Inactivation of phagocytosis-stimulating activity of tuftsin by polymorphonuclear neutrophils: A possible role of leucine aminopeptidase as an ecto-enzyme

The subcellular localization of the tuftsin-inactivating activity was studied using guinea-pig polymorphonuclear neutrophils and the following results were obtained. 1. The tuftsin-inactivating activity was present in the membrane function but not in the cytosol and the granular fractions. 2. Intact neutrophils inactivated tuftsin rapidly. However, when neutrophils were modified chemically by a poorly permeant reagent, diazotized sulfanilic acid, the tuftsin-inactivating activity decreased sifnificantly without any inhibition of marker enzymes of cytosol, microsome, granulesa and mitochondria, suggesting that the tuftsin-inactivating activity is located on the plasma membrane as an ecto-enzyme. 3. When neutrophils were modified by diazotized sulfanilic acid at different concentrations, the tuftsin-inactivating activity of neutrophils was inhibited in proportion to the degree of inhibition of the activity of leucine aminopeptidase, an ecto-enzyme. 4, Hydrolysis of L-leucyl-β-napthylamide, a synthetic substrate of leucine aminopeptidase, was inhibited competitively by tuftsin. 5. Treatmetn of neutrophils with serine protease inhibitors affected neither tuftsin-inactivating nor leucine aminopeptidase activity at all, indicating no involvement of serine proteases, which is said to be located on the cell surface membrane, in the tuftsin-inactivating activity of neutrophils. The possibility was deduced from the above results that leucine aminopeptidase may act as a tuftsin-inactivating enzyme.     

Nonopsonic phagocytosis of Mycobacterium kansasii by human neutrophils depends on cholesterol and is mediated by CR3 associated with glycosylphosphatidylinositol-anchored proteins

Receptors involved in the phagocytosis of microorganisms under nonopsonic conditions have been little studied in neutrophils. Complement receptor type 3 (CR3) is a pattern recognition receptor able to internalize zymosan and C3bi-coated particles. We report that Abs directed against CR3 strongly inhibited nonopsonic phagocytosis of Mycobacterium kansasii in human neutrophils. In these cells CR3 has been found associated with several GPI-anchored proteins localized in cholesterol-rich microdomains (rafts) of the plasma membrane. Cholesterol sequestration by nystatin, filipin, or beta-cyclodextrin as well as treatment of neutrophils with phosphatidylinositol phospholipase C to remove GPI-anchored proteins from the cell surface markedly inhibited phagocytosis of M. kansasii, without affecting phagocytosis of zymosan or serum-opsonized M. kansasii. Abs directed against several GPI-anchored proteins inhibited phagocytosis of M. kansasii, but not of zymosan. N:-acetyl-D-glucosamine, which is known to disrupt interactions between CR3 and GPI proteins, also strongly diminished phagocytosis of these mycobacteria. In conclusion, phagocytosis of M. kansasii involved CR3, GPI-anchored receptors, and cholesterol. In contrast, phagocytosis of zymosan or opsonized particles involved CR3, but not cholesterol or GPI proteins. We propose that CR3, when associated with a GPI protein, relocates in cholesterol-rich domains where M. kansasii are internalized. When CR3 is not associated with a GPI protein, it remains outside of these domains and mediates phagocytosis of zymosan and opsonized particles, but not of M. kansasii.     

Possible involvement of aminopeptidase, an ecto-enzyme, in the inactivation of bradykinin by intact neutrophils

The subcellular localization of the bradykinin-inactivating activity was studied using guinea-pig neutrophils and the following results were obtained. The bradykinin-inactivating activities were found to be present in the cytosol and membrane fractions but not in the granular and nuclear fractions. The bradykinin-inactivating activity of the cytosol fraction was inhibited by N-carbobenzoxy-Gly-Pro, an inhibitor of prolyl endopeptidase, whereas that of the membrane fraction was inhibited by bestatin, an inhibitor of aminopeptidase. Prolyl endopeptidase and aminopeptidase activities were located predominantly in the cytosol and membrane fractions, respectively, and their activities were inhibited by their respective inhibitors. Prolyl endopeptidase and aminopeptidase activities measured with synthetic substrates were competitively inhibited by bradykinin, suggesting that bradykinin is a possible substrate for prolyl endopeptidase and aminopeptidase. Intact neutrophils inactivated bradykinin rapidly. However, when neutrophils were modified chemically by diazotized sulfanilic acid, a poorly permeant reagent which inactivates ecto-enzymes selectively, both the bradykinin-inactivating activity and aminopeptidase activity of neutrophils decreased significantly without any inhibition of cytosol prolyl endopeptidase. The possibility that aminopeptidase, an ecto-enzyme, would be responsible for the inactivation of bradykinin by intact neutrophils was deduced from the results above, although both cytosol prolyl endopeptidase and membrane aminopeptidase could inactivate bradykinin.     

Leucine aminopeptidase as an echo-enzyme of polymorphonuclear neutrophils

Intact polymorphonuclear neutrophils were modified chemically by a poorly permeable reagent, diazotized sulfanilic acid, and the changes in the activity of 5'-nucleotidase, alkaline phosphodiesterase, and leucine aminopeptidase were examined. Among three plasma membrane enzymes, 5'-nucleotidase activity was hardly detected in the human neutrophils. The activity of alkaline phosphodiesterase was observed in all the neutrophils examined, but was not inhibited by diazotized sulfanilic acid in the guinea-pig neutrophils. On the other hand, the activity of leucine aminopeptidase was not only found but also inhibited by diazotized sulfanilic acid without the inhibition of lactate dehydrogenase, a cytosol enzyme, in all the neutrophils, suggesting that leucine aminopeptidase is located generally on the plasma membrane as an ecto-enzyme in the neutrophils.     

Leucine aminopeptidase as an ecto-enzyme of polymorphonuclear neutrophils

Intact polymorphonuclear neutrophils were modified chemically by a poorly permeable reagent, diazotized sulfanilic acid, and the changes in the activity of 5′-nucleotidase, alkaline phosphodiesterase, and leucine aminopeptidase were examined. Among three plasma membrane enzymes, 5′-nucleotidase activity was hardly detected in the human neutrophils. The activity of alkaline phosphodiesterase was observed in all the neutrophils examined, but was not inhibited by diazotized sulfanilic acid in the guinea-pig neutrophils. On the other hand, the activity of leucine aminopeptidase was not only found but also inhibited by diazotized sulfanilic acid without the inhibition of lactate dehydrogenase, a cytosol enzyme, in all the neutrophils, suggesting that leucine aminopeptidase is located generally on the plasma membrane as an ecto-enzyme in the neutrophils.     

Effect of phagocytosis on receptor distribution and endocytic activity in macrophages

Phagocytosis requires the internalization of a significant fraction of the plasma membrane and results in the intracellular deposition of large particles. We evaluated the effect of phagocytosis on the cellular distribution of recycling receptors and uptake of ligand to determine whether phagocytosis affects receptor behavior. Phagocytosis of zymosan, latex particles, or IgG-coated red blood cells by rabbit alveolar macrophages did not decrease the number of cell surface receptors for transferrin, alpha 2-macroglobulin X protease complexes, maleylated proteins, or mannosylated proteins. The number of surface receptors for transferrin was also unaltered in J774 cells, a macrophage-like cell line. In both cell types extensive phagocytosis did not affect the rate of receptor-mediated endocytosis or the distribution of receptors between the endosome and the cell surface. However, fluid phase pinocytosis was reduced by phagocytosis. The major reduction appeared to be not in the rate of internalization but rather in the delivery of fluid to the lysosome. These results demonstrate that internalization of a significant amount of the plasma membrane during phagocytosis does not diminish the number of receptors on the cell surface and has no effect on receptor-mediated ligand uptake.     

Stimulation of neutrophils by tumor necrosis factor

Human recombinant tumor necrosis factor (TNF) was shown to be a weak direct stimulus of the neutrophil respiratory burst and degranulation. The stimulation, as measured by iodination, H2O2 production, and lysozyme release, was considerably increased by the presence of unopsonized zymosan in the reaction mixture, an effect which was associated with the increased ingestion of the zymosan. TNF does not act as an opsonin but, rather, reacts with the neutrophil to increase its phagocytic activity. TNF-dependent phagocytosis, as measured indirectly by iodination, is inhibited by monoclonal antibodies (Mab) 60.1 and 60.3, which recognize different epitopes on the C3bi receptor/adherence-promoting surface glycoprotein of neutrophils. Other neutrophil stimulants, namely N-formyl-methionyl-leucyl-phenylalanine, the Ca2+ ionophore A23187, and phorbol myristic acetate, also increase iodination in the presence of zymosan; as with TNF, the effect of these stimulants is inhibited by Mab 60.1 and 60.3, whereas, in contrast to that of TNF, their stimulation of iodination is unaffected by an Mab directed against TNF. TNF may be a natural stimulant of neutrophils which promotes adherence to endothelial cells and to particles, leading to increased phagocytosis, respiratory burst activity, and degranulation.     

Complement receptor 3 (CD11b/CD18) mediates type I and type II phagocytosis during nonopsonic and opsonic phagocytosis,respectively

Two types of opsonic phagocytosis have been defined depending on the receptor engaged: FcgammaRs mediate type I phagocytosis of IgG-coated particles; complement receptor 3 (CR3) mediates type II phagocytosis of complement-coated particles. In addition to opsonic phagocytosis, CR3 also mediates nonopsonic phagocytosis of zymosan (Z) and Mycobacterium kansasii through engagement of distinct sites. Using Chinese hamster ovary cells stably expressing human CR3, we studied CR3-mediated ingestion of nonopsonized particles, Z or M. kansasii, compared with opsonized zymosan (OZ). We show that 1) while OZ sinks into cells, Z is engulfed by pseudopodia as visualized by electron microscopy; 2) in contrast to OZ, nonopsonic phagocytosis of Z and M. kansasii depends on Rac and Cdc42 but not on Rho activity; and 3) CR3-mediated phagocytosis of Z depends on the kinase activity of the Src family tyrosine kinase Hck, while OZ internalization does not. Therefore, CR3 mediates type I phagocytosis under nonopsonic conditions and type II under opsonic conditions. This is the first evidence that a single receptor can mediate both types of phagocytosis depending on the ligand used.     

Inhibition of platelet aggregation by synthetic phosphatidylcholines: possible involvement of vesiculation of platelet plasma membranes

The purpose of this investigation was to determine which enzyme activities are true canine neutrophil plasma membrane markers. Three enzymes thought to be present on plasma membranes were chosen for study: 5'-nucleotidase, magnesium-dependent adenosine triphosphatase (Mg2+-ATPase), and leucine aminopeptidase. Both 5'-nucleotidase and Mg2+-ATPase were found to be ectoenzymes in the canine neutrophil but additional Mg2+-ATPase activity was located intracellularly. An endogenous inhibitor of 5'-nucleotidase was found in the cytosol of canine neutrophils. The specific 5'-nucleotidase inhibitor, adenosine 5'-[alpha, beta-methylene] diphosphate also inhibited the canine enzyme in intact cells. Leucine aminopeptidase was located solely in the myeloperoxidase-containing granules of the canine neutrophil. Plasma membrane, as identified by the presence of Mg2+-ATPase and 5'-nucleotidase activities, was separated from other cell organelles by Percoll-density gradient centrifugation of a 10 000 X g supernatant of nitrogen cavitated neutrophils.     

Possible exposure of leucine aminopeptidase on the cell surface of rabbit blood neutrophils by digitonin treatment

The sensitivity of leucine aminopeptidase to diazotized sulfanilic acid (DSA) was compared between neutrophils from blood and peritoneal exudates of rabbit. The leucine aminopeptidase activity of peritoneal neutrophils was inhibited about 40% by DSA, whereas that of blood neutrophils was not inhibited at all by the reagent. However, pretreatment of blood neutrophils with digitonin in the presence and in the absence of divalent cations rendered leucine aminopeptidase sensitive to DSA to the same extent as peritoneal neutrophils, without affecting the cell viability and lactate dehydrogenase activity. These findings seem to indicate that the leucine aminopeptidase of blood neutrophils, which is normally inaccessible to DSA, was exposed on the cell surface by digitonin treatment.     

Lysosomal enzyme secretion from human neutrophils mediated by cyclic CMP: inhibition of cyclic GMP accumulation and neutrophil function by glucocorticosteroids.

The effects of several glucocorticosteroids on cyclic GMP accumulation, guanylate cyclase activity, calcium influx, lysosomal enzyme secretion, and phagocytosis were studied in human neutrophils. Contact between neutrophils and serum-treated zymosan particles, in the presence of calcium at pH 7.4, triggered these cellular events within five minutes. Each of these neutrophil functions was markedly inhibited by methylprednisolone sodium succinate, triamcinolone acetonide hemisuccinate and paramethasone acetate but was unaffected by two mineralo-corticosteroids. Human neutrophil soluble guanylate cyclase activity was not changed by the glucocorticoids. Inhibition of phagocytosis by, and lysosomal enzyme secretion from, neutrophils by glucocorticosteroids may be the result of a reduction in cyclic GMP accumulation within these cells. The data suggest that glucocorticosteroids inhibit cyclic GMP accumulation in neutrophils by reducing the influx of extracellular calcium into the cells, thereby limiting the availability of intracellular calcium for metabolic processes associated with the accumulation of cyclic GMP.     

Myeloperoxidase deficiency enhances zymosan phagocytosis associated with up-regulation of surface expression of CD11b in mouse neutrophils

Myeloperoxidase (MPO), a major component of neutrophils, catalyzes the production of hypochlorous acid (HOCl) from hydrogen peroxide and chloride anion. Phagocytosis is a critical event induced by neutrophils for host defense and inflammation. Interestingly, we found that MPO-deficient (MPO^?/?) neutrophils engulfed larger amounts of zymosan than wild-type neutrophils. Blocking of the CD11b subunit of complement receptor 3 (CR3) as well as inhibition of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) dramatically reduced zymosan phagocytosis. In contrast, blocking of dectin-1, toll-like receptor 2 (TLR2), or spleen tyrosine kinase (Syk) had no significant effects on phagocytosis. Western blotting analysis showed that inhibition of FAK decreased the phosphorylation of ERK1/2, indicating that ERK1/2 is a downstream regulator of FAK in neutrophils. Importantly, we found that cell surface expression of CD11b and phosphorylation of ERK1/2 was significantly higher in zymosan-stimulated MPO^?/? neutrophils than in zymosan-stimulated wild-type neutrophils. Pretreatment with the MPO inhibitor 4-aminobenzoic acid hydrazide dramatically enhanced both zymosan phagocytosis and the surface expression of CD11b in wild-type neutrophils, but not in MPO^?/? neutrophils. Collectively, these results strongly suggest that up-regulation of the CD11b/FAK/ERK signaling pathway due to absence of MPO enhances the zymosan phagocytic activity of mouse neutrophils.     

Serine proteases in the spiny lobster olfactory organ: their functional expression along a developmental axis, and the contribution of a CUB-serine protease

Several serine proteases and protease inhibitors have been identified in the crustacean olfactory organ, which is comprised of the lateral flagellum of the antennule and its aesthetascs sensilla that house olfactory receptor neurons and their supporting cells. The function of these proteases in the olfactory organ is unknown, but may include a role in perireception (e.g., odor activation or inactivation) or in the development or survival of olfactory receptor neurons. To examine directly the function of proteases in the olfactory organ of the Caribbean spiny lobster Panulirus argus, we used different tissue fractions from the lateral flagellum in an enzyme activity assay with a variety of protease substrates and inhibitors. Trypsin-like serine protease activity occurs throughout the lateral flagellum but is enriched in the cell membranes from aesthetascs. Cysteine- and metalloprotease activities also occur in olfactory tissue, but are more abundant in tissue fractions other than aesthetascs. To assess the contribution of one of the olfactory serine proteases--CUB-serine protease (Csp)--Csp was immunoprecipitated using an antibody; results with the remaining fraction suggest that Csp accounts for at least 40% of the total serine protease activity in the olfactory organ. The amount of total serine protease activity follows a developmental axis in the lateral flagellum. Total protease activity is lowest in the proximal zone, which lacks aesthetascs, and the proliferation zone, where olfactory receptor neurons and associated cells are born, and highest in aesthetascs of the distally-located senescence zone, which has the oldest olfactory tissue.     

Perturbation of beta-glucan receptors on human neutrophils initiates phagocytosis and leukotriene B4 production

Normal human neutrophils were stimulated with the yeast cell wall product, zymosan, and examined for two biologic responses, ingestion of particles and production of leukotriene B4 (LTB4), under conditions that were comparable and optimal for the quantitation of each response. Monolayers of adherent neutrophils ingested unopsonized zymosan particles, at particle-to-cell ratios of 12.5:1 to 125:1, in a dose- and time-related manner. At a ratio of 125:1, the percentages of neutrophils ingesting greater than or equal to 1 and greater than or equal to 3 zymosan particles reached plateau levels of 55 +/- 6 and 32 +/- 9% (mean +/- SD, n = 8), respectively, within 30 min. At this same ratio, neutrophils during gravity sedimentation with zymosan particles synthesized LTB4 in a time-dependent manner for at least 45 min. The maximum amount of immunoreactive LTB4 released into supernatants was 3.8 +/- 1.2 ng per 10(6) neutrophils (mean +/- SD, n = 5) and the corresponding total immunoreactive LTB4 was 6.2 +/- 1.9 ng per 10(6) neutrophils. Treatment of 2 x 10(7) suspended neutrophils with 250 micrograms of trypsin for 20 min before concurrent assessment of neutrophil phagocytosis and LTB4 production reduced both of these responses by about 50%. Pretreatment of neutrophils with 800 micrograms/ml of soluble yeast beta-glucan inhibited their ingestion of zymosan by 84% (mean +/- SD, n = 3), with 50% inhibition occurring with 100 micrograms/ml of soluble beta-glucan; 800 micrograms/ml of soluble yeast alpha-mannan had no inhibitory effect. Pretreatment of neutrophils with 400 micrograms/ml of soluble yeast beta-glucan inhibited neutrophil synthesis of LTB4 by 90%, with 50% occurring with 200 micrograms/ml; 400 micrograms/ml of soluble yeast alpha-mannan had no inhibitory effect. The presence of 1.25 micrograms/ml of cytochalasin B during incubation with zymosan particles reduced neutrophil phagocytosis from 65 to 6%, and neutrophil synthesis of LTB4 from total levels of 6.0 +/- 0.3 ng/10(6) cells to zero (mean +/- SD, n = 3). Pretreatment with either cytochalasin B or vinblastine did not alter neutrophil generation of LTB4 induced by calcium ionophore. Neutrophils pretreated with vinblastine, at 4 x 10(-6) to 4 x 10(-4) M, and then maintained at one-half these concentrations during incubation with unopsonized zymosan particles exhibited no diminution in particle ingestion, but were markedly reduced in zymosan-induced synthesis of LTB4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Serine proteases in the spiny lobster olfactory organ: Their functional expression along a developmental axis,and the contribution of a CUB‐serine protease

Several serine proteases and protease inhibitors have been identified in the crustacean olfactory organ, which is comprised of the lateral flagellum of the antennule and its aesthetascs sensilla that house olfactory receptor neurons and their supporting cells. The function of these proteases in the olfactory organ is unknown, but may include a role in perireception (e.g., odor activation or inactivation) or in the development or survival of olfactory receptor neurons. To examine directly the function of proteases in the olfactory organ of the Caribbean spiny lobster Panulirus argus, we used different tissue fractions from the lateral flagellum in an enzyme activity assay with a variety of protease substrates and inhibitors. Trypsin‐like serine protease activity occurs throughout the lateral flagellum but is enriched in the cell membranes from aesthetascs. Cysteine‐ and metalloprotease activities also occur in olfactory tissue, but are more abundant in tissue fractions other than aesthetascs. To assess the contribution of one of the olfactory serine proteases—CUB‐serine protease (Csp)—Csp was immunoprecipitated using an antibody; results with the remaining fraction suggest that Csp accounts for at least 40% of the total serine protease activity in the olfactory organ. The amount of total serine protease activity follows a developmental axis in the lateral flagellum. Total protease activity is lowest in the proximal zone, which lacks aesthetascs, and the proliferation zone, where olfactory receptor neurons and associated cells are born, and highest in aesthetascs of the distally‐located senescence zone, which has the oldest olfactory tissue. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2004     

Differential Effects of Serum Heat Treatment on Chemotaxis and Phagocytosis by Human Neutrophils

Neutrophils, in cooperation with serum, are vital gatekeepers of a host’s microbiome and frontline defenders against invading microbes. Yet because human neutrophils are not amenable to many biological techniques, the mechanisms governing their immunological functions remain poorly understood. We here combine state-of-the-art single-cell experiments with flow cytometry to examine how temperature-dependent heat treatment of serum affects human neutrophil interactions with “target” particles of the fungal model zymosan. Assessing separately both the chemotactic as well as the phagocytic neutrophil responses to zymosan, we find that serum heat treatment modulates these responses in a differential manner. Whereas serum treatment at 52°C impairs almost all chemotactic activity and reduces cell-target adhesion, neutrophils still readily engulf target particles that are maneuvered into contact with the cell surface under the same conditions. Higher serum-treatment temperatures gradually suppress phagocytosis even after enforced cell-target contact. Using fluorescent staining, we correlate the observed cell behavior with the amounts of C3b and IgG deposited on the zymosan surface in sera treated at the respective temperatures. This comparison not only affirms the critical role of complement in chemotactic and adhesive neutrophil interactions with fungal surfaces, but also unmasks an important participation of IgGs in the phagocytosis of yeast-like fungal particles. In summary, this study presents new insight into fundamental immune mechanisms, including the chemotactic recruitment of immune cells, the adhesive capacity of cell-surface receptors, the role of IgGs in fungal recognition, and the opsonin-dependent phagocytosis morphology of human neutrophils. Moreover, we show how, by fine-tuning the heat treatment of serum, one can selectively study chemotaxis or phagocytosis under otherwise identical conditions. These results not only refine our understanding of a widely used laboratory method, they also establish a basis for new applications of this method.     

POTASSIUM AND AMINO ACID TRANSPORT IN HUMAN LEUKOCYTES EXPOSED TO PHAGOCYTIC STIMULI

Influxes of potassium and amino acids were measured in suspensions of human polymorphonuclear leukocytes (PMNs) under resting conditions and after various phagocytic stimuli. Both ouabain-sensitive (or pump) and ouabain-insensitive (or leak) influxes of K were determined. In 5 mM external K, mean total K influx was 0.69 nmol/10⁶ cells x min, of which 52% was ouabain-sensitive. Ouabain binding was irreversible, and, as in erythrocytes, was inhibited by K. At external concentrations of 0.1 mM, influxes of lysine and leucine were entirely carrier-mediated, with means of 0.021 nmol/10⁶ cells x min, and 0.019 nmol/10⁶ cells x min, respectively. After incubation of PMNs with zymosan or latex particles, the K pump was reduced more than 60%, whereas amino acid influxes were inhibited only by 30%. PMNs were also exposed to cytochalasin B before challenge by particles: the drug prevented phagocytosis but not surface binding of zymosan, nor did it influence transport of K or amino acids. After pretreatment of PMNs with cytochalasin B, interaction of zymosan with their surface resulted in the same degree of inhibition of influxes of K and amino acids as when the cells were permitted to phagocytose the particles. In contrast, exposure of PMN to latex particles, which do not bind to cytochalasin B-treated cells, after pretreatment of cells with cytochalasin B did not result in inhibition of influxes. Treatment of cells with colchicine had no effect on either membrane transport or its inhibition after exposure to various phagocytic stimuli. These results indicate that the surface membranes of PMNs are functionally heterogeneous with respect to the association of transport sites for the different solutes. Moreover, loss of specific membrane functions from phagocytosing cells may result from the surface-at-tachment phase of particle-cell interactions, since the interactions of zymosan particles with PMNs in the absence of phagocytosis also inhibited transport of solutes.     

Biochemical studies on liver functions in primary cultured hepatocytes of adult rats. III. Changes of enzyme activities on cell membranes during culture

When liver cells were dispersed with collagenase, their 5'-nucleotidase activity decreased to half the initial level, but it increased to the original level again on culture of the cells for a few days. The activity of another membrane enzyme, alkaline phosphatase, did not decrease on dispersion of the cells, but it increased about 10-fold on culture of the cells. These inductions did not require any hormone, but the effects were greater at a high cell density. These enzymes are located in both the plasma membranes and the cytoplasm, but the enzymes in these two locations can be distinguished by differences in their pH optima, substrate specificities, and susceptibilities to inhibitors. The increases were found to be due to increases in the activity of only the enzymes in the plasma membranes. The increases in enzyme activities were inhibited by actinomycin D, cycloheximide, and puromycin. The activities of leucine aminopeptidase and aminopeptidase B, other membrane enzymes, remained constant during dispersion and culture of the cells. These results show that enzymes in the cell membranes are affected in different ways by cell dispersion with collagenase and subsequent culture of the cells.     

Reaction of serine proteases with substituted isocoumarins: discovery of 3,4-dichloroisocoumarin, a new general mechanism based serine protease inhibitor

The mechanism-based inactivations of a number of serine proteases, including human leukocyte (HL) elastase, cathepsin G, rat mast cell proteases I and II, several human and bovine blood coagulation proteases, and human factor D by substituted isocoumarins and phthalides which contain masked acyl chloride or anhydride moieties, are reported. 3,4-Dichloroisocoumarin, the most potent inhibitor investigated here, inactivated all the serine proteases tested but did not inhibit papain, leucine aminopeptidase, or beta-lactamase. 3,4-Dichloroisocoumarin was fairly selective toward HL elastase (kobsd/[I] = 8920 M-1 s-1); the inhibited enzyme was quite stable to reactivation (kdeacyl = 2 X 10(-5) s-1), while enzymes inhibited by 3-acetoxyisocoumarin and 3,3-dichlorophthalide regained full activity upon standing. The rate of inactivation was decreased dramatically in the presence of reversible inhibitors or substrates, and ultraviolet spectral measurements indicate that the isocoumarin ring structure is lost upon inactivation. Chymotrypsin A gamma is totally inactivated by 1.2 equiv of 3-chloroisocoumarin or 3,4-dichloroisocoumarin, and approximately 1 equiv of protons is released upon inactivation. These results indicate that these compounds react with serine proteases to release a reactive acyl chloride moiety which can acylate another active site residue. These are the first mechanism-based inhibitors reported for many of the enzymes tested, and 3,4-dichloroisocoumarin should find wide applicability as a general serine protease inhibitor.     

Effects of aminopeptidase inhibitors actinonin and amastatin on chemotactic and phagocytic responses of human neutrophils

Actinonin and amastatin are low-molecular-weight inhibitors of aminopeptidases associated with cell surfaces. The purpose of this study was to determine their effects on human neutrophil functions such as chemotaxis and phagocytosis. Both actinonin and amastatin enhanced chemotaxis to the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine. On the other hand, the effects of both agents on neutrophil phagocytosis were varied. Bacterial attachment to neutrophils was slightly affected by these agents. However, actinonin enhanced the internalization of bacteria by neutrophils. Neutrophil leucine aminopeptidase activity was also determined and was found to be weakly inhibited by these agents.