共查询到20条相似文献,搜索用时 15 毫秒
1.
In this paper a detailed study of the effect of nitration of tyrosine residues by tetranitromethane on H+ conduction and other reactions catalyzed by the H+-ATPase complex in phosphorylating submitochondrial particles, uncoupled particles, and the purified complex is presented. Tetranitromethane treatment of submitochondrial particles results in marked inhibition of ATP hydrolysis, ATP-33Pi exchange, and proton conduction by the H+-ATPase complex. These effects are caused by nitration of tyrosine residues of H+-ATPase complex as shown by the appearance of the absorption peak at 360 nm (specific for nitrotyrosine formation) and inhibition of ATP hydrolysis and ATP-33Pi exchange in the complex purified from tetranitromethane-treated particles. H+ conduction in phospholipid vesicles inlaid with F0 is also inhibited by tetranitromethane treatment. These observations indicate that tyrosine residue(s) of F0 are critically involved in energy-linked proton translocation in the ATP-ase complex. 相似文献
2.
Sodium-transloating ATPase in the fermentative bacteriumStreptococcus faecalis exchanges sodium for potassium ions. Sodium ions stimulate its activity, but K+ ions have no significant effect at present. Although the molecular nature of the sodium ATPase is not clear, the enzyme is distinct from other ion-motive ATPases (E1E2 type and F1F0 type) as judged by its resistance to vanadate as well as dicyclohexylcarbodiimde. The sodium ATPase is induced when cells are grown on media rich in sodium, particularly under conditions that limit the generation of a proton potential or block the constitutive sodium/proton antiporter, indicating that an increase in the cytoplasmic sodium level serves as the signal. The enzyme is not induced in response to K+ deprivation. The sodium ATPase may have evolved to cope with a sodium-rich environment under conditions that limit the magnitude of the proton potential. 相似文献
3.
The membrane sector (F0) of H+-ATPase was prepared by trypsin and urea treatment of F1-F0 and reconstituted with purified F1. The oligomycin sensitivity of the reconstituted F1-F0 complex obtained by treating F1 or F0 with Mg2+ before binding is much higher than that obtained without Mg2+ treatment. The greater change in the intrinsic fluorescence of the reconstituted F1-F0 complex obtained by Mg2+ treatment suggests that conformational changes may occur during the reconstitution. We deduce that Mg2+ binds to membrane lipids, thus decreasing membrane fluidity and changing the physical state of the lipids to provide a suitable microenvironment for conformational changes in F0. The data also suggest that the conformational change in the F0 portion of the F1-F0 complex can be transmitted to the F1 portion, the conformation of which is in turn altered, resulting in the formation of an F1-F0 complex with high oligomycin sensitivity. On the other hand, Mg2+ may act on F1 directly to induce a suitable conformational change which is then trnsmitted to F0, resulting in the formation of an H+-ATPase with greater sensitivity to oligomycin.Abbreviations STED
0.25 M sucrose, 10 mM Tris-SO4, 0.2 mM EDTA, and 1 mM dithiothreitol, pH 8.0
- NADH
nicotinamide adenine dinucleotide, reduced form
- olig.
oligomycin
- OSCP
oligomycin sensitivity conferring protein
- F6
coupling factor 6
- F1
coupling factor one (or F1-ATPase)
- F1
+Mg
2+ and F1
–Mg
2+
the F1 treated and untreated with 1 mM Mg2+ respectively
- F0
the membrane sector proteins of the H+-ATPase
- TUF0
trypsin-urea – F0
- EUF0
EDTA-urea – F0
- F0
+Mg
2+ and F0
–Mg
2+
the F0 treated and untreated with 1 mM Mg2+ respectively
- (F1 · F0)+Mg
2+ and (F1 · F0)–Mg
2+
the reconstituted F1 · F0 complex containing Mg2+-treated F1 and F0 and untreated F1 and F0 respectively
- F1 · F0
+Mg
2+ and F1 · F0
–Mg
2+
the reconstituted H+-ATPase complex derived from the binding of purified F1 to the F0 treated and untreated with Mg2+ respectively
- F1
+Mg
2+ · F0 and F1
–Mg
2+ · F0
the reconstituted H+-ATPase derived from the binding of F0 to the purified F1 treated and untreated with Mg2+ respectively 相似文献
4.
Renoguanylin (REN) is a recently described member of the guanylin family, which was first isolated from eels and is expressed in intestinal and specially kidney tissues. In the present work we evaluate the effects of REN on the mechanisms of hydrogen transport in rat renal tubules by the stationary microperfusion method. We evaluated the effect of 1 μM and 10 μM of renoguanylin (REN) on the reabsorption of bicarbonate in proximal and distal segments and found that there was a significant reduction in bicarbonate reabsorption. In proximal segments, REN promoted a significant effect at both 1 and 10 μM concentrations. Comparing control and REN concentration of 1 μM, JHCO3−, nmol cm− 2 s− 1 − 1,76 ± 0,11control × 1,29 ± 0,08REN 10 μM; P < 0.05, was obtained. In distal segments the effect of both concentrations of REN was also effective, being significant e.g. at a concentration of 1 μM (JHCO3−, nmol cm− 2 s− 1 − 0.80 ± 0.07control × 0.60 ± 0.06REN 1 μM; P < 0.05), although at a lower level than in the proximal tubule. Our results suggest that the action of REN on hydrogen transport involves the inhibition of Na+/H+exchanger and H+-ATPase in the luminal membrane of the perfused tubules by a PKG dependent pathway. 相似文献
5.
猪心线粒体Fo的纯化、重建及其质子转运功能 总被引:1,自引:0,他引:1
比较了猪心线粒体FoF1-ATPase膜部分Fo的四种纯化方法.结果表明,用NaBr从亚线粒体除去FoF1-ATPase的水溶性部分F1-ATPase后,再以CHAPS增溶,并经蔗糖梯度离心,可获得高纯度的Fo.SDS-聚丙烯酰胺凝胶电泳鉴定表明,纯化的Fo含有b、OSCP(寡霉素敏感授予蛋白)、d、a、e、F6、IF1、A6L和c等9种亚基.用去污剂稀释法将纯化的Fo在脂质体上重建后,重建Fo表现较高的被动转运质子活性.这为在体外深入研究Fo的活性、构象与膜脂的关系,以及Fo与F1-ATPase的组装等提供了很好的实验模型. 相似文献
6.
The effect of chemical modifiers of amino acid residues on the proton conductivity of H+-ATPase in inside out submitochondrial particles has been studied. Treatment of submitochondrial particles prepared in the presence of EDTA (ESMP) with the arginine modifiers, phenylglyoxal or butanedione, or the tyrosine modifier, tetranitromethane, caused inhibition of the ATPase activity. Phenylglyoxal and tetranitromethane also caused inhibition of the anaerobic release of respiratory H+ in ESMP as well as in particles deprived of F1 (USMP). Butanedione treatment caused, on the contrary, acceleration of anaerobic proton release in both particles. The inhibition of proton release caused by phenylglyoxal and tetranitromethane exhibited in USMP a sigmoidal titration curve. The same inhibitory pattern was observed with oligomycin and withN,N-dicyclohexylcarbodiimide. In ESMP, relaxation of H+ exhibited two first-order phases, both an expression of the H+ conductivity of the ATPase complex. The rapid phase results from transient enhancement of H+ conduction caused by respiratory H+ itself. Oligomycin,N,N-dicyclohexylcarbodiimide, and tetranitromethane inhibited both phases of H+ release, and butanedione accelerated both. Phenylglyoxal inhibited principally the slow phase of H+ conduction. In USMP, H+ release followed simple first-order kinetics. Oligomycin depressed H+ release, enhanced respiratory H+, and restored the biphasicity of H+ release. Phenylglyoxal and tetranitromethane inhibited H+ release in USMP without modifying its first-order kinetics. Butanedione treatment caused biphasicity of H+ release from USMP, introducing a very rapid phase of H+ release. Addition of soluble F1 to USMP also restored biphasicity of H+ release. A mechanism of proton conduction by F
o
is discussed based on involvement of tyrosine or other hydroxyl residues, in series with the DCCD-reactive acid residue. There are apparently two functionally different species of arginine or other basic residues: those modified by phenylglyoxal, which facilitate H+ conduction, and those modified by butanedione, which retard H+ diffusion. 相似文献
7.
Robert H. Fillingame 《Journal of bioenergetics and biomembranes》1992,24(5):485-491
The F0 sector of the ATP synthase complex facilitates proton translocation through the membrane, and via interaction with the F1 sector, couples proton transport to ATP synthesis. The molecular mechanism of function is being probed by a combination of mutant analysis and structural biochemistry, and recent progress on theEscherichia coli F0 sector is reviewed here. TheE. coli F0 is composed of three types of subunits (a, b, andc) and current information on their folding and organization in F0 is reviewed. The structure of purified subunitc in chloroform-methanol-H2O resembles that in native F0, and progress in determining the structure by NMR methods is reviewed. Genetic experiments suggest that the two helices of subunitc must interact as a functional unit around an essential carboxyl group as protons are transported. In addition, a unique class of suppressor mutations identify a transmembrane helix of subunita that is proposed to interact with the bihelical unit of subunitc during proton transport. The role of multiple units of subunitc in coupling proton translocation to ATP synthesis is considered. The special roles of Asp61 of subunitc and Arg210 of subunita in proton translocation are also discussed. 相似文献
8.
Malyan AN 《Photosynthesis research》2006,88(1):9-18
A modified ‘cold chase’ technique was used to study tight [14C]ADP and [14C]ATP binding to noncatalytic sites of chloroplast ATP synthase (CF0F1). The binding was very low in the dark and sharply increased with light intensity. Dissociation of labeled nucleotides incorporated into noncatalytic sites of CF0F1 or CF1 reconstituted with EDTA-treated thylakoid membranes was also found to be light-dependent. Time dependence of nucleotide dissociation is described by the first order equation with a k
d of about 5 min−1. The exposure of thylakoid membranes to 0.7–24.8 μM nucleotides leads to filling of up to two noncatalytic sites of CF0F1. The sites differ in their specificity: one preferentially binds ADP, whereas the other – ATP. A much higher ATP/ADP ratio of nucleotides bound at noncatalytic sites of isolated CF1 dramatically decreases upon its reconstitution with EDTA-treated thylakoid membranes. It is suggested that the decrease is caused by conformational changes in one of the α subunits induced by its interaction with the δ subunit and/or subunit I–II when CF1 becomes bound to a thylakoid membrane. 相似文献
9.
ATPases with unusual membrane-embedded rotor subunits were found in both F1F0 and A1A0 ATP synthases. The rotor subunit c of A1A0 ATPases is, in most cases, similar to subunit c from F0. Surprisingly, multiplied c subunits with four, six, or even 26 transmembrane spans have been found in some archaea and these multiplication events were
sometimes accompanied by loss of the ion-translocating group. Nevertheless, these enzymes are still active as ATP synthases.
A duplicated c subunit with only one ion-translocating group was found along with “normal” F0 c subunits in the Na+ F1F0 ATP synthase of the bacterium Acetobacterium woodii. These extraordinary features and exceptional structural and functional variability in the rotor of ATP synthases may have
arisen as an adaptation to different cellular needs and the extreme physicochemical conditions in the early history of life. 相似文献
10.
Summary [14C]Phthalate is transported into L1210 cells via two separate routes, an anion exchange system whose primary substrates are folate compounds, and a second less active system which is sensitive to bromosulfophthalein. When the principal uptake component was blocked by a specific irreversible inhibitor of this system, the remaining route (at pH 7.4) appeared to be saturable and was inhibited by several anions in addition to bromosulfophthalein (K
i
=2 m), including 8-anilino-1-naphthalein sulfonate (K
i
=25 m), unlabeled phthalate (K
i
=500 m), and chloride (K
i
=3500 m). A pronounced effect by pH was also observed. Influx and total uptake of phthalate both increased progressively with decreasing pH and reached values that were 20-fold higher at pH 6.0, compared with pH 7.4. This pH-dependent increase could be blocked, however, by the addition of compounds (nigericin and carbonylcyanidem-chlorophenylhydrazone) which, in combination, collapse proton gradients. Phthalate efflux was relatively insensitive to changes in extracellular pH but could be inhibited (up to 90%) by bromosulfophthalein. Several other anions also inhibited efflux, but to a lesser extent, while chloride, phthalate, lactate, glycolate and acetate enhanced efflux up to 1.8-fold. Efflux also increased at pH 6.0, but not at pH 7.5, upon addition of nigericin and carbonylcyanidem-chlorophenylhydrazone. These results suggest that phthalate is a nonphysiological substrate for a carrier system which mediates transport via an anion/H+ symport mechanism. This system is not the lactate/H+ symport carrier of L1210 cells since: (A) phthalate and lactate influx were inhibited to differing degrees by various anions; and (B) lactic anhydride inhibited the influx and efflux of lactate but had no effect on the transmembrane movement of phthalate. The specificity of this system suggests that its primary anion substrate may be chloride. 相似文献
11.
David G. Griffiths Michael J. Pringle James B. Hughes D. Rao Sanadi 《Journal of bioenergetics and biomembranes》1984,16(5-6):465-475
Electron transport particles and purified H+-ATPase (F1-Fo) vesicles from beef heart mitochondria have been treated with two classes of thiol reagent, viz. membrane-impermeable organomercurials and a homologous series ofN-polymethylene carboxymaleimides (Mal-(CH2)
x
-COOH or AMx). The effect of such treatment on ATP-driven reactions (ATP-Pi exchange and proton translocation) has been examined and compared to the effects on rates of ATP hydrolysis. The organomercurials inhibited ATP-Pi exchange and one of them (p-chloromercuribenzoate) inhibited ATPase activity. Of the maleimide series (AMx), AM10 and AM11 inhibited both ATP-Pi exchange and ATP-driven membrane potential, but not ATPase activity. The other members of the series were essentially inactive.N-Ethylmaleimide was intermediate in its efficacy. Passive H+ conductance through the membrane sector Fo was 50% blocked by AM10, slightly blocked by AM2 andN-ethylmaleimide, and unaffected by the other members of the AMx series. The data imply that one -SH near the membrane surface and one -SH about 12 Å from the surface are functional in proton translocation through the H+-ATPase. 相似文献
12.
The ATP hydrolysis rate and the ATP hydrolysis-linked proton translocation by the F0F1-ATPase of beef heart submitochondrial particles were examined in the presence of several divalent metal cations. All Me–ATP complexes tested sustained ATP hydrolysis, although to a different extent. However, only Mg- and Mn-ATP-dependent hydrolysis could sustain a high level of proton pumping activity, as determined by acridine fluorescence quenching. Moreover, the K
m of the Me-ATP hydrolysis-induced proton pumping activity was very similar to the K
m value of Me-ATP hydrolysis. Both oligomycin and DCCD caused the full recovery of the fluorescence, providing clear evidence for the association of Mg-ATP hydrolysis with proton translocation through the F0F1-ATPase complex. In contrast, with other Me-ATP complexes, including Ca-ATP as substrate, the proton pumping activity was undetectable, implicating an uncoupling nature for these substrates. Attempts to demonstrate the involvement of the subunit of the enzyme in the coupling mechanism failed, suggesting that the participation of at least the N-terminal segment of the subunit in the coupling mechanism of the mitochondrial enzyme is unlikely. 相似文献
13.
Van Walraven HS Scholts MJ Zakharov SD Kraayenhof R Dilley RA 《Journal of bioenergetics and biomembranes》2002,34(6):455-464
It was shown before (Wooten, D. C., and Dilley, R. A. (1993) J. Bioenerg. Biomembr. 25, 557–567; Zakharov, S. D., Li, X., Red'ko, T. P., and Dilley, R. A. (1996) J. Bioenerg. Biomembr. 28, 483–493) that pH dependent reversible Ca2+ binding near the N- and C-terminal end of the 8 kDa subunit c modulates ATP synthesis driven by an applied pH jump in chloroplast and E. coli ATP synthase due to closing a proton gate proposed to exist in the F0 H+ channel of the F0F1 ATP synthase. This mechanism has further been investigated with the use of membrane vesicles from mutants of the cyanobacterium Synechocystis 6803. Vesicles from a mutant with serine at position 37 in the hydrophilic loop of the c-subunit replaced by the charged glutamic acid (strain plc 37) has a higher H+/ATP ratio than the wild type and therefore shows ATP synthesis at low values of
H
+. The presence of 1 mM CaCl2 during the preparation and storage of these vesicles blocked acid–base jump ATP formation when the pH of the acid side (inside) was between pH 5.6 and 7.1, even though the pH of the acid–base jump was thermodynamically in excess of the necessary energy to drive ATP formation at an external pH above 8.28. That is, in the absence of added CaCl2, ATP formation did occur under those conditions. However, when the base stage pH was 7.16 and the acid stage below pH 5.2, ATP was formed when Ca2+ was present. This is consistent with Ca2+ being displaced by H+ ions from the F0 on the inside of the thylakoid membrane at pH values below about 5.5. Vesicles from a mutant with the serine of position 3 replaced by a cysteine apparently already contain some bound Ca2+ to F0. Addition of 1 mM EGTA during preparation and storage of those vesicles shifted the otherwise already low internal pH needed for onset of ATP synthesis to higher values when the external pH was above 8. With both strains it was shown that the Ca2+ binding effect on acid–base induced ATP synthesis occurs above an internal pH of about 5.5. These results were corroborated by 45Ca2+- ligand blot assays on organic solvent soluble preparations containing the 8 kDa F0 subunit c from the S-3-C mutant ATP synthase, which showed 45Ca2+ binding as occurs with the pea chloroplast subunit III. The phosphorylation efficiency (P/2e), at strong light intensity, of Ca2+ and EGTA treated vesicles from both strains were almost equal showing that Ca2+ or EGTA have no other effect on the ATP synthase such as a change in the proton to ATP ratio. The results indicate that the Ca2+ binding to the F0 H+ channel can block H+ flux through the channel at pH values above about 5.5, but below that pH protons apparently displace the bound Ca2+, opening the CF0 H+ channel between the thylakoid lumen and H+ conductive channel. 相似文献
14.
José de JesÚs Garcia M. Tuena de Gómez-Puyou A. Gómez-Puyou 《Journal of bioenergetics and biomembranes》1995,27(1):127-136
The effect of trifluoperazine (TFP) on the ATPase activity of soluble and paniculate F1ATPase and on ATP synthesis driven by succinate oxidation in submitochondrial particles from bovine heart was studied at pH 7.4 and 8.8. At the two pH. TFP inhibited ATP hydrolysis. Inorganic phosphate protected against the inhibiting action of TFP. The results on the effect of various concentrations of phosphate in the reversal of the action of TFP on hydrolysis at pH 7.4 and 8.8 showed that H2PO
4
–
is the species that competes with TFP. The effect of TFP on oxidative phosphorylation was studied at concentrations that do not produce uncoupling or affect the aerobic oxidation of succinate (<15M). TFP inhibited oxidative phosphorylation to a higher extent at pH 8.8 than at pH 7.4; this was through a diminution in theV
max, and an increase in theK
m for phosphate. Data on phosphate uptake during oxidative phosphorylation at several pH showed that H2PO
4
–
is the true substrate for oxidative phosphorylation. Thus, in both synthesis and hydrolysis of ATP, TFP and H2PO
4
–
interact with a common site. However, there is a difference in the sensitivity to TFP of ATP synthesis and hydrolysis; this is more noticeable at pH 8.8, i.e. ATPase activity of soluble F1 remains at about 40% of the activity of the control in a concentration range of TFP of 40–100M, whereas in oxidative phosphorylation 14M TFP produces a 60% inhibition of phosphate uptake. 相似文献
15.
Gotthold Gäbel Susanne Vogler Holger Martens 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1991,161(4):419-426
Summary Unidirectional 22Na+ and 36Cl– fluxes were determined in short-circuited, stripped rumen mucosa from sheep by using the Ussing chamber technique. In both CO2/HCO
–
3
-containing and CO2/HCO
–
3
-free solutions, replacement of gluconate by short-chain fatty acids (SCFA, 39 mM) significantly enhanced mucosal-toserosal Na+ absorption without affecting the Cl– transport in the same direction. Short-chain fatty acid stimulation of Na+ transport was at least partly independent of Cl– and could almost completely be abolished by 1 mM mucosal amiloride, while stimulation of Na+ transport was enhanced by lowering the mucosal pH from 7.3 to 6.5. Similar to the SCFA action, raising the PCO2 in the mucosal bathing solution led to an increase in the amiloride-sensitive mucosal-to-serosal Na+ flux. Along with its effect on sodium transport, raising the PCO2 also stimulated chloride transport. The results are best explained by a model in which undissociated SCFA and/or CO2 permeate the cell membrane and produce a raise in intracellular H+ concentration. This stimulates an apical Na+/H+ exchange, leading to increased Na+ transport. The stimulatory effect of CO2 on Cl– transport is probably mediated by a Cl–/HCO
–
3
exchange mechanism in the apical membrane. Binding of SCFA anions to that exchange as described for the rat distal colon (Binder and Mehta 1989) probably does not play a major role in the rumen.Abbreviations
DIDS
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid
-
G
t
transepithelial conductance (mS·cm-2)
-
HSCFA
undissociated short-chain fatty acids
-
J
ms
mucosal-to-serosal flux (Eq · cm-2 · h-1)
-
J
net
net flux (Eq · cm-2 · h-1)
-
J
sm
serosal-to-mucosal flux (Eq · cm-2 · h-1)
-
PD
transepithelial potential difference (mV)
-
SCFA
dissociated short-chain fatty acids
-
SCFA
short-chain fatty acids 相似文献
16.
Zaitseva L. G. Ovchinnikova T. V. Vodovozova E. L. Molotkovsky Yul. G. Polyakov N. B. Titov M. I. Esipov S. E. Grinkevich V. A. 《Russian Journal of Bioorganic Chemistry》2002,28(5):371-384
The subunits of the F0 membrane sector of bovine heart mitochondrial H+-ATPase that contact the lipids of the mitochondrial inner membrane were identified with the use of specially synthesized proteoliposomes that contained active mitochondrial H+-ATPase and a photoreactive lipid, which was 1-acyl-2-[12-[di-azocyclopentadiene-2-carbonylamino)-[12-14C]dodecanoyl]-sn-glycero-3-phosphocholine, 1-acyl-2-[11-([125I]diazoiodocyclopentadiene-2-carbonyloxy)undecanoyl]-sn-glycero-3-phosphocholine, or 1-acyl-2-[12-(diazocyclopentadiene-2-carbonylamino)dodecanoyl]-sn-glycero-3-phosphocholine, where acyl is a mixture of the residues of palmitic (70%) and stearic (30%) acids. An analysis of the cross-linked products obtained upon the UV-irradiation of these proteoliposomes indicated that subunits c and a of the F0 membrane sector contact the lipids. The crosslinked products were identified by SDS-PAGE and MALDI mass spectrometry. 相似文献
17.
Gutiérrez-Nájera N Muñoz-Clares RA Palacios-Bahena S Ramírez J Sánchez-Nieto S Plasencia J Gavilanes-Ruíz M 《Planta》2005,221(4):589-596
Fumonisin B1 (FB1) is an amphipathic toxin produced by the pathogenic fungus Fusarium verticillioides which causes stem, root and ear rot in maize (Zea mays L.). In this work, we studied the action of FB1 on the plasma membrane H+-ATPase (EC 3.6.1.34) from germinating maize embryos, and on the fluidity and lipid peroxidation of these membranes. In maize embryos the toxin at 40 M inhibited root elongation by 50% and at 30 M decreased medium acidification by about 80%. Irrespective of the presence and absence of FB1, the H+-ATPase in plasma membrane vesicles exhibited non-hyperbolic saturation kinetics by ATPH-Mg, with Hill number of 0.67. Initial velocity studies revealed that FB1 is a total uncompetitive inhibitor of this enzyme with an inhibition constant value of 17.5±1 M. Thus FB1 decreased Vmax and increased the apparent affinity of the enzyme for ATP-Mg to the same extent. Although FB1 increased the fluidity at the hydrophobic region of the membrane, no correlation was found with its effect on enzyme activity, since both effects showed different FB1-concentration dependence. Peroxidation of membrane lipids was not affected by the toxin. Our results suggest that, under in vivo conditions, the plasma membrane H+-ATPase is a potentially important target of the toxin, as it is inhibited not only by FB1 but also by its structural analogs, the sphingoid intermediates, which accumulate upon the inhibition of sphinganine N-acyltransferase by this toxin. 相似文献
18.
Structure and function of the membrane-integral components of the mitochondrial H+-ATPase 总被引:1,自引:0,他引:1
J. Houštěk J. Kopecký P. Svoboda Z. Drahota 《Journal of bioenergetics and biomembranes》1982,14(1):1-13
Conclusions Based on our present knowledge about the composition of mitochondrial F0, it is evident that its mode of interaction with F1 is more complex in comparison with bacteria and chloroplasts. As far as the H+-channel is concerned, no definite conclusion about the involvement of other subunits besides the DCCD-binding protein can be drawn at present. This holds for mitochondria as well as for chloroplasts and bacteria. Experimental evidence is accumulating in favor of the oligomeric and asymmetrical arrangement of the H+-channel. Extraction of its few polar amino acid residues by specific agents reveals the fundamental functional importance of these residues in the path of protons across the membrane. In particular, the use of DCCD was of primary importance for elucidation of the structural features underlying the protonophoric activity. It may be hoped that application of similar new approaches in combination with studies of the intact phosphorylating assembly will help us to clarify the molecular mechanism of ATP synthesis.Abbreviations DCCD
N,N-dicyclohexylcarbodiimide
- SDS
sodium dodecyl sulfate 相似文献
19.
Sandbichler AM Pelster B 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2004,174(8):601-610
Mechanisms of acid release and intracellular pH (pHi) homeostasis were analysed in goldfish (Carassius auratus) gill cells in primary culture. The rate of acid secretion was measured using a cytosensor microphysiometer, and pHi was determined using the fluorescent probe 2,7-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein (BCPCF). Amiloride, a Na+ channel and Na+/H+ exchanger (NHE) inhibitor, had no effect on pHi, but acid secretion of the gill cells was significantly impaired. In the presence of amiloride, the intracellular acidification (achieved using the NH4Cl pulse technique) was more severe than in the absence of amiloride, and recovery from the acidosis was slowed down. Accordingly, acid secretion of gill cells was severely reduced in the absence of extracellular Na+. Under steady-state conditions, 4,4-diisothiocyanatodihydro-stilbene-2,2-disulfonic acid (DIDS), a HCO3–-transport inhibitor, caused a slow acidification of pHi, and acid secretion was significantly reduced. No recovery from intracellular acidification was observed in the presence of DIDS. Bafilomycin A1, an inhibitor of V-ATPase, had no effect on steady-state pHi and recovery from an intracellular acidification, whereas the rate of acid secretion under steady-state conditions was slightly reduced. Immunohistochemistry clearly revealed the presence of the V-ATPase B-subunit in goldfish gill lamellae. Taken together, these results suggest that a Na+-dependent HCO3– transport is the dominant mechanism besides an NHE and V-ATPase to control pHi in goldfish gill cells.Communicated by G. Heldmaier 相似文献
20.
Yoshitaka Saito Terutaka Ozawa Akinori Nishiyama 《The Journal of membrane biology》1987,98(2):135-144
Summary In the isolated, superfused mouse lacrimal gland, intracellular Na+ activities (aNa
i
) of the acinar cells were directly measured with double-barreled Na+-selective microelectrodes. In the nonstimulated conditionaNa
i
was 6.5±0.5 mM and membrane potential (V
m
) was –38.9±0.4 mV. Addition of 1 mM ouabain or superfusion with a K+-free solution slightly depolarized the membrane and caused a gradual increase inaNa
i
. Stimulation with acetylcholine (ACh, 1 M) caused a membrane hyperpolarization by about 20 mV and an increase inaNa
i
by about 9 mM in 5 min. The presence of amiloride (0.1 mM) reduced the ACh-induced increase inaNa
i
by approximately 50%, without affectingV
m
and input resistance in both nonstimulated and ACh-stimulated conditions. Acid loading the acinar cells by an addition/withdrawal of 20 mM NH4Cl or by replacement of Tris+-buffer saline solution with HCO
3
–
/CO2-buffered solution increasedaNa
i
by a few mM. Superfusion with a Cl–-free NO
3
–
solution or 1 mM furosemide or 0.5 mM bumetanide-containing solution had little effect on the restingaNa
i
levels, however, it reduced the ACh-induced increase inaNa
i
by about 30%. Elimination of metabolite anions (glutamate, fumarate and pyruvate) from the superfusate reduced both the restingaNa
i
and the ACh-induced increase inaNa
i
.The present results suggest the presence of multiple Na+ entry mechanisms activated by ACh, namely, Na+/H+ exchange, Na-K-Cl cotransport and organic substrate-coupled Na+ transport mechanisms. 相似文献