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1.
Summary To control the intracellular free Ca2+ concentration from the cell exterior, pollen tubes ofLilium longiflorum were treated with a Ca2+ ionophore, A23187. Cytoplasmic streaming was inhibited when the free Ca2+ concentration of the external medium ([Ca2+]) was raised to 5×10–6 M or higher. At [Ca2+] below 1×10–6 M, the rhodamine-phalloidin stained actin filaments appeared straight and thin. However, at [Ca2+] which inhibited cytoplasmic streaming, the actin filaments appeared fragmented. In pollen tubes, Ca2+ regulation of cytoplasmic streaming may be linked not only to myosin (Shimmen 1987) but also to actin.Abbreviations ATP
adenosine-5-triphosphoric acid
- [Ca2+]
concentration of free Ca2+
- EGTA
ethyleneglycol-bis-(-aminoethylether)N,N,N,N-tetraacetic acid
- HEPES
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
- PIPES
piperazine-N,N-bis(2-ethanesulfonic acid)
- Rh-ph
rhodamine-conjugated phalloidin 相似文献
2.
Yasuhiro Anraku Naoyuki Umemoto Ryogo Hirata Yoh Wada 《Journal of bioenergetics and biomembranes》1989,21(5):589-603
Our current work on a vacuolar membrane proton ATPase in the yeastSaccharomyces cerevisiae has revealed that it is a third type of H+-translocating ATPase in the organism. A three-subunit ATPase, which has been purified to near homogeneity from vacuolar membrane vesicles, shares with the native, membrane-bound enzyme common enzymological properties of substrate specificities and inhibitor sensitivities and are clearly distinct from two established types of proton ATPase, the mitochondrial F0F1-type ATP synthase and the plasma membrane E1E2-type H+-ATPase. The vacuolar membrane H+-ATPase is composed of three major subunits, subunita (M
r
=67 kDa),b (57kDa), andc (20 kDa). Subunita is the catalytic site and subunitc functions as a channel for proton translocation in the enzyme complex. The function of subunitb has not yet been identified. The functional molecular masses of the H+-ATPase under two kinetic conditions have been determined to be 0.9–1.1×105 daltons for single-cycle hydrolysis of ATP and 4.1–5.3×105 daltons for multicycle hydrolysis of ATP, respectively.N,N-Dicyclohexylcarbodiimide does not inhibit the former reaction but strongly inhibits the latter reaction. The kinetics of single-cycle hydrolysis of ATP indicates the formation of an enzyme-ATP complex and subsequent hydrolysis of the bound ATP to ADP and Pi at a 7-chloro-4-nitrobenzo-2-oxa-1,3-diazolesensitive catalytic site. Cloning of structural genes for the three subunits of the H+-ATPase (VMA1, VMA2, andVMA3) and their nucleotide sequence determination have been accomplished, which provide greater advantages for molecular biological studies on the structure-function relationship and biogenesis of the enzyme complex. Bioenergetic aspects of the vacuole as a main, acidic compartment ensuring ionic homeostasis in the cytosol have been described.Abbreviations CCCP
carbonyl cyanidem-chlorophenyl hydrazone
- DCCD
N,N-dicyclohexylcarbondiimide
- DES
diethylstilbestrol
- DIDS
4,4-diisothiocyano-2,2-stilbene disulfonic acid
- NBD-Cl
7-chloro-4-nitrobenzo-2-oxa-1,3-diazole
- Pi
inorganic phosphate
- SDS
sodium dodecylsulfate
- SF6847
3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile
- SITS
4-acetamide-4-isothiocyanatostilbene-2,2-disulfonic acid
- ZW3-14
N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate 相似文献
3.
G. Cai A. Moscatelli C. Del Casino V. Chevrier M. Mazzi A. Tiezzi M. Cresti 《Protoplasma》1996,190(1-2):68-78
Summary In the pollen and pollen tube of higher plants, the distribution of the microtubular cytoskeleton has been extensively studied. Even though the pattern of microtubules is known, one of the most remarkable deficiencies is the absence of data on the localization of microtubule-nucleation sites in the pollen tubes. In order to get insights about the localization of centrosome-like structures in the pollen tube ofNicotiana tabacum L., we have used the monoclonal antibody 6C6 to search for pericentriolar antigen(s). The antibody was initially raised against a component of animal centrosomes and has been already employed to locate centrosomal structures in other plant cell types. By immunoblotting analysis, a polypeptide of Mr 77,000 was identified specifically in the membrane-associated protein fraction of the pollen tube, and is absent from the soluble protein pool. Immunofluorescence observations have shown the polypeptide to be located in the apical part of the pollen tube (about 40–50 m from the tip) in association with the cortical area. A purified plasma membrane fraction from the growing pollen tubes has been obtained, using H+-ATPase activity as an organelle marker. The plasma membrane fraction was shown to be enriched in the Mr 77,000 polypeptide, which can be extracted from membranes by treatment with the detergent CHAPS at a concentration of 0.5%. These data open new research perspectives on the localization and analysis of putative cortical microtubule nucleation sites in the pollen tube.Abbreviations ATP
adenosine-5-triphosphate
- CHAPS
3-[(3-cholamidopropyl)-dimethylammonio]-propanesulfonate
- DTT
dithiothreitol
- EDTA
ethylenediaminetetracetic acid
- EGTA
ethylene glycolbis(-amino-ethyl ether) N,N,N,N-tetraacetic acid
- HEPES
4-(2-hydroxyethyl)-1-piperazineethane sulphonic acid
- MES
2-(N-morpholino)ethane sulphonic acid
- MT
microtubule
- SDS-PAGE
sodium-dodecyl-sulphate polyacrylamide gel electrophoresis
- PMSF
phenylmethyl-sulphonyl-fluoride
- TAME
tosyl-arginine-methylester 相似文献
4.
Mónica R. Calera Rachel Mata Ana Luisa Anaya Blas Lotina-Hennsen 《Photosynthesis research》1995,45(2):105-110
5-O--d-galactopyranosyl-7-methoxy-3,4-dihydroxy-4-phenylcoumarin isolated from Exostema caribaeum (Rubiaceae) has been found to act as an energy-transfer inhibitor in spinach chloroplasts. ATP synthesis and phosphorylating (coupled) electron flow were inhibited by 89 and 72%, respectively, at a concentration of 400 M. H+-uptake, basal and uncoupled electron transport were not affected by the coumarin. The light-activated Mg+2-ATPase activity from bound membrane thylakoid chloroplasts was slightly inhibited by the coumarin. Also, the heat-activated Ca+2-ATPase activity of the isolated coupling factor protein was insensitive to this compound. In chloroplasts partially stripped of coupling factor 1 by an EDTA treatment, the coumarin showed a restoration of the proton uptake process. These results suggest that the 4-phenylcoumarin under investigation inhibited phosphorylation in chloroplasts by specifically blocking the transport of protons through a membrane-bound component or a carrier channel (CFO) located in a hydrophobic region at or near the functional binding site for the coupling factor 1.Abbreviations CF1
chloroplast coupling factor 1
- CFO
coupling factor zero
- DCCD
dicyclohexylcarbodiimide
- DTT
dithiothreitol
- EDTA
ethylene-diaminetetraacetic acid
- HEPES
N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid
- MES
2-(N-morpholino) ethanesulphonic acid
- TCA
trichloroacetic acid
Taken in part from PhD thesis of M.R. Calera. 相似文献
5.
The bacteriocin butyricin 7423 inhibited the activity of the membrane H+-ATPase (BF0, F1) of vegetative cells of Clostridium pasteurianum but not that of its soluble BF1 component. In vitro studies with the H+-ATPases of mutant strains selected for diminished sensitivity (a) to butyricin 7423 and (b) to dicyclohexylcarbodi-imide, confirmed that butyricin 7423 interacts with the BF0 component of this enzyme complex. Even so, certain other mutant strains displaying decreased sensitivity to butyricin 7423 possessed H+-ATPases which in vitro showed undiminished sensitivity to inhibition by the bacteriocin. Furthermore, from the changes in intracellular ATP concentration and in the rates and net extent of efflux of intracellular 86Rb+ ions that were provoked by exposure of the parent and several of the mutant strains to butyricin 7423, it was concluded that its primary bactericidal action was not attributable to stoichiometric inhibition of the membrane H+-ATPase. High extracellular concentrations of K+ ions enabled Cl. pasteurianum to survive exposure to low concentrations of this membrane-active bacteriocin.Non-standard abbreviations H+-ATPase
proton translocating adenosine 5-triphosphatase (EC 3.6.1.3)
- DCCD
dicyclohexylcarbodiimide 相似文献
6.
A large, transient depolarization of the plasma membrane precedes the rapid blue-light (BL)-induced growth suppression in etiolated seedlings of Cucumis sativus L. The mechanism of this voltage transient was investigated by applying inhibitors of ion channels and the plasma-membrane H+-ATPase, by manipulating extracellular ion concentrations, and by measuring cell input resistance and ATP levels. The depolarizing phase was not affected by Ca2+-channel blockers (verapamil, La3+) or by reducing extracellular free Ca2+ by treatment with ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA). However, these treatments did reduce the rate of repolarization, indicating an inward movement of Ca2+ is involved. No effects of the K+-channel blocker tetraethylammonium (TEA+) were detected. Vanadate and KCN, used to inhibit the H+-ATPase, reduced or completely inhibited the BL-induced depolarization. Levels of ATP increased by 11–26% after 1–2 min of BL. Input resistance of trichome cells, measured with double-barreled microelectrodes, remained constant during the onset of the depolarization but decreased as the membrane voltage became more positive than -90 mV. The results indicate that the depolarization mechanism initially involves inactivation of the H+-ATPase with subsequent transient activation of one or more types of ion channels.Abbreviations and Symbols BL
blue light
- CI
current injection
- EGTA
ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid
- TEA+
tetraethylammonium
- Vm
membrane voltage
We wish to thank Drs. Adam Bertl and Clifford L. Slayman, Yale School of Medicine, New Haven, Conn., USA, for helpful discussions. This work was supported by a Natural Sciences and Engineering Research Council of Canada Scholarship (E.P.S.) and National Science Foundation Grant DMB-8351030 (D.J.C.). 相似文献
7.
H. Paschinger 《Archives of microbiology》1977,113(3):285-291
The Na level inside cells of Anacystis nidulans is lower than in the external medium reflecting an effective Na extrusion. Na efflux is an active process and is driven by a Na+/H+-antiport system. The necessary H+-gradient is generated by a proton translocating ATPase in the plasmalemma. This ATPase (electrogenic proton pump) also produces the membrane potential (about -110 mV) responsible for K accumulation. N,N-dicyclohexylcarbodiimide (DCCD) inhibits the ATPase and the H+-gradient completely, but the membrane potential is only reduced (<-70 mV), since K efflux initiated by DCCD maintains the potential partly by diffusion potential.With DCCD, active Na efflux is inhibited thus revealing Na uptake and leading by equilibration to the membrane potential to a 5–20 fold accumulation. Na uptake depends on the DCCD concentration with an optimum at (1–2)×10-4 M DCCD. Pretreatment with DCCD for a few minutes followed by replacement of the medium suffices to induce Na uptake.DCCD induced Na influx is about 5 times faster in light than in darkness, and the steady state is reached much earlier in light; a 5 fold increase by light was also found for Rb uptake with untreated cells. Valinomycin stimulates the influx of Rb to about the same rate in light and dark. Therefore light may unspecifically increase the permeability of the plasma-lemma probably via the ATP level. Similarly to DCCD also 3×10-3 M N-ethylmaleimide induces Na uptake.Abbreviations Used DCCD
N,N-dicyclohexylcarbodiimide
- NEM
N-ethylmaleimide
- CCCP
carbonylcyanide m-chlorophenylhydrazone
- Pipes
piperazine-N,N-bis(2-ethanesulfonic acid)
- DCMU
3-(3,4-dichlorophenyl)-1,1-dimethylurea 相似文献
8.
Aminophylline, an inhibitor of cyclic nucleotide phosphodiesterase (EC 3.1.4.17), inhibits elongation and correlated H+ and K+ transport in embryos of Haplopappus gracilis and in pea internode segments. Moreover, the drug strongly inhibits the stimulation of these processes by fusicoccin and indole-3-acetic acid and reduces passive permeability of the membrane. The possible mechanisms of action of aminophylline are discussed.Abbreviations cAMP
adenosine 3:5-cyclic monophosphate
- FC
fusicoccin
- IAA
indole-3-acetic acid
- MES
2-N-morpholinoethanesulfonic acid
- PDE
cyclic nucleotide phosphodiesterase 相似文献
9.
Partial purification of myosin from lily pollen tubes by monitoring with in vitro motility assay 总被引:1,自引:0,他引:1
Summary Myosin in pollen tubes ofLilium longiflorum was partially purified, using an in vitro motility assay as a monitor. The main components in the partially purified preparation had molecular masses of 110, 120, and 140 kDa in SDS-PAGE. They became bound to actin filaments in an ATP-dependent manner. Among the components, only that of 120 kDa became bound to ATP and was concluded to be the heavy chain of pollen tube myosin.Abbreviations ATP
adenosine-5-triphosphate
- DTT
dithiothreitol
- EB
extraction buffer
- EGTA
ethyleneglycol-bis-(-aminoethylether) N, N, N, N-tetraacetic acid
- PAGE
polyacrylamide gel electrophoresis
- PIPES
piperazine-N,N-bis-(2-ethanesulfonic acid)
- PMSF
phenylmethylsulfonyl fluoride
- SDS
sodium dodecylsulfate
- TBS
Tris buffered saline
- TEB
Tris-EGTA buffer 相似文献
10.
Acidification of weakly buffered suspensions of the cyanobacteria Anacystis nidulans, Nostoc sp. strain MAC, Dermocarpa sp. and Anabaena variabilis was observed after the application of oxygen pulses to anaerobic cells. The acidification was caused by proton extrusion from the oxygen pulsed cells since it was eliminated by the uncoupler (H+ ionophore) carbonyl cyanide m-chlorophenylhydrazone. Results with the inhibitors dicyclohexylcarbodiimide or 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, orthovanadate and cyanide indicated the association of various fractions of the observed proton extrusion with different activities of the cell membrane, viz. a H+-translocating reversible F0F1-ATPase, a unidirectional H+-translocating ATP hydrolase, and a respiratory electron transport system, respectively. Further parameters investigated were the pH dependence and the H+/O stoichiometry of the H+ extrusion from oxygen pulsed cyanobacteria. H+/O ratios at neutral pH were between 4 (Anacystis nidulans) and 0.3 (Dermocarpa) with uninhibited, actively phosphorylating cells and between 2 (Anacystis nidulans) and 0.4 (Dermocarpa) with ATPase-inhibited (ATP-depleted) cells, respectively. It is significant that with all four cyanobacteria tested a major fraction of the observed H+ ejection remained unaffected by ATPase inhibitors even at concentration which completely abolished all oxidative phosphorylation. Vanadate had a major effect on the H+ extrusion from Anabaena only. From this it is concluded that in the cyanobacterial species investigated part of the H+ extrusion from oxygen pulsed cells is directly linked to some H+-translocating respiratory electron transport chain present in the cell membrane.Abbreviations CCCP
carbonyl cyanide m-chlorophenylhydrazone
- DCCD
N, N-dicyclohexylcarbodiimide
- DCMU
N-(3,4-dichlorophenyl-)N,N-dimethylurea
- NBD
7-chloro-4-nitrobenzoxa-1,3-diazole
- TPP+
tetraphenylphosphonium
- Mes
2-(N-morpholino)ethanesulfonic acid
- Pipes
piperazine-N,N-bis-(2-ethanesulfonic acid)
- Hepes
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
- Taps
tris (hydroxymethyl)-methyl-aminopropanesulfonic acid
- Ches
2-(N-cyclohexylamino)-ethanesulfonic acid
- Caps
3-cyclohexylamino)-1-propanesulfonic acid; according to most textbooks (e.g. Nicholls 1982) the terms proton electrochemical potential (
) and protonmotive force (pmf, p), both of which equivalently describe the energetic state of energy-transducing membranes, were used synonymously and expressed in mV units throughout this article (however, cf. Lowe and Jones 1984)
Dedicated to Prof. G. Drews on the occasion of his 60th birthday 相似文献
11.
Summary Two-dimensional crystalline arrays of Ca2+-ATPase molecules develop after treatment of sarcoplasmic reticulum vesicles with Na3VO4 in a Ca2+-free medium. The influence of membrane potential upon the rate of crystallization was studied by ion substitution using oxonol VI and 3,3-diethyl-2,2-thiadicarbocyanine (Di–S–C2(5)) to monitor inside positive or inside negative membrane, potentials, respectively. Positive transmembrane potential accelerates the rate of crystallization of Ca2+-ATPase, while negative potential disrupts preformed Ca2+-ATPase crystals, suggesting an influence of transmembrane potential upon the conformation of Ca2+-ATPase. 相似文献
12.
Jos M. J. Lamers Pieter D. Verdouw Jaime Mas-Oliva 《Molecular and cellular biochemistry》1987,78(2):169-176
Summary The Ca2+ channel blockers felodipine and bepridil are known to affect selectively functions of calmodulin. We studied their effects on calmodulin binding and ATPase activities of calmodulin-containing and calmodulin-depleted rabbit heart sarcolemma. Both drugs as well as the specific anti-calmodulin drug calmidazolium at a concentration of 50 µM, inhibited the Ca2+-stimulated calmodulin binding to calmodulin-depleted sarcolemma. Within the concentration range of 3 to 100 µM all three drugs also progressively inhibited Ca2+ pumping ATPase in calmodulin containing sarcolemma, although the enzyme was assayed at saturating Ca2+ (100 µM). The inhibitory potency of calmidazolium and bepridil, but not that of felodipine, increased when the membrane protein concentration in the ATPase assay was lowered. At low membrane protein concentration 30 µM calmidazolium completely blocked calmodulin-dependent Ca2+ pumping ATPase, whereas the inhibition caused by 30 µM felodipine or bepridil remained partially. A similar inhibition pattern of the drugs was found in the calmodulin binding experiments. Within a concentration range of 3 to 30 µM, all three drugs had negligible effects on the basal Ca2+ pumping ATPase which was measured in calmodulin-depleted sarcolemma. In conclusion, the characteristics of the anti-calmodulin action of felodipine on the rabbit heart sarcolemmal Ca2+ pumping ATPase are not different from those of bepridil. Both drugs may inhibit the enzyme by interference with the Ca2+-stimulated binding of calmodulin.Abbreviations Ca2+ pumping ATPase
Ca2+ stimulated Mg2+-dependent ATP hydrolyzing activity
- Na+ pumping ATPase
Na+-stimulated K+- and Mg2+-dependent ATP hydrolyzing activity
- Tris-maleate
tris (hydroxymethyl) aminomethane hydrogen maleate
- Hepes
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
- Mes
2-(N-morpholino) ethane sulfonic acid and Egta, ethylene glycol bis (p-amino ethylether)-N,N,N,N tetraacetic acid 相似文献
13.
Summary When K+ of high concentration (50 mM) was applied toNitella cells, the cytoplasmic streaming stopped instantly as in the case of electrical stimulation. Recovery of the streaming after chemical stimulation was much slower than after electrical stimulation. When the endoplasm content was modified by centrifugation, streaming recovery was accelerated in the centrifugal cell fragments rich in endoplasm and deccelerated in those poor in it. The recovery was also accelerated either by permeabilizing the plasmalemma in the presence of EGTA in the external solution or by removing the tonoplast by vacuolar perfusion with the EGTA-containing medium. We concluded that the streaming was recovered due to decrease of the cytoplasmic Ca2+ concentration, which seems to be accelerated by sequestering of Ca2+ by endoplasmic components. The slow recovery of the streaming after KCl-stimulated cessation is assumed to be caused by continuous influx of Ca2 + during the prolonged membrane depolarization.Abbreviations ATP
adenosine 5-triphosphoric acid
- EGTA
ethyleneglycol-bis-(-aminoethyl ether)N,N-tetraacetic acid
- PIPES
piperazine-N,N-bis(2-ethanesulfonic acid) 相似文献
14.
D. Rao Sanadi James B. Hughes Saroj Joshi 《Journal of bioenergetics and biomembranes》1981,13(5-6):425-431
Addition of Cd2+ or phenylarsine oxide (PhAsO) to respiring rat liver mitochondria results first in acidification of the medium (H+ efflux) followed by disappearance of H+ (discharge of the pH gradient or uncoupling). The first phase of H+ efflux is dependent upon the presence of K+ in the medium, and is not seen in the presence of valinomycin, which is consistent with the conclusion that H+ efflux is linked to membrane potential-dependent uptake of K+. These effects are abolished by low levels of 2,3-dimercaptopropanol but potentiated by excess of 2-mercaptoethanol, showing involvement of a dithiol type of group in the response. Mersalyl produces only the H+ efflux, and subsequent addition of Cd2+ or PhAsO produces collapse of the pH.Abbreviations BAL
British Anti-Lewisite or 2,3-dimercaptopropanol
- 2-ME
2-mercaptoethanol
- PhAsO
phenylarsine oxide
- FCCP
carbonylcyanide trifluoromethoxyphenylhydrazone
- HEPES
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid 相似文献
15.
Servé W. M. Kengen Judith J. Mosterd Rob L. H. Nelissen Jan T. Keltjens Chris van der Drift Godfried D. Vogels 《Archives of microbiology》1988,150(4):405-412
The enzymatic conversion of formaldehyde to CH3S-CoM in crude extracts of Methanobacterium thermoautotrophicum was used as a means to investigate the methyl-tetrahydromethanopterin: HS-CoM methyltransferase reaction. All components necessary for formaldehyde conversion were shown to be present in a soluble protein fraction. This soluble cell fraction still contained a major amount of corrinoids. Apart from tetrahydromethanopterin no other soluble cofactors were required for formaldehyde conversion. The dependence of the system on catalytic amounts of ATP was shown to be specific. Several nucleoside triphosphates or ADP were unable to substitute for ATP. Remarkably, various strong reducing systems, especially titanium(III)citrate could replace ATP to a large extent. The ATP-dependent formaldehyde conversion to CH3S-CoM was inhibited in the presence of nitrous oxide, detergents or 2,3-dialdehyde-ATP. The results support a role for a corrinoid protein in the methyl-tetrahydromethanopterin: HS-CoM methyltransferase reaction at which ATP is involved in the activation of this protein, probably in the conversion of inactive B12a or B12r to active B12s.Abbreviations HS-CoM
Coenzyme M, 2-mercaptoethanesulfonate
- CH3S-CoM
methylcoenzyme M, 2-(methylthio)ethanesulfonate
- H4MPT
5,6,7,8-tetrahydromethanopterin
- BES
2-bromoethanesulfonate
- BCE
boiled cell-free extract
- DTT
dithiothreitol
- TCS
3,3,4,5-tetrachlorosalicylanilide
- DNTB
2,2-dinitro-5,5-dithiobenzoic acid
- TES
N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate
- HEPES
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
- PIPES
piperazine-N,N-bis[2-ethanesulfonic acid]
- AMP-PNP
5-adenylyl imidophosphate 相似文献
16.
P. A. Dibrov R. L. Lazarova V. P. Skulachev M. L. Verkhovskaya 《Journal of bioenergetics and biomembranes》1989,21(3):347-357
Addition of Na+ to the K+-loadedVibrio alginolyticus cells, creating a 250-fold Na+ gradient, is shown to induce a transient increase in the intracellular ATP concentration, which is abolished by the Na+/H+ antiporter, monensin. The pNa-supported ATP synthesis requires an additional driving force supplied by endogenous respiration or, alternatively, by a K+ gradient (high [K+] inside). In the former case, ATP formation is resistant to the protonophorous uncoupler. Dicyclohexylcarbodiimide and diethylstilbestrol, but not vanadate, completely inhibit Na+ pulse-induced ATP formation. The data agree with the assumption that Na+-ATP-synthase is involved in oxidative phosphorylation inV. alginolyticus. Interrelation of H+ and Na+ cycles in bacteria is discussed.Abbreviations
and
electrochemical gradients of H+ and Na+, respectively
-
transmembrane electric potential difference
- pH, pNa, and pK
concentration gradients of H+, Na+, and K+, respectively
- CCCP
carbonyl cyanidem-chlorophenylhydrazone
- DCCD
N,N-dicyclohexylcarbodiimide
- DES
diesthylstilbestrol
- HQNO
2-heptyl-4-hydroxyquinolineN-oxide
- Tricine
N[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine 相似文献
17.
Phosphorylation of the plasma-membrane H+-ATPase of oat roots by a calcium-stimulated protein kinase
When plasma-membrane vesicles isolated from oat (Avena sativa L.) root cells were incubated with [-32P]ATP, the H+-ATPase was found to be phosphorylated at serine and threonine residues. Phosphotyrosine was not detected. Endogenous ATPase kinase activity was also observed in plasma-membrane vesicles isolated from potato (Solanum tuberosum L.) root cells as well as from yeast (Saccharomyces cerevisiae). Identity of the phosphorylated oat root Mr=100 000 polypeptide as the ATPase was confirmed using conventional glycerol density-gradient centrifugation to purify the native enzyme and by a new procedure for purifying the denatured polypeptide using reversephase high-performance liquid chromatography. Kinase-mediated phosphorylation of the oat root plasma-membrane H+-ATPase was stimulated by the addition of low concentrations of Ca2+ and by a decrease in pH, from 7.2 to 6.2. These results demonstrate that kinase-mediated phosphorylation of the H+-ATPase is a plausible mechanism for regulating activity. They further indicate that changes in the cytoplasmic [Ca2+] and pH are potentially important elements in modulating the kinase-mediated phosphorylation.Abbreviations EDTA
ethylenediaminetetraacetic acid
- EGTA
ethylene glycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid
- Mr
relative molecular mass
- RP-HPLC
reverse-phase high-performance liquid chromatography
- SDS-PAGE
sodium dodecyl sulfate polyacrylamide gel electrophoresis 相似文献
18.
D. Siebers C. Lucu K. Böttcher K. Jürss 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1994,164(1):16-22
Isolated posterior gills (no. 7) of shore crabsCarcinus maenas acclimated to brackish water of a salinity of 10 S were bathed and perfused with 50% sea water (200 mmol·l-1 Na+), and the internal perfusate collected during subsequent periods of 5 min. During a single passage through the gill the pH of the perfusion medium decreased from ca. 8.1 to ca. 7.7, a result implying that the gill possesses structures which recognize unphysiologically high pH values in the haemolymph and regulates them down to physiological values of ca. 7.7. The calculated apparent proton fluxes from the epithelial cells into the haemolymph space amounted to 17.9 mol·g fw-1·h-1, a value of only 3.8% of net Na+ fluxes observed under comparable conditions. When 0.1 mmol·l-1 KCN, an inhibitor of mitochondrial cytochrome oxidase, or 5 mmol·l-1 ouabain, a specific inhibitor of Na+/K+-ATPase were applied in the internal perfusate, down-regulation of pH was no longer observed and the gill was completely depolarized, i.e. transepithelial potential differences dropped from-7.8 to 0 mV (haemolymph space negative to bath). Regulation of pH was completely inhibited by antagonists of carbonic anhydrase (0.1 mmol·l-1 acetazolamide or 0.01 mmol·l-1 ethoxyzolamide) applied in the perfusate. Inhibitors of Na+/H+ exchange, 0.1 mmol·l-1 amiloride applied in the external bathing medium or in the internal perfusate, and symmetrical 0.01 mmol·l-1 5-(N-ethyl-N-isopropyl)amiloride, as well as inhibitors of Cl-/HCO3
- exchange and Na+/HCO3
- cotransport, 0.5 mmol·l-1 4,4-diisothiocyanatostilbene-2,2-disulphonate or 0.3 mmol·l-1 4-acetamido-4-isothiocyanatostilbene 2,2-disulphonate applied on both sides of the gill, and inhibitors of H+-ATPase, 0.05 mmol·l-1
N-ethylmaleimide and 0.1 mmol·l-1
N,N-dicyclohexylcarbodiimide —applied on both sides of the gill — did not alter the acidification of the perfusate observed in controls. Using artificial salines buffered to pH 8.1 with 0.75 mmol·l-1 tris (hydroxymethyl) aminomethane instead of 2 mmol·l-1 HCO3
-, apparent proton fluxes were reduced to 11% of controls, a result suggesting that pH regulation by crab gills needs the presence of HCO3
-. The findings obtained suggest that pH regulation by crab gills depends on the oxidative metabolism of the intact branchial epithelium and that carbonic anhydrase plays a central role in this process. Na+/H+ exchange, anion exchange or cotransport and active proton secretion seem not to be involved. While unimpaired active ion uptake is a prerequisite for pH regulation, ion transport itself is independent of it.Abbreviations acetazolamide
(N-[sulphamoyl-1, 3, 4-thiadiazol-2-yl]-acetamide)
- amiloride
3,5-diamino-6-chloropyrazinoyl-guanidine
- CA
carbonic anhydrase
- DBI
dextrane-bound inhibitor thiadiazolesulphonamide
- DCCD N
N dicyclohexylcarbodiimide
- DIDS
4,4-diisothiocyanato-stilbene-2,2-disulphonate
- EIPA
5-(N-ethyl-N-isopropyl) amiloride
- ethoxyzolamide
6-ethoxy-2-benzothiazole-sulphonamide
- fw
fresh weight
-
J
H
+
apparent proton flux
- NEM
N-ethylmaleimide
- PD
transepithelial potential difference
- PEG-STZ
polyethylene-glycol-thiadiazolesulphonamide
- STTS
4-acetamido-4-isothiocyanatostibene 2,2-disulphonate
- SW
sea water
- TRIS
tris(hydroxymethyl)aminomethane 相似文献
19.
Summary Microtubules inNicotiana tabacum pollen tube subprotoplasts reassembled in wave-like to concentric cortical arrays. Crosslinks between microtubules were either 15 or 80 nm in length. Cortical actin filaments showed different distributions; no colocalization like that in pollen tubes was observed. Degradation of actin filaments by cytochalasin D had no influence on microtubule organization. Degradation of microtubules and/or actin filaments did not affect outgrowth of the subprotoplasts. Organization of the microtubules occurred independent of the presence of the generative cell and/or the vegetative nucleus. No relation of actin filament and microtubule organization with organelle distribution could be detected.Abbreviations AFs
actin filaments
- DAPI
4,6-diamidino-2-phenylindole
- EGTA
ethylene glycol bis (2-amino ethylether) N,N,N,N-tetraacetic acid
- FITC
fluorescein isothiocyanate
- MTs
microtubules
- SPPs
subprotoplasts
- TRITC
tetramethyl rhodamine B isothiocyanate 相似文献
20.
N. C. Jørgensen 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1995,165(6):450-457
Simultaneous net uptake of Na+ and net extrusion of H+, both inhibited by amiloride, could be stimulated in red blood cells of the frog, Rana temporaria, either by intracellular acidification or cellular shrinkage. Net transports of Na+ and H+ were transient, dying out after 10–20 min (20°C) when stimulated by intracellular acidification but developing more slowly and proceeding for more than 60 min (20°C) when stimulated by cellular shrinkage. Evidence is presented suggesting a coupling between the transports of Na+ and H+ with an exchange ratio of 1:1 Na+/H+ exchange, stimulated by intracellular acidification, was able to readjust intracellular pH also when operating in parallel to a fully working anion exchanger in CO2/HCO
3
-
-buffered media. Inhibition of anion exchange resulted in reduced cellular net uptake of Na+.Abbreviations
DIDS
4,4-diisothiocyanatostilbene-2,2-disulphonate
-
DMSO
dimethylsulphoxide
-
IU
international unit
-
pH
e
extracellular pH
-
pH
i
intracellular pH
-
RBC
red blood cell 相似文献