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1.
The NADPH oxidase is an electron transport chain in "professional" phagocytic cells that transfers electrons from NADPH in the cytoplasm, across the wall of the phagocytic vacuole, to form superoxide. The electron transporting flavocytochrome b is activated by the integrated function of four cytoplasmic proteins. The antimicrobial function of this system involves pumping K+ into the vacuole through BKCa channels, the effect of which is to elevate the vacuolar pH and activate neutral proteases. A number of homologous systems have been discovered in plants and lower animals as well as in man. Their function remains to be established.  相似文献   

2.
Andrew R Cross 《BBA》2004,1657(1):1-22
The NADPH oxidase is an electron transport chain in “professional” phagocytic cells that transfers electrons from NADPH in the cytoplasm, across the wall of the phagocytic vacuole, to form superoxide. The electron transporting flavocytochrome b is activated by the integrated function of four cytoplasmic proteins. The antimicrobial function of this system involves pumping K+ into the vacuole through BKCa channels, the effect of which is to elevate the vacuolar pH and activate neutral proteases. A number of homologous systems have been discovered in plants and lower animals as well as in man. Their function remains to be established.  相似文献   

3.
The NADPH oxidase of neutrophils, essential for innate immunity, passes electrons across the phagocytic membrane to form superoxide in the phagocytic vacuole. Activity of the oxidase requires that charge movements across the vacuolar membrane are balanced. Using the pH indicator SNARF, we measured changes in pH in the phagocytic vacuole and cytosol of neutrophils. In human cells, the vacuolar pH rose to ~9, and the cytosol acidified slightly. By contrast, in Hvcn1 knock out mouse neutrophils, the vacuolar pH rose above 11, vacuoles swelled, and the cytosol acidified excessively, demonstrating that ordinarily this channel plays an important role in charge compensation. Proton extrusion was not diminished in Hvcn1-/- mouse neutrophils arguing against its role in maintaining pH homeostasis across the plasma membrane. Conditions in the vacuole are optimal for bacterial killing by the neutral proteases, cathepsin G and elastase, and not by myeloperoxidase, activity of which was unphysiologically low at alkaline pH.  相似文献   

4.
A. W. Segal 《Protoplasma》1995,184(1-4):86-103
Summary Phagocytic cells of the immune system contain an oxidase that is important for the killing and digestion of engulfed microbes. This is an electron transport chain that transfers electrons from NADPH in the cytosol to oxygen to form superoxide and hydrogen peroxide in the phagocytic vacuole. Absence or abnormality of this oxidase results in the syndrome of CGD, characterised by a profound predisposition to infection. The electron transport chain consists of a flavocytochrome b located in the plasma membrane and membrane of the specific granules. It is composed of a and b-subunits, with apparent molecular masses of 23 kDa and 76–92 kDa, respectively. The b-subunit is a member of the FNR family of reductases with FAD and NADPH binding sites. Based upon the crystal structure of FNR we have constructed a model of the more hydrophilic C terminal half of this b-subunit, which acts as a guide to the organisation of the molecule, and provides a template on which to map mutations in CGD. The location of the heme is uncertain. Electron transport is dependent upon an activation complex of cytosolic proteins including p40 phox , p47 phox , and p67 phox , and the small GTP binding protein, p21 rac . This oxidase system is important for the killing and digestion of bacteria and fungi. This might be accomplished in a number of ways. The oxidase produces superoxide and hydrogen which might be toxic themselves. The hydrogen peroxide can act as substrate for myeloperoxidase which can oxidise chloride and iodide to chlorine and iodine and their hypohalous acids. The proteins contained within the cytoplasmic granules are also very important in the killing process. These are neutral proteinases that require a neutral or slightly alkaline pH for optimal activity. The oxidase transports electrons, unaccompanied by protons, across the wall of the phagocytic vacuole, resulting in an elevation of the vacuolar pH, thereby optimising conditions for killing and digestion of engulfed organisms by these neutral proteinases.  相似文献   

5.
The activities of NADPH and NADH oxidase were determined in homogenates of isolated pure parenchymal and nonparenchymal rat liver cells at neutral (7.4) and acid (5.5) pH. The NADPH oxidase at pH 7.4 is about equally active in parenchymal and nonparenchymal cells and in both cell types is rather insensitive to KCN (1 mm) inhibition. By lowering the pH to 5.5, the NADPH oxidase of the nonparenchymal cells is stimulated (twofold) while the activity in parenchymal cells is decreased. The NADH consumption at neutral pH in parenchymal cells is 75% inhibited by KCN, while this activity in nonparenchymal cells is relatively insensitive to KCN. The NADH oxidase in both parenchymal and nonparenchymal liver cells is less active when the pH is lowered from 7.4 to 5.5. The distribution of NAD(P)H oxidases between parenchymal and nonparenchymal liver cells and the effect of pH on their activities suggest that in the nonparenchymal cells, the NADPH oxidase might play a role in the synthesis of H2O2 within the phagocytic vacuole. A scheme is proposed which describes the metabolic events involved in H2O2 formation and catabolism of endo(phago)cytosed particles in nonparenchymal liver cells.  相似文献   

6.
The fungal vacuole: composition, function, and biogenesis.   总被引:86,自引:4,他引:82  
The fungal vacuole is an extremely complex organelle that is involved in a wide variety of functions. The vacuole not only carries out degradative processes, the role most often ascribed to it, but also is the primary storage site for certain small molecules and biosynthetic precursors such as basic amino acids and polyphosphate, plays a role in osmoregulation, and is involved in the precise homeostatic regulation of cytosolic ion and basic amino acid concentration and intracellular pH. These many functions necessitate an intricate interaction between the vacuole and the rest of the cell; the vacuole is part of both the secretory and endocytic pathways and is also directly accessible from the cytosol. Because of the various roles and properties of the vacuole, it has been possible to isolate mutants which are defective in various vacuolar functions including the storage and uptake of metabolites, regulation of pH, sorting and processing of vacuolar proteins, and vacuole biogenesis. These mutants show a remarkable degree of genetic overlap, suggesting that these functions are not individual, discrete properties of the vacuole but, rather, are closely interrelated.  相似文献   

7.
Charge compensation during the phagocyte respiratory burst   总被引:4,自引:0,他引:4  
The phagocyte NADPH oxidase produces superoxide anion (O(2)(.-)) by the electrogenic process of moving electrons across the cell membrane. This charge translocation must be compensated to prevent self-inhibition by extreme membrane depolarization. Examination of the mechanisms of charge compensation reveals that these mechanisms perform several other vital functions beyond simply supporting oxidase activity. Voltage-gated proton channels compensate most of the charge translocated by the phagocyte NADPH oxidase in human neutrophils and eosinophils. Quantitative modeling of NADPH oxidase in the plasma membrane supports this conclusion and shows that if any other conductance is present, it must be miniscule. In addition to charge compensation, proton flux from the cytoplasm into the phagosome (a) helps prevent large pH excursions both in the cytoplasm and in the phagosome, (b) minimizes osmotic disturbances, and (c) provides essential substrate protons for the conversion of O(2)(*-) to H(2)O(2) and then to HOCl. A small contribution by K+ or Cl- fluxes may offset the acidity of granule contents to keep the phagosome pH near neutral, facilitating release of bactericidal enzymes. In summary, the mechanisms used by phagocytes for charge compensation during the respiratory burst would still be essential to phagocyte function, even if NADPH oxidase were not electrogenic.  相似文献   

8.
Liver fibrosis is a reversible wound‐healing response that occurs after liver injury. NADPH oxidases (NOXs) and reactive oxygen species (ROS) which are expressed in hepatocytes (HCs), hepatic stellate cells (HSCs), and Kupffer cells (KCs) play an important role in the development of hepatic fibrosis. In in vitro studies, we had shown that ursolic acid (UA) could reverse liver fibrosis by inhibiting the activation of NOX‐mediated fibrotic signaling networks in HSCs. In this study, we verified that UA could alleviate CCl4‐induced liver fibrosis by reducing the expression of NOXs/ROS in HCs, HSCs, KCs. Meanwhile, the phagocytic index α and clearance index K which represent phagocytosis of KCs were unchanged. Together, all our data demonstrated that UA induced the proliferation of HCs, promoted apoptosis in HSCs, and prevented activation of KCs in vivo by reducing the expression of NOXs/ROS in HCs, HSCs, KCs. Besides, UA had no effect on the host defense function.  相似文献   

9.
The NADPH oxidase is the main weapon of phagocytic white blood cells that are the first line of defence of our body against invading pathogens, and patients lacking a functional oxidase suffer from severe and recurrent infections. The oxidase is a multisubunit enzyme complex that transports electrons from cytoplasmic NADPH to molecular oxygen in order to generate superoxide free radicals. Electron transport across the plasma membrane is electrogenic and is associated with the flux of protons through voltage-activated proton channels. Both proton and electron currents can be recorded with the patch-clamp technique, but whether the oxidase is a proton channel or a proton channel modulator remains controversial. Recently, we have used the inside-out configuration of the patch-clamp technique to record proton and electron currents in excised patches. This approach allows us to measure the oxidase activity under very controlled conditions, and has provided new information about the enzymatic activity of the oxidase and its coupling to proton channels. In this chapter I will discuss how the unique characteristics of the electron and proton currents associated with the redox activity of the NADPH oxidase have extended our knowledge about the thermodynamics and the physiological regulation of this remarkable enzyme.  相似文献   

10.
Part of the catalytic function of xanthine oxidase (XO) involves the transfer of two electrons from a substrate to a molybdenum ion on the enzyme followed by equilibration of these electrons among other electron resting sites on the enzyme. The electrons are removed from the enzyme at a flavin by oxygen to form hydrogen peroxide. This paper considers mechanisms which allow the electrons to equilibrate between the different resting sites on the enzyme. The mechanisms are chosen to be consistent with known properties of the enzyme (relative reduction potentials, electron transfer rates, and the estimated separation of these resting sites). Tunneling appears to be a good candidate to account for most of the electron transport. It is shown that the XO electron transport system is similar in many respects to sections of mitochondrial electron transport chains and can serve as a nice model for parts of these more complicated biological electron transport systems.  相似文献   

11.
The changes in membrane structure of rabbit polymorphonuclear (PMN) leukocytes during bacterial phagocytosis was investigated with scanning electron microscope (SEM), thin-section, and freeze-fracture techniques. SEM observations of bacterial attachment sites showed the involvement of limited areas of PMN membrane surface (0.01-0.25μm(2)). Frequently, these areas of attachment were located on membrane extensions. The membrane extensions were present before, during, and after the engulfment of bacteria, but were diminished in size after bacterial engulfment. In general, the results obtained with SEM and thin-section techniques aided in the interpretation of the three-dimensional freeze-fracture replicas. Freeze-fracture results revealed the PMN leukocytes had two fracture faces as determined by the relative density of intramembranous particles (IMP). Membranous extensions of the plasma membrane, lysosomes, and phagocytic vacuoles contained IMP's with a distribution and density similar to those of the plasma membrane. During phagocytosis, IMPs within the plasma membrane did not undergo a massive aggregation. In fact, structural changes within the membranes were infrequent and localized to regions such as the attachment sites of bacteria, the fusion sites on the plasma membrane, and small scale changes in the phagocytic vacuole membrane during membrane fusion. During the formation of the phagocytic vacuole, the IMPs of the plasma membrane appeared to move in with the lipid bilayer while maintaining a distribution and density of IMPs similar to those of the plasma membranes. Occasionally, IMPs were aligned to linear arrays within phagocytic vacuole membranes. This alignment might be due to an interaction with linearly arranged motile structures on the side of the phagocytic vacuole membranes. IMP-free regions were observed after fusion of lysosomes with the phagocytic vacuoles or plasma membrane. These IMP-free areas probably represent sites where membrane fusion occurred between lysosomal membrane and phagocytic vacuole membrane or plasma membrane. Highly symmetrical patterns of IMPs were not observed during lysosomal membrane fusion.  相似文献   

12.
Ricardo Murphy 《BBA》2006,1757(8):996-1011
The phagocyte NADPH oxidase produces superoxide anion (O2·−) by the electrogenic process of moving electrons across the cell membrane. This charge translocation must be compensated to prevent self-inhibition by extreme membrane depolarization. Examination of the mechanisms of charge compensation reveals that these mechanisms perform several other vital functions beyond simply supporting oxidase activity. Voltage-gated proton channels compensate most of the charge translocated by the phagocyte NADPH oxidase in human neutrophils and eosinophils. Quantitative modeling of NADPH oxidase in the plasma membrane supports this conclusion and shows that if any other conductance is present, it must be miniscule. In addition to charge compensation, proton flux from the cytoplasm into the phagosome (a) helps prevent large pH excursions both in the cytoplasm and in the phagosome, (b) minimizes osmotic disturbances, and (c) provides essential substrate protons for the conversion of O2·− to H2O2 and then to HOCl. A small contribution by K+ or Cl fluxes may offset the acidity of granule contents to keep the phagosome pH near neutral, facilitating release of bactericidal enzymes. In summary, the mechanisms used by phagocytes for charge compensation during the respiratory burst would still be essential to phagocyte function, even if NADPH oxidase were not electrogenic.  相似文献   

13.
Qiu QS 《遗传学报》2012,39(4):167-171
Membrane fusion is the last step in trafficking pathways during which membrane vesicles fuse with target organelles to deliver cargos. It is a central cellular reaction that plays important roles in signal transduction, protein sorting and subcellular compartmentation. Recent progress in understanding the roles of ion transporters in vacuole fusion in yeast is summarized in this article. It is becoming increasingly evident that the vacuolar proton pump V-ATPase and vacuolar Na+/H+ antiporter ScNhx1p are key components of the vacuole fusion machinery in yeast. Yeast ScNhx1p regulates vacuole fusion by controlling the luminal pH. V-ATPases serve a dual role in vacuolar integrity in which they regulate both vacuole fusion and fission reactions in yeast. Fission defects are epistatic to fusion defects. Vacuole fission depends on the proton translocation activity of the V-ATPase; by contrast, the fusion reaction does not need the transport activity but requires the physical presence of the proton pump. V0, the membrane-integral sector of the V-ATPase, forms trans-complexes between the opposing vacuoles in the terminal phase of vacuole fusion where the V0trans-complexes build a continuous proteolipid channel at the fusion site to mediate the bilayer fusion.  相似文献   

14.
The t-tubules of mammalian ventricular myocytes are invaginations of the surface membrane that form a complex network within the cell, with restricted diffusion to the bulk extracellular space. The trans-sarcolemmal flux of many ions, including Ca(2+), occurs predominantly across the t-tubule membrane and thus into and out of this restricted diffusion space. It seems possible, therefore, that ion concentration changes may occur in the t-tubule lumen, which would alter ion flux across the t-tubule membrane. We have used a computer model of the ventricular myocyte, incorporating a t-tubule compartment and experimentally determined values for diffusion between the t-tubule lumen and bulk extracellular space, and ion fluxes across the t-tubule membrane, to investigate this possibility. The results show that influx and efflux of different ion species across the t-tubule membrane are similar, but not equal. Changes of ion concentration can therefore occur close to the t-tubular membrane, thereby altering trans-sarcolemmal ion flux and thus cell function, although such changes are reduced by diffusion to the bulk extracellular space. Slowing diffusion results in larger changes in luminal ion concentrations. These results provide a deeper understanding of the role of the t-tubules in normal cell function, and are a basis for understanding the changes that occur in heart failure as a result of changes in t-tubule structure and ion fluxes.  相似文献   

15.
Ion channels can function in three physiological modes through their ability to: 1) accommodate osmotically significant fluxes over short periods; 2) propagate signals along or across membranes; 3) control the membrane potential. With respect to mineral nutrition it is via the control of the membrane potential that ion channels are probably most significant. In this paper the physiology and prospects for molecular biology of plant ion channels are discussed. It is concluded that identifying and altering the primary structures that determine functional characteristics of plant ion channel genes could result in changes in the transport characteristics of higher plants.  相似文献   

16.
Water loss from plants is determined by the aperture of stomatal pores in the leaf epidermis, set by the level of vacuolar accumulation of potassium salt, and hence volume and turgor, of a pair of guard cells. Regulation of ion fluxes across the tonoplast, the key to regulation of stomatal aperture, can only be studied by tracer flux measurements. There are two transport systems in the tonoplast. The first is a Ca2+-activated channel, inhibited by phenylarsine oxide (PAO), responsible for the release of vacuolar K+(Rb+) in response to the “drought” hormone, abscisic acid (ABA). This channel is sensitive to pressure, down-regulated at low turgor and up-regulated at high turgor, providing a system for turgor regulation. ABA induces a transient stimulation of vacuolar ion efflux, during which the flux tracks the ion content (volume, turgor), suggesting ABA reduces the set-point of a control system. The second system, which is PAO-insensitive, is responsible for an ion flux from vacuole to cytoplasm associated with inward water flow following a hypo-osmotic transfer. It is suggested that this involves an aquaporin as sensor, and perhaps also as responder; deformation of the aquaporin may render it ion-permeable, or, alternatively, the deformed aquaporin may signal to an associated ion channel, activating it. Treatment with inhibitors of aquaporins, HgCl2 or silver sulfadiazine, produces a large transient increase in ion release from the vacuole, also PAO-insensitive. It is suggested that this involves the same aquaporin, either rendered directly ion-permeable, or signalling to activate an associated ion channel.  相似文献   

17.
Leishmania have a digenetic life cycle, involving a motile, extracellular stage (promastigote) which parasitises the alimentary tract of a sandfly vector. Bloodfeeding activity by an infected sandfly can result in transmission of infective (metacyclic) promastigotes to mammalian hosts, including humans. Leishmania promastigotes are rapidly phagocytosed but may survive and transform into non-motile amastigote forms which can persist as intracellular parasites. Leishmania amastigotes multiply in an acidic intracellular compartment, the parasitophorous vacuole. pH plays a central role in the developmental switch between promastigote and amastigote stages, and amastigotes are metabolically most active when their environment is acidic, although the cytoplasm of the amastigote is regulated at near-neutral pH by an active process of proton extrusion. A steep proton gradient is thus maintained across the amastigote surface and all membrane processes must be adapted to function under these conditions. Amastigote uptake systems for glucose, amino acids, nucleosides and polyamines are optimally active at acidic pH. Promastigote uptake systems are kinetically distinct and function optimally at more neutral environmental pH, indicating that membrane transport activity is developmentally regulated. The nutrient environment encountered by amastigotes is not well understood but the parasitophorous vacuole can fuse with endosomes, phagosomes and autophagosomes, suggesting that a diverse range of macromolecules will be present. The parasitophorous vacuole is a hydrolytic compartment in which such material will be rapidly degraded to low molecular weight components which are typical substrates for membrane transporters. Amastigote surface transporters must compete for these substrates with equivalent host transporters in the membrane of the parasitophorous vacuole. The elaboration of accumulative transporters with high affinity will be beneficial to amastigotes in this environment. The influence of environmental pH on membrane transporter function is discussed, with emphasis on the potential role of a transmembrane proton gradient in active, high affinity transport.  相似文献   

18.
The effects of pH on the resting and action potentials and onthe fluxes of potassium, sodium, and chloride across the membranesof internodal cells of Chara australis have been investigated. Experiments were carried out in an artificial pond water (A.P.W.)of standard composition: CaCl2, 01 mM; KCl, 0.1 mM; NaCl, 1.0mM. The resting potential decreased as the pH was lowered from6.5, being depolarized by about 75 mV at pH 4.5. Measurementsof the ion fluxes as a function of pH suggested that this depolarizationwas caused by an increase in the permeability to sodium anda decrease in permeability to potassium at pH 4.5. Action potentialsof constant peak value can be elicited for some time at pH 4.5,but after 20 min or so the cell becomes refractory. All theseeffects on resting and action potentials are fully reversible.We briefly speculate about the mechanism of these pH effects.  相似文献   

19.
Acanthamoeba myosins IA and IB were localized by immunofluorescence and immunoelectron microscopy in vegetative and phagocytosing cells and the total cell contents of myosins IA, IB, and IC were quantified by immunoprecipitation. The quantitative distributions of the three myosin I isoforms were then calculated from these data and the previously determined localization of myosin IC. Myosin IA occurs almost exclusively in the cytoplasm, where it accounts for approximately 50% of the total myosin I, in the cortex beneath phagocytic cups and in association with small cytoplasmic vesicles. Myosin IB is the predominant isoform associated with the plasma membrane, large vacuole membranes and phagocytic membranes and accounts for almost half of the total myosin I in the cytoplasm. Myosin IC accounts for a significant fraction of the total myosin I associated with the plasma membrane and large vacuole membranes and is the only myosin I isoform associated with the contractile vacuole membrane. These data suggest that myosin IA may function in cytoplasmic vesicle transport and myosin I-mediated cortical contraction, myosin IB in pseudopod extension and phagocytosis, and myosin IC in contractile vacuole function. In addition, endogenous and exogenously added myosins IA and IB appeared to be associated with the cytoplasmic surface of different subpopulations of purified plasma membranes implying that the different myosin I isoforms are targeted to specific membrane domains through a mechanism that involves more than the affinity of the myosins for anionic phospholipids.  相似文献   

20.
Although acidification of phagocytic vacuoles has received a broadened interest with the development of pH-sensitive fluorescent probes to follow the pH changes of vacuoles and acidic vesicles in living cells, the mechanism responsible for the acidification of such vacuoles still remains in doubt. In previous studies of the digestive vacuole system in the ciliate Paramecium caudatum we observed and described a unique population of apparently nonlysosomal vesicles that quickly fused with the newly released vacuole before the vacuole became acid and before lysosomes fused with the vacuole. In this paper we report the following: (a) these vesicles, named acidosomes, are devoid of acid phosphatase; (b) these vesicles accumulate neutral red as well as acridine orange, two observations that demonstrate their acid content; (c) cytochalasin B given 15 s after exposure of the cells to indicator dye-stained yeast will inhibit the acidification of yeast-containing vacuoles; and that (d) we observed using electron microscopy, that fusion of acidosomes with the vacuole is inhibited by cytochalasin B. We conclude that the mechanism for acidification of phagocytic vacuoles in Paramecium resides, at least partially if not entirely, in the acidosomes.  相似文献   

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