We conclude that cathepsin D, and not cathepsin E, causes desquamation by degrading desmosomes.

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

The distribution of intramembrane particles (IMP) as revealed by freeze-fracture electron microscopy has been analyzed following treatment of mouse L cells and fusiondeficient L cell derivatives with several concentrations of polyethylene glycol (PEG). In cell cultures treated with concentrations of PEG below the critical level for fusion, no aggregation of IMP was observed. When confluent cultures of the parental cells are treated with 50% PEG, >90% of the cells fuse, and cold-induced IMP aggregation is extensive. In contrast, identical treatment of fusion-deficient cell lines shows neither extensive fusion nor IMP redistribution. At higher concentrations of PEG, however, the PEG-resistant cells fuse extensively and IMP aggregation is evident. Thus the decreased ability of the fusion-deficient cells to fuse after treatment with PEG is correlated with the failure of IMP aggregation to occur. A technique for quantifying particle distribution was developed that is practical for the accurate analysis of a large number of micrographs. The variance from the mean number of particles in randomly chosen areas of fixed size was calculated for each cell line at each concentration of PEG. Statistical analysis confirms visual observation of highly aggregated IMP, and allows detection of low levels of aggregation in parental cells that were less extensively fused by exposure to lower concentrations of PEG. When low levels of fusion were induced in fusion-deficient cells, however, no IMP aggregation could be detected.

Cultured resident murine maaophages are incubated in the continuous presence of the fluorescent endocytic marker Lucifer Yellow and a phorbol ester that activates protein kinase C. Under these steady-state labeling conditions the fluorescent tracer was, for the most part, in a tubularlreticular compartment. Enzyme cytochemical localization of acid phosphatase in the same cells showed essentially a one-to-one correlation between the Lucifer Yellow-and acid phosphatase-

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