Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1978 May;20(2):398–405. doi: 10.1128/iai.20.2.398-405.1978

Allosteric Transformation of Reduced Nicotinamide Adenine Dinucleotide (Phosphate) Oxidase Induced by Phagocytosis in Human Polymorphonuclear Leukocytes

Lawrence R DeChatelet 1,2, Pamela S Shirley 1, Linda C McPhail 1, David B Iverson 1, George J Doellgast 1
PMCID: PMC421869  PMID: 27457

Abstract

We used sensitive isotopic and fluorometric assay procedures to investigate reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]oxidation in a particulate fraction derived from normal and chronic granulomatous disease leukocytes. Granules isolated from normal resting cells showed allosteric kinetics with regard to oxidation of either NADH or NADPH, so that no enzyme activity was observed at physiological concentrations of substrate. If the granules were isolated from cells that had previously phagocytized zymosan, normal hyperbolic kinetics were obtained, so that activity could now be observed at low levels of substrate. The activity towards NADPH was always substantially greater than that towards NADH at any given concentration of substrate. This alteration in kinetics with phagocytosis was not observed with the other granule enzymes, acid phosphatase or β-glucuronidase, and thus appeared to be specific for the reduced pyridine nucleotide oxidase(s). In contrast, granules isolated from cells of patients with chronic granulomatous disease showed allosteric kinetics regardless of whether they were obtained from resting or phagocytizing cells, so that NADPH oxidation was not measurable at physiological concentrations of substrate. This defect in the oxidation of NADPH by granules isolated from phagocytizing chronic granulomatous disease cells was observed over the pH range of 4.0 to 7.0. These data suggest that initiation of the respiratory burst by pahgocytosis normally requires an allosteric transformation in a reduced pyridine nucleotide oxidase, which in turn allows expression of enzymatic activity at physiological concentrations of substrate. The defect in chronic granulomatous disease appears to lie in an inability to achieve this transformation, and the enzyme remains in the inactive, allosteric form.

Full text

PDF
398

Selected References

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

  1. Babior B. M., Curnutte J. T., Kipnes B. S. Pyridine nucleotide-dependent superoxide production by a cell-free system from human granulocytes. J Clin Invest. 1975 Oct;56(4):1035–1042. doi: 10.1172/JCI108150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baehner R. L., Karnovsky M. J., Karnovsky M. L. Degranulation of leukocytes in chronic granulomatous disease. J Clin Invest. 1969 Jan;48(1):187–192. doi: 10.1172/JCI105967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Briggs R. T., Drath D. B., Karnovsky M. L., Karnovsky M. J. Localization of NADH oxidase on the surface of human polymorphonuclear leukocytes by a new cytochemical method. J Cell Biol. 1975 Dec;67(3):566–586. doi: 10.1083/jcb.67.3.566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Curnutte J. T., Karnovsky M. L., Babior B. M. Manganese-dependent NADPH oxidation by granulocyte particles. The role of superoxide and the nonphysiological nature of the manganese requirement. J Clin Invest. 1976 Apr;57(4):1059–1067. doi: 10.1172/JCI108348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Curnutte J. T., Kipnes R. S., Babior B. M. Defect in pyridine nucleotide dependent superoxide production by a particulate fraction from the cranulocytes of patients with chronic granulomatous disease. N Engl J Med. 1975 Sep 25;293(13):628–632. doi: 10.1056/NEJM197509252931303. [DOI] [PubMed] [Google Scholar]
  8. DeChatelet L. R., Cooper M. R. A modified procedure for the determination of leukocyte alkaline phosphatase. Biochem Med. 1970 Aug;4(1):61–68. doi: 10.1016/0006-2944(70)90103-1. [DOI] [PubMed] [Google Scholar]
  9. DeChatelet L. R., McPhail L. C., Mullikin D., McCall C. E. An isotopic assay for NADPH oxidase activity and some characteristics of the enzyme from human polymorphonuclear leukocytes. J Clin Invest. 1975 Apr;55(4):714–721. doi: 10.1172/JCI107981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. DeChatelet L. R., McPhail L. C., Mullikin D., McCall C. E. Reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate diaphorase activity in human polymorphonuclear leukocytes. Infect Immun. 1974 Sep;10(3):528–534. doi: 10.1128/iai.10.3.528-534.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. DeChatelet L. R. Oxidative bactericidal mechanisms of polymorphonuclear leukocytes. J Infect Dis. 1975 Mar;131(3):295–303. doi: 10.1093/infdis/131.3.295. [DOI] [PubMed] [Google Scholar]
  12. Harlan J., DeChatelet L. R., Iverson D. B., McCall C. E. Magnesium-dependent adenosine triphosphatase as a marker enzyme for the plasma membrane of human polymorphonuclear leukocytes. Infect Immun. 1977 Feb;15(2):436–443. doi: 10.1128/iai.15.2.436-443.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hohn D. C., Lehrer R. I. NADPH oxidase deficiency in X-linked chronic granulomatous disease. J Clin Invest. 1975 Apr;55(4):707–713. doi: 10.1172/JCI107980. [DOI] [PMC free article] [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. Iverson D., DeChatelet L. R., Spitznagel J. K., Wang P. Comparison of NADH and NADPH oxidase activities in granules isolated from human polymorphonuclear leukocytes with a fluorometric assay. J Clin Invest. 1977 Feb;59(2):282–290. doi: 10.1172/JCI108639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kaplan E. L., Laxdal T., Quie P. G. Studies of polymorphonuclear leukocytes from patients with chronic granulomatous disease of childhood: bactericidal capacity for streptococci. Pediatrics. 1968 Mar;41(3):591–599. [PubMed] [Google Scholar]
  17. 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]
  18. McPhail L. C., DeChatelet L. R., Shirley P. S. Further characterization of NADPH oxidase activity of human polymorphonuclear leukocytes. J Clin Invest. 1976 Oct;58(4):774–780. doi: 10.1172/JCI108528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. McPhail L. C., DeChatelet L. R., Shirley P. S., Wilfert C., Johnston R. B., Jr, McCall C. E. Deficiency of NADPH oxidase activity in chronic granulomatous disease. J Pediatr. 1977 Feb;90(2):213–217. doi: 10.1016/s0022-3476(77)80632-x. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Rossi F., Romeo D., Patriarca P. Mechanism of phagocytosis-associated oxidative metabolism in polymorphonuclear leucocytes and macrophages. J Reticuloendothel Soc. 1972 Aug;12(2):127–149. [PubMed] [Google Scholar]
  23. Segal A. W., Peters T. J. Characterisation of the enzyme defect in chronic granulomatous disease. Lancet. 1976 Jun 26;1(7974):1363–1365. doi: 10.1016/s0140-6736(76)93021-x. [DOI] [PubMed] [Google Scholar]
  24. Wilkinson R. W., Powars D. R., Hochstein P. New evidence for the role of NADH oxidase in phagocytosis by human granulocytes. Biochem Med. 1975 May;13(1):83–88. doi: 10.1016/0006-2944(75)90142-8. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES