 1990 Jan;87(2):682–685. doi: 10.1073/pnas.87.2.682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Busse R., Mülsch A. Calcium-dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin. FEBS Lett. 1990 Jun 4;265(1-2):133–136. doi: 10.1016/0014-5793(90)80902-u. [DOI] [PubMed] [Google Scholar]
  5. Geiser J. R., van Tuinen D., Brockerhoff S. E., Neff M. M., Davis T. N. Can calmodulin function without binding calcium? Cell. 1991 Jun 14;65(6):949–959. doi: 10.1016/0092-8674(91)90547-c. [DOI] [PubMed] [Google Scholar]
  6. Harper J. F., Sussman M. R., Schaller G. E., Putnam-Evans C., Charbonneau H., Harmon A. C. A calcium-dependent protein kinase with a regulatory domain similar to calmodulin. Science. 1991 May 17;252(5008):951–954. doi: 10.1126/science.1852075. [DOI] [PubMed] [Google Scholar]
  7. Ignarro L. J. Biosynthesis and metabolism of endothelium-derived nitric oxide. Annu Rev Pharmacol Toxicol. 1990;30:535–560. doi: 10.1146/annurev.pa.30.040190.002535. [DOI] [PubMed] [Google Scholar]
  8. Ikeda A., Okuno S., Fujisawa H. Studies on the generation of Ca2+/calmodulin-independent activity of calmodulin-dependent protein kinase II by autophosphorylation. Autothiophosphorylation of the enzyme. J Biol Chem. 1991 Jun 25;266(18):11582–11588. [PubMed] [Google Scholar]
  9. Ladant D. Interaction of Bordetella pertussis adenylate cyclase with calmodulin. Identification of two separated calmodulin-binding domains. J Biol Chem. 1988 Feb 25;263(6):2612–2618. [PubMed] [Google Scholar]
  10. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  11. Lamas S., Marsden P. A., Li G. K., Tempst P., Michel T. Endothelial nitric oxide synthase: molecular cloning and characterization of a distinct constitutive enzyme isoform. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6348–6352. doi: 10.1073/pnas.89.14.6348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Means A. R. Molecular mechanisms of action of calmodulin. Recent Prog Horm Res. 1988;44:223–262. doi: 10.1016/b978-0-12-571144-9.50012-0. [DOI] [PubMed] [Google Scholar]
  13. Moncada S., Palmer R. M., Higgs E. A. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev. 1991 Jun;43(2):109–142. [PubMed] [Google Scholar]
  14. O'Neil K. T., DeGrado W. F. How calmodulin binds its targets: sequence independent recognition of amphiphilic alpha-helices. Trends Biochem Sci. 1990 Feb;15(2):59–64. doi: 10.1016/0968-0004(90)90177-d. [DOI] [PubMed] [Google Scholar]
  15. Olwin B. B., Edelman A. M., Krebs E. G., Storm D. R. Quantitation of energy coupling between Ca2+, calmodulin, skeletal muscle myosin light chain kinase, and kinase substrates. J Biol Chem. 1984 Sep 10;259(17):10949–10955. [PubMed] [Google Scholar]
  16. Olwin B. B., Storm D. R. Calcium binding to complexes of calmodulin and calmodulin binding proteins. Biochemistry. 1985 Dec 31;24(27):8081–8086. doi: 10.1021/bi00348a037. [DOI] [PubMed] [Google Scholar]
  17. Picton C., Klee C. B., Cohen P. Phosphorylase kinase from rabbit skeletal muscle: identification of the calmodulin-binding subunits. Eur J Biochem. 1980 Oct;111(2):553–561. doi: 10.1111/j.1432-1033.1980.tb04971.x. [DOI] [PubMed] [Google Scholar]
  18. Sacks D. B., Porter S. E., Ladenson J. H., McDonald J. M. Monoclonal antibody to calmodulin: development, characterization, and comparison with polyclonal anti-calmodulin antibodies. Anal Biochem. 1991 May 1;194(2):369–377. doi: 10.1016/0003-2697(91)90243-m. [DOI] [PubMed] [Google Scholar]
  19. Schleicher M., Watterson D. M. Analysis of differences between coomassie blue stain and silver stain procedures in polyacrylamide gels: conditions for the detection of calmodulin and troponin C. Anal Biochem. 1983 Jun;131(2):312–317. doi: 10.1016/0003-2697(83)90175-6. [DOI] [PubMed] [Google Scholar]
  20. Sharma R. K., Wang J. H. Purification and characterization of bovine lung calmodulin-dependent cyclic nucleotide phosphodiesterase. An enzyme containing calmodulin as a subunit. J Biol Chem. 1986 Oct 25;261(30):14160–14166. [PubMed] [Google Scholar]
  21. Slavin W. Atomic absorption spectrometry. Methods Enzymol. 1988;158:117–145. doi: 10.1016/0076-6879(88)58052-7. [DOI] [PubMed] [Google Scholar]
  22. Stuehr D. J., Cho H. J., Kwon N. S., Weise M. F., Nathan C. F. Purification and characterization of the cytokine-induced macrophage nitric oxide synthase: an FAD- and FMN-containing flavoprotein. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7773–7777. doi: 10.1073/pnas.88.17.7773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Watterson D. M., Sharief F., Vanaman T. C. The complete amino acid sequence of the Ca2+-dependent modulator protein (calmodulin) of bovine brain. J Biol Chem. 1980 Feb 10;255(3):962–975. [PubMed] [Google Scholar]
  24. Xie Q. W., Cho H. J., Calaycay J., Mumford R. A., Swiderek K. M., Lee T. D., Ding A., Troso T., Nathan C. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science. 1992 Apr 10;256(5054):225–228. doi: 10.1126/science.1373522. [DOI] [PubMed] [Google Scholar]
  25. Yui Y., Hattori R., Kosuga K., Eizawa H., Hiki K., Kawai C. Purification of nitric oxide synthase from rat macrophages. J Biol Chem. 1991 Jul 5;266(19):12544–12547. [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES