B. B. Paul

The role of the phagocyte in host-parasite interactions: XIX. Leukocytic glutathione reductase and its involvement in phagocytosis

Abstract Glutathione reductase activity was found in guinea pig peritoneal polymorphonuclear leuckocytes. The cofactor requirement for the enzyme was found to be NADPH. This was determined by the requirement of GSSG for oxidation of the reduced cofactor and the stimulatory effect of KCN on GSH peroxidase which was also found in these cells. Enzyme activity was inhibited by the sulfhydryl antagonist N -ethyl maleimide. The addition of GSSG to the reaction mixture resulted in an increase in cellular GSH content. The activity of the enzyme was confined to the 20,000 g supernatant fraction. Phagocytosis caused a significant increase in GSSG reductase activity within 15 sec after particle addition. The NADPH oxidase stimulation which has been associated with the phagocytic process did not occur until some minutes after the addition of latex particles. Since NADP + is a limiting factor for the hexose monophosphate shunt and GSSG reductase can increase the NADP + content of the cell, one can now postulate that increased activity of this enzyme is an early event involved in the phagocytosis-associated increase in this metabolic pathway.

Chlorination by the Myeloperoxidase-H2O2-CI-Antimicrobial System at Acid and Neutral pH

Summary The oxidation of Cl- ion by H2O2 yielding Cl+ is catalyzed by myelope-roxidase (MPO) prepared from human PMN. Both H2O2 and CI- in this reaction serve as substrates. Affinity (apparent) studies between MPO and CI- and MPO and H2O2 were carried out. Both (apparent) affinities vary according to pH. The higher the pH, the less the affinity for Cl-. In contrast, the affinity between MPO and H2O2 increases with pH. The dissociation constant of the MPO-CI- enzyme substrate complex is affected by the second substrate, H2O2. The effect of H2O2 on the MPO-C1- affinity is competitive. Likewise, the MPO-H2O2 affinity is decreased with increasing Cl- concentration. This influence is also competitive. From these interactions, it may be concluded that the optimal pH for chlori-nation is dependent on the [H2O2]/[C1-] ratio. The relationship of pH, H2O2, and CI- concentrations may be expressed by the following formula: pH = log [CI-] x560/ [H2O2]. Using the formula, it can be shown that optimal chlorination may occur between pH 4.0 and 7.4. We wish to thank Paul Wallace and Gerry Heatley for technical assistance, Roberta Lemon for preparing the manuscript, and George Daynes for photography. This investigation was supported by Grant CA-1567 awarded by the National Cancer Institute, DHEW, and USPHS Grant HD-1805, NIH.

Peroxidase Mediated Antimicrobial Activities of Alveolar Macrophage Granules

The 20,000g pellet obtained by centrifugation of a homogenate of rabbit alveolar macrophages has antibacterial activity in the presence of a hydrogen peroxide-generating system and iodide. Peroxidase activity has been demonstrated in this fraction. Addition of 3-amino-1,2,4-triazole diminished the antibacterial activity of the pellet-hydrogen peroxide-iodide system.

Direct involvement of nadph oxidase with the stimulated respiratory and hexose monophosphate shunt activities in phagocytizing leukocytes

Abstract Guinea pig peritoneal exudate polymorphonuclear leukocytes (PMN) have KCN-sensitive and insensitive NADPH oxidation activity. These activities can be localized to different homogenate fractions. NADPH oxidation activity of the 19 000 g pellet is KCN-insensitive. Myeloperoxidase (MPO) activity which is also confined to this fraction is completely inhibited by 1 mM KCN. This observation indicates that KCN-insensitive NADPH oxidation is not due to MPO. KCN-sensitive NADPH oxidation activity is found in the 19 000 g supernatant fraction, as is catalase. Purified beef liver catalase does not oxidize NADPH, thus indicating that the NADPH oxidation by the supernatant is not due to the presence of catalase. NADPH oxidation activity of both the 19 000 g pellet and supernatant fractions collected from phagocytizing cells is significantly greater than that found in the corresponding fractions collected from resting cells. The increased oxidative burst associated with phagocytosis can be accounted for by the stimulated NADPH oxidase activity.

The role of the phagocyte in host-parasite interactions. XXIII. Relation of bactericidal activity to peroxidase-associated decarboxylation and deamination

Abstract Peroxidase-mediated decarboxylation and deamination of appropriate substrates appear to be associated with bactericidal activity of the myeloperoxidase-H2O2-chloride antimicrobial system. Products resulting from these reactions, possibly aldehydes, may be the actual bactericidal agents.

The role of the phagocyte in host-parasite interactions: XXV. Metabolic and bactericidal activities of leukocytes from pregnant women☆☆☆

Abstract The hexose monophosphate shunt (HMS) activity of phagocytizing cells collected from pregnant women is approximately twice that found in nonpregnant women. HMS activity in resting leukocytes from bacteriuric pregnant women was lower than that found in leukocytes from normal pregnant women. Phagocytizing cells showed increased activity. The myeloperoxidase (MPO) activity of leukocytes from pregnant women increased significantly during phagocytosis. This increase was not observed in nonpregnant women. Granules containing 20,000 × g MPO isolated from leukocytes from pregnant women have bactericidal activity in the presence of nonbactericidal concentrations of H 2 O 2 in a chloride medium. These granules also decarboxylate alanine in the presence of H 2 O 2 in a chloride medium. Freshly isolated strains of bacteria are not killed as effectively as stock strains. This appears to be due to decreased phagocytosis and/or resistance of the fresh isolates to the MPO-H 2 O 2 -chloride bactericidal system.

Positive correlation of optical density at 650 nm. with lecithin/ sphingomyelin ratios in amniotic fluid

In this study, we have attempted to correlate optical density measurements of amniotic fluids with L/S ratios. We may conclude, with over a 98 per cent accuracy, that fluids having optical density readings of 0.15 and above, at 650 nm. will have an L/S ratio over 2.0. Fluids having optical density readings up to 0.05 will have L/S ratios of about 1.3. Finally, amniotic fluids having optical densities greater than 0.05 and less than 0.15 will have L/S ratios of approximately 1.5.

Chlorination, Decarboxylation and Bactericidal Activity Mediated by the MPO-H2O2-Cl− System

1. MPO, H2O2 and C1- form a complex that undergoes intramolecular rearrangement yielding the chlorinium ion. 2. The chlorinium ion can interact with MPO, bacteria and amino acids. 3. The reaction can occur at a wide range of pH, H2O2 concentration and C1- concentration. 4. The chlorinium ion can attack different protein molecules to cause structural changes. 5. Preincubation of MPO with bacteria results in greater bactericidal activity. 6. Diffusible bactericidal agents are also produced by the MPO-H2O2-C1- system.

The role of the phagocyte in host-parasite interactions. XXXIX. Stimulation of bactericidal activity of myeloperoxidase-containing leukocytic fractions by estrogens.

Abstract Estriol and estrone have been shown to stimulate bactericidal activity of myeloperoxidase (MPO)-containing 20,000 × g granule pellets obtained from guinea pig peritoneal polymorphonuclear leukocytes. Beta estradiol and progesterone were without effect. An MPO-mediated amino acid decarboxylation reaction, optimal at acidic pH and postulated as a mechanism involved with the bactericidal activity, was also stimulated by estriol and estrone but not by beta estradiol and progesterone. In contrast, no stimulation of MPO-mediated guaiacol oxidation at pH 7.0 was observed with any of these hormones. The stimulation of bactericidal activity by estriol and estrone was not observed if highly purified human MPO was substituted for the guinea pig MPO-containing 20,000 × g granule pellets. These observations suggest that estriol and estrone may be stimulating MPO-mediated bactericidal activity through an indirect mechanism. It is possible that hyperbactericidal leukocyte activity observed in pregnancy may be related, at least in part, to stimulation of its MPO-mediated bactericidal activity and amino acid decarboxylation by estriol and/or estrone.

Mouse spleen lymphocyte bactericidal and peroxidase activities: enhancement by whole body x-irradiation.

Summary A 20,000 g pellet obtained from the homogenate of CD1 mouse spleen cells has measurable peroxidase and bactericidal activities. Whole body x-irradiation stimulates the in vitro peroxidative and bactericidal activities of the spleen cells. These cells do not phagocytize. However, addition of polystyrene latex particles to spleen cells from non- or x-irradiated mice, increases glucose oxidation. The extent of this stimulation (three- to fourfold) is less than that of PMN (six- to tenfold). Interaction between particles and lymphocyte surface may be the cause of this stimulation. The increased bactericidal activity due to x-ray is attributed mainly to an increase in the peroxidase activity of the lymphocytes. Surface (membrane) action rather than phagocytosis may be involved in the bactericidal process. The spleen lymphocyte peroxidase is distinct from myeloperoxidase in several respects. We thank Alicia Cruz and John Dirgo for excellent technical assistance, and Gerri Groppi for preparing this manuscript.