Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1975 Dec 1;67(3):566–586. doi: 10.1083/jcb.67.3.566

Localization of NADH oxidase on the surface of human polymorphonuclear leukocytes by a new cytochemical method

PMCID: PMC2111669  PMID: 407

Abstract

The ultrastructural localization of NADH oxidase, a possible enzyme in the increased oxidative activity of polymorphonuclear leukocytes (PMN) during phagocytosis, was studied. A new cytochemical technique for the localization of H2O2, a product of NADH oxidase activity, was developed. Cerous ions, in the presence of peroxide, form an electron- dense precipitate. Resting and phagocytically stimulated PMN were exposed to cerous ions at pH 7.5 to demonstrate sites of NADH- dependent, cyanide-insensitive H2O2 production. Resting PMN exhibites slight activity on the plasma membrane; phagocytizing PMN had extensive deposits of reaction product localized within the phagosome and on the plasma membrane. Peroxide involvement was demonstrated by the inhibitory effect of catalase on cerium precipitation; the surface localization of the enzyme responsible was confirmed by using nonpenetrating inhibitors of enzymatic activity. A correlative study was performed with an NADH-dependent, tetrazolium-reduction system. As with cerium, formazan deposition on the surface of the cell was NADH dependent, cyanide insensitive, and stimulated by phagocytosis. Superoxide dismutase did not inhibit tetrazolium reduction, as observed cytochemically, indicating direct enzymatic dye reduction without superoxide interposition. These findings, combined with oxygen consumption studies on resting and stimulated PMN in the presence or absence of NADH, indicate that NADH oxidase is a surface enzyme in human PMN. It is internalized during phagocytosis and retains its peroxide-generating capacity within the phagocytic vacuole.

Full Text

The Full Text of this article is available as a PDF (5.9 MB).

Selected References

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

  1. Babior B. M., Kipnes R. S., Curnutte J. T. Biological defense mechanisms. The production by leukocytes of superoxide, a potential bactericidal agent. J Clin Invest. 1973 Mar;52(3):741–744. doi: 10.1172/JCI107236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baehner R. L., Karnovsky M. L. Deficiency of reduced nicotinamide-adenine dinucleotide oxidase in chronic granulomatous disease. Science. 1968 Dec 13;162(3859):1277–1279. doi: 10.1126/science.162.3859.1277. [DOI] [PubMed] [Google Scholar]
  3. Baehner R. L., Nathan D. G. Leukocyte oxidase: defective activity in chronic granulomatous disease. Science. 1967 Feb 17;155(3764):835–836. doi: 10.1126/science.155.3764.835. [DOI] [PubMed] [Google Scholar]
  4. Baehner R. L., Nathan D. G. Quantitative nitroblue tetrazolium test in chronic granulomatous disease. N Engl J Med. 1968 May 2;278(18):971–976. doi: 10.1056/NEJM196805022781801. [DOI] [PubMed] [Google Scholar]
  5. Berg H. C. Sulfanilic acid diazonium salt: a label for the outside of the human erythrocyte membrane. Biochim Biophys Acta. 1969 Jun 3;183(1):65–78. doi: 10.1016/0005-2736(69)90130-8. [DOI] [PubMed] [Google Scholar]
  6. Briggs R. T., Karnovsky M. L., Karnovsky M. J. Cytochemical demonstration of hydrogen peroxide in polymorphonuclear leukocyte phagosomes. J Cell Biol. 1975 Jan;64(1):254–260. doi: 10.1083/jcb.64.1.254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. CAGAN R. H., KARNOVSKY M. L. ENZYMATIC BASIS OF THE RESPIRATORY STIMULATION DURING PHAGOCYTOSIS. Nature. 1964 Oct 17;204:255–257. doi: 10.1038/204255a0. [DOI] [PubMed] [Google Scholar]
  8. Curnutte J. T., Babior B. M. Biological defense mechanisms. The effect of bacteria and serum on superoxide production by granulocytes. J Clin Invest. 1974 Jun;53(6):1662–1672. doi: 10.1172/JCI107717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Curnutte J. T., Whitten D. M., Babior B. M. Defective superoxide production by granulocytes from patients with chronic granulomatous disease. N Engl J Med. 1974 Mar 14;290(11):593–597. doi: 10.1056/NEJM197403142901104. [DOI] [PubMed] [Google Scholar]
  10. DePierre J. W., Karnovsky M. L. Ecto-enzyme of granulocytes: 5'-nucleotidase. Science. 1974 Mar 15;183(4129):1096–1098. doi: 10.1126/science.183.4129.1096. [DOI] [PubMed] [Google Scholar]
  11. DePierre J. W., Karnovsky M. L. Ecto-enzymes of the guinea pig polymorphonuclear leukocyte. I. Evidence for an ecto-adenosine monophosphatase, adenosine triphosphatase, and -p-nitrophenyl phosphates. J Biol Chem. 1974 Nov 25;249(22):7111–7120. [PubMed] [Google Scholar]
  12. Graham R. C., Jr, Karnovsky M. J., Shafer A. W., Glass E. A., Karnovsky M. L. Metabolic and morphological observations on the effect of surface-active agents of leukocytes. J Cell Biol. 1967 Mar;32(3):629–647. doi: 10.1083/jcb.32.3.629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. HARRIS H. Chemotaxis of granulocytes. J Pathol Bacteriol. 1953 Jul;66(1):135–146. doi: 10.1002/path.1700660117. [DOI] [PubMed] [Google Scholar]
  14. Holmes B., Page A. R., Good R. A. Studies of the metabolic activity of leukocytes from patients with a genetic abnormality of phagocytic function. J Clin Invest. 1967 Sep;46(9):1422–1432. doi: 10.1172/JCI105634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. KARNOVSKY M. L. Metabolic basis of phagocytic activity. Physiol Rev. 1962 Jan;42:143–168. doi: 10.1152/physrev.1962.42.1.143. [DOI] [PubMed] [Google Scholar]
  16. Karnovsky M. L. Chronic granulomatous disease--pieces of a cellular and molecular puzzle. Fed Proc. 1973 Apr;32(4):1527–1533. [PubMed] [Google Scholar]
  17. Klebanoff S. J., Hamon C. B. Role of myeloperoxidase-mediated antimicrobial systems in intact leukocytes. J Reticuloendothel Soc. 1972 Aug;12(2):170–196. [PubMed] [Google Scholar]
  18. Klebanoff S. J. Intraleukocytic microbicidal defects. Annu Rev Med. 1971;22:39–62. doi: 10.1146/annurev.me.22.020171.000351. [DOI] [PubMed] [Google Scholar]
  19. Klebanoff S. J. Iodination of bacteria: a bactericidal mechanism. J Exp Med. 1967 Dec 1;126(6):1063–1078. doi: 10.1084/jem.126.6.1063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  21. Mandell G. L., Sullivan G. W. Pyridine nucleotide oxidation by intact human polymorphonuclear neutrophils. Biochim Biophys Acta. 1971 Apr 6;234(1):43–45. doi: 10.1016/0005-2728(71)90127-7. [DOI] [PubMed] [Google Scholar]
  22. McCall C. E., DeChatelet L. R., Butler R., Brown D. Enhanced phagocytic capacity. The biologic basis for the elevated histochemical nitroblue tetrazolium reaction. J Clin Invest. 1974 Nov;54(5):1227–1234. doi: 10.1172/JCI107866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. McCord J. M., Fridovich I. The reduction of cytochrome c by milk xanthine oxidase. J Biol Chem. 1968 Nov 10;243(21):5753–5760. [PubMed] [Google Scholar]
  24. Michell R. H., Pancake S. J., Noseworthy J., Karnovsky M. L. Measurement of rates of phagocytosis: the use of cellular monolayers. J Cell Biol. 1969 Jan;40(1):216–224. doi: 10.1083/jcb.40.1.216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nathan D. G., Baehner R. L., Weaver D. K. Failure of nitro blue tetrazolium reduction in the phagocytic vacuoles of leukocytes in chronic granulomatous disease. J Clin Invest. 1969 Oct;48(10):1895–1904. doi: 10.1172/JCI106156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nathan D. G. Editorial: NBT reduction by human phagocytes. N Engl J Med. 1974 Jan 31;290(5):280–281. doi: 10.1056/NEJM197401312900512. [DOI] [PubMed] [Google Scholar]
  27. Niukian K., Eichel B., Shahrik H. A. Histochemical disclosure of endogenous and H2O2-dependent reducing enzyme activities in leukocytes and other cytological entities from the human oral cavity. Arch Oral Biol. 1973 Apr;18(4):505–516. doi: 10.1016/0003-9969(73)90071-x. [DOI] [PubMed] [Google Scholar]
  28. OREN R., FARNHAM A. E., SAITO K., MILOFSKY E., KARNOVSKY M. L. Metabolic patterns in three types of phagocytizing cells. J Cell Biol. 1963 Jun;17:487–501. doi: 10.1083/jcb.17.3.487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Patriarca P., Cramer R., Dri P., Fant L., Basford R. E., Rossi F. NADPH oxidizing activity in rabbit polymorphonuclear leukocytes: localization in azurophilic granules. Biochem Biophys Res Commun. 1973 Aug 6;53(3):830–837. doi: 10.1016/0006-291x(73)90168-x. [DOI] [PubMed] [Google Scholar]
  30. Patriarca P., Cramer R., Moncalvo S., Rossi F., Romeo D. Enzymatic basis of metabolic stimulation in leucocytes during phagocytosis: the role of activated NADPH oxidase. Arch Biochem Biophys. 1971 Jul;145(1):255–262. doi: 10.1016/0003-9861(71)90034-8. [DOI] [PubMed] [Google Scholar]
  31. Patriarca P., Zatti M., Cramer R., Rossi F. Stimulation of the respiration of polymorphonuclear leucocytes by phospholipase C. Life Sci I. 1970 Aug 1;9(15):841–849. doi: 10.1016/0024-3205(70)90046-9. [DOI] [PubMed] [Google Scholar]
  32. Paul B. B., Strauss R. R., Jacobs A. A., Sbarra A. J. Direct involvement of NADPH oxidase with the stimulated respiratory and hexose monophosphate shunt activities in phagocytizing leukocytes. Exp Cell Res. 1972 Aug;73(2):456–462. doi: 10.1016/0014-4827(72)90071-7. [DOI] [PubMed] [Google Scholar]
  33. Paul B., Sbarra A. J. The role of the phagocyte in host-parasite interactions. 13. The direct quantitative estimation of H2O2 in phagocytizing cells. Biochim Biophys Acta. 1968 Feb 1;156(1):168–178. doi: 10.1016/0304-4165(68)90116-5. [DOI] [PubMed] [Google Scholar]
  34. RAJAGOPALAN K. V., HANDLER P. HEPATIC ALDEHYDE OXIDASE. II. DIFFERENTIAL INHIBITION OF ELECTRON TRANSFER TO VARIOUS ELECTRON ACCEPTORS. J Biol Chem. 1964 Jun;239:2022–2026. [PubMed] [Google Scholar]
  35. ROBERTS J., QUASTEL J. H. OXIDATION OF REDUCED TRIPHOSPHOPYRIDINE NUCLEOTIDE BY GUINEA PIG POLYMORPHONUCLEAR LEUCOCYTES. Nature. 1964 Apr 4;202:85–86. doi: 10.1038/202085a0. [DOI] [PubMed] [Google Scholar]
  36. Romeo D., Zabucchi G., Rossi F. Reversible metabolic stimulation of polymorphonuclear leukocytes and macrophages by concanavalin A. Nat New Biol. 1973 May 23;243(125):111–112. [PubMed] [Google Scholar]
  37. Root R. K., Stossel T. P. Myeloperoxidase-mediated iodination by granulocytes. Intracellular site of operation and some regulating factors. J Clin Invest. 1974 May;53(5):1207–1215. doi: 10.1172/JCI107667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rozenszajn L., Shoham D. The demonstration of dehydrogenases and diaphorases in cells of peripheral blood and bone marrow. Blood. 1967 May;29(5):737–746. [PubMed] [Google Scholar]
  39. Salin M. L., McCord J. M. Superoxide dismutases in polymorphonuclear leukocytes. J Clin Invest. 1974 Oct;54(4):1005–1009. doi: 10.1172/JCI107816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Segal A. W., Levi A. J. The mechanism of the entry of dye into neutrophils in the nitroblue tetrazolium (NBT) test. Clin Sci Mol Med. 1973 Dec;45(6):817–826. doi: 10.1042/cs0450817. [DOI] [PubMed] [Google Scholar]
  41. Segal A. W. Nitroblue-tetrazolium tests. Lancet. 1974 Nov 23;2(7891):1248–1252. doi: 10.1016/s0140-6736(74)90758-2. [DOI] [PubMed] [Google Scholar]
  42. Sims K. L., Weitsen H. A., Bloom F. E. Histochemical localization of brain succinic semialdehyde dehydrogenase--a -aminobutyric acid degradative enzyme. J Histochem Cytochem. 1971 Jul;19(7):405–415. doi: 10.1177/19.7.405. [DOI] [PubMed] [Google Scholar]
  43. Stossel T. P., Pollard T. D., Mason R. J., Vaughan M. Isolation and properties of phagocytic vesicles from polymorphonuclear leukocytes. J Clin Invest. 1971 Aug;50(8):1745–1747. doi: 10.1172/JCI106664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Takanaka K., O'Brien P. J. Mechanisms of hydrogen peroxide formation in leukocytes: the NAD(P)H oxidase activity of myeloperoxidase. Biochem Biophys Res Commun. 1975 Feb 17;62(4):966–971. doi: 10.1016/0006-291x(75)90417-9. [DOI] [PubMed] [Google Scholar]
  45. VANSTEVENINCK J., WEED R. I., ROTHSTEIN A. LOCALIZATION OF ERYTHROCYTE MEMBRANE SULFHYDRYL GROUPS ESSENTIAL FOR GLUCOSE TRANSPORT. J Gen Physiol. 1965 Mar;48:617–632. doi: 10.1085/jgp.48.4.617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. VENABLE J. H., COGGESHALL R. A SIMPLIFIED LEAD CITRATE STAIN FOR USE IN ELECTRON MICROSCOPY. J Cell Biol. 1965 May;25:407–408. doi: 10.1083/jcb.25.2.407. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES