Localization of D-amino acid oxidase on the cell surface of human polymorphonuclear leukocytes

Abstract

The ultrastructural localization of o-amino acid oxidase (DAO) was studied cytochemically by detecting sites of hydrogen peroxide production in human polymorphonuclear leukocytes (PMNs). Reaction product, which forms when cerous ions react with HzO2 to form an electron-dense precipitate, was demonstrated on the cell surface and within the phagosomes of phagocytically stimulated cells when D-amino acids were provided as substrate. Resting cells showed only slight activity. The competitive inhibitor O,L-2-hydroxybutyrate greatly reduced the D-amino acid-stimulated reaction while KCN did not. The cell surface reaction was abolished by nonpenetrating inhibitors of enzyme activity while that within the phagosome was not eliminated. Dense accumulations of reaction product were formed in cells which phagocytosed Staphylococcus aureus in the absence of exogenous substrate. No reaction product formed with Proteus vulgaris while an intermediate amount formed when Escherichia coli were phagocytosed. Variation in the amount of reaction product with the different bacteria correlated with the levels of o-amino acids in the bacterial cell walls which are available for the DAO of PMNs. An alternative approach utilizing ferricyanide as an electron acceptor was also used. This technique verified the results obtained with the cerium reaction, i.e., the DAO is located in the cell surface and is internalized during phagocytosis and is capable of HzO2 production within the phagosome. The present finding that DAO is localized on the cell surface further supports the concept that the plasma membrane is involved in peroxide formation in PMNs.KEY WORDS D-amino acid oxidase cell surface polymorphonuclear ieukocytes phagocytosis Phagocytosis by polymorphonuclear leukocytes (PMNs) 1 is accompanied by changes in cellular Abbreviations used in this paper: ATZ, 3-amino-1,2,4-triazole; DAB, 3-3' diaminobenzidine; DAO, D-amino acid oxidase; FCS, fetal calf serum; HBSS, Hanks' balanced salt solution; PCMBS, parachloromercuribenzenesulfonate; PMNs, polymorphonuclear leukocytes; and PS, polystyrene beads. metabolism which include increases in oxygen consumption, hexose monophosphate shunt activity, and production of hydrogen peroxide (24,16,18). Recently, Briggs et al. (3) demonstrated cytochemically that PMNs have a surface NADH oxidase which is stimulated by phagocytosis or other perturbations of the plasma membrane. The NADH oxidase internalization may be an important event since this enzyme can generate H202 within the phagosome for utilization by myeloperoxidase for bactericidal activity (17).Human PMNs have other enzymes which are J. CELL BIOLOGY 9 The Rockefeller University Press 9 0021-9525/78

Histochemical staining of cells containing flavoenzyme D-amino acid oxidase based on its enzymatic activity: Application of a coupled peroxidation method.

“…This visible product showed high contrast in comparison with other histochemical methods including the tetrazolium method (12,16,27,38,40,46,47) and the previous coupled-peroxidation method (16) . The cerium method (3,37,41,42) is useful for electron microscopic demonstration of the oxidase activity, but is inapplicable to light microscopy because of the nonvisible product.…”

“…20) . They are based on three general means of detection : the reduction of tetrazolium (12,16,27,38,40,46,47) or ferricyanide (37) during oxidation of the substrate; the coupled peroxidation methods for hydrogen peroxide detection (14,16,(42)(43)(44) or the direct detection of hydrogen peroxide with cerous chloride (3,37,41,42) ; and immunocytochemical methods (39,43). However, it seems that they are not practical as to sensitivity or simplicity.…”

Protective Role ofd-Amino Acid Oxidase against Staphylococcus aureus Infection

“…Robinson et al reported that DAO is located on the cell surface and DAO is internalized with foreign objects, such as bacteria (26). Reports that purified porcine DAO has antibacterial activity against Escherichia coli in vitro and that its activity was enhanced by addition of D-amino acids, as well as by binding of DAO to bacterial cell walls, have appeared in the literature (10,29).…”

Lead citrate method of guanylate cyclase and its application to tissue receiving rapid freeze substitution fixation.

“…On this viewpoint attempts were reported, introducing cadmium, strontioum, barium, and cerium (52). Of these metals, cerium is shown to be excellent in the precise localization of reaction product at the ultrastructural level (45)(46)(47)(48). Its disadvantage is that it is rather difficult for developing color in light microscopy (41 Unlike ACase, which is considered to be totally particulate, the major portion of GCase appears to reside in the soluble fraction after the homogenization of most tissues, but there was still some detectable activity in the particulate fraction (39).…”