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1.
Vincent C. K. Chiu Donald Mouring Brant D. Watson Duncan H. Haynes 《The Journal of membrane biology》1980,56(2):121-132
Summary The binding of the anionic fluorescent probe 1-anilino-8-naphthalene-sulfonate (ANS–) was used to estimate the surface potential of fragmented sarcoplasmic reticulum (SR) derived from rabbit skeletal muscle. The method is based on the observation that ANS– is an obligatory anion whose equilibrium constant for binding membranes is proportional to the electrostatic function of membrane surface potential, exp(e0/kT, where 0 is the membrane surface potential,e is the electronic charge, andkT has its usual meaning. The potential measured is characteristic of the ANS– bindings of phosphatidylcholine head groups and is about one-third as large as the average surface potential predicted by the Gouy-Chapman theory. At physiological ionic strength the surface potentials, measured by ANS–, referred to as the aqueous phase bathing the surface, were in the range –10 to –15 mV. This was observed for the outside and inside surfaces of the Ca2+-ATPase-rich fraction of theSR and for both surfaces of theSR fraction rich in acidic Ca2+ binding proteins. The inside and outside surfaces were differentiated on the basis of ANS– binding kinetics observed in stopped-flow rapid mixing experiments. A mechanism by which changes in Ca2+ concentration could give rise to an electrostatic potential across the membrane and possibly result in changes in Ca2+ permeability.The dependence of the surface potential on the monovalent ion concentration in the medium was used together with the Gouy-Chapman theory to determine the lower limits for the surface charge density for the inside and outside surfaces of the two types ofSR. Values for the Ca2+-ATPase richSR fraction were between 2.9×103 and 3.8×103 esu/cm2, (0.96×10–6 and 1.26×10–6 C/cm2) with no appreciable transmembrane asymmetry. A small amount of asymmetry was observed in the values for the inside and outside surfaces of the fraction rich in acidic binding proteins which were ca. 6.6×103 and ca. 2.2×103 esu/cm2 (2.2×10–6 and 0.73×10–6 C/cm). The values could be accounted for by the known composition of negatively-charged phospholipids in theSR. The acidic Ca2+ binding proteins were shown to make at most a small contribution to the surface charge, indicating that their charge must be located at least several tens of Å from the membrane surface. The experiments gave evidence for a Donnan effect on the K+ distribution in the fraction rich in acidic binding proteins. This could be accounted for by the known concentration of acidic binding proteins in thisSR fraction.The equilibrium constant for ANS– was shown to be more sensitive to changes in the divalent cation concentration than to changes in the monovalent cation concentration, as predicted by the Gouy-Chapman theory. Use of these findings together with the stopped-flow rapid mixing techniques constitutes a method for rapid and continuous monitoring of changes in ion concentrations in theSR lumen. 相似文献
2.
Michael R. Blatt Alonso Rodriguez-Navarro Clifford L. Slayman 《The Journal of membrane biology》1987,98(2):169-189
Summary Active transport of potassium in K+-starvedNeurospora was previously shown to resemble closely potassium uptake in yeast,Chlorella, and higher plants, for which K+ pumps or K+/H+-ATPases had been proposed. ForNeurospora, however, potassium-proton cotransport was demonstrated to operate, with a coupling ratio of 1 H+ to 1 K+ taken inward so that K+, but not H+, moves against its electrochemical gradient (Rodriguez-Navarro et al.,J. Gen. Physiol.
87:649–674).In the present experiments, the current-voltage (I–V) characteristic of K+–H+ cotransport in spherical cells ofNeurospora has been studied with a voltage-clamp technique, using difference-current methods to dissect it from other ion-transport processes in theNeurospora plasma membrane. Addition of 5-200 M K+ to the bathing medium causes 10–150 mV depolarization of the unclamped membrane, and yields a sigmoidI–V curve with a steep slope (maximal conductance of 10–30 S/cm2) for voltages of –300 to –100 mV, i.e., in the normal physiologic range. Outside that range the apparentI–V curve of the K+-H+ symport saturates for both hyperpolarization and depolarization. It fails to cross the voltage axis at its predicted reversal potential, however, an effect which can be attributed to failure of theI–V difference method under reversing conditions.In the absence of voltage clamping, inhibitors—such as cyanide or vanadate—which block the primary proton pump inNeurospora also promptly inhibit K+ transport and K+-H+ currents. But when voltage clamping is used to offset the depolarizing effects of pump blockade, the inhibitors have no immediate effect on K+-H+ currents. Thus, the inhibition of K+ transport usually observed with these agents reflects the kinetic effect of membrane depolarization rather than any direct chemical action on the cotransport system itself.Detailed study of the effects of [K+]o and pHo on theI–V curve for K+-H+ symport has revealed that increasing membrane potential systematicallydecreases the apparent affinity of the transporter for K+, butincreases affinity for protons (K
m
range: for [K+]o, 15–45 M; for [H+]o, 10–35 nM). This behavior is consistent with two distinct reaction-kinetic models, in which (i) a neutral carrier binds K+ first and H+ last in the forward direction of transport, or (ii) a negatively charged carrier (–2) binds H+ first and K+ last. 相似文献
3.
A. A. Jasaitis I. I. Severina V. P. Skulachev S. M. Smirnova 《Journal of bioenergetics and biomembranes》1972,3(5):388-396
Generation of a membrane potential in the respiratory chain-deficient particles of beef heart mitochondria has been studied. For detection of membrane potential, phenyl dicarbaundecaborane (PCB–,) and anilinonaphthalene sulphonate (ANS–) probes were used. The respiratory chain-deficient submitochondrial particles were prepared after Arion and Racker (E-SMP), the procedure including complete disappearance of membrane structures and subsequent reconstitution of membrane vesicles as judged by the electron microscopy study. E-SMP were found to be deficient in cytochromesa,a
3 and transhydrogenase, the cytochromeb,c
1 andc content being lowered. Addition of NADH, succinate and tetramethyl-p-phenylenediamine+ascorbate did not induce either any oxygen consumption or membrane potential formation. Treatment of E-SMP with NADPH+NAD+ or with NADH+CoQ0 did not entail generation of membrane potential, in contrast to that of parent, pyrophosphate submitochondrial particles (PP-SMP).E-SMP displayed an oligomycin-sensitive ATPase activity which could be increased by reconstitution of E-SMP with coupling factor F1. Addition of ATP resulted in an uptake of PCB– and enhancement of ANS– fluorescence, the facts testifying to the formation of the membrane potential with plus inside E-SMP. Membrane potential formation was arrested by oligomycin, rutamycin, and uncouplers. Addition of respiratory chain inhibitors (antimycin+rotenone+ cyanide), complete reduction of respiratory carriers by dithionite and oxidation by ferricyanide were without effect on ATP-supported formation of membrane potential in E-SMP. It was concluded that utilization of ATP energy for the membrane potential generation does not depend on the state of the respiratory carriers and can be demonstrated under the conditions when none of redox chain coupling sites were functioning.Abbreviations PCB–
phenyl dicarbaundecaborane
- ANS–
anilinonaphthalene sulfonate
- E-SMP
the respiratory chain-deficient submitochondrial particles
- PP-SMP
pyrophosphate submitochondrial particles 相似文献
4.
This review will focus on the recent advance in the study of effect of transmembrane Ca2+ gradient on the function of membrane proteins. It consits of two parts: 1. Transmembrane Ca2+ gradient and sarcoplasmic reticulum Ca2+-ATPase; 2. Effect of transmembrane Ca2+ gradient on the components and coupling of cAMP signal transduction pathway. The results obtained indicate that a proper transmembrane Ca2+ gradient may play an important role in modulating the conformation and activity of SR Ca2+-ATPase and the function of membrane proteins involved in the cAMP signal transduction by mediating the physical state change of the membrane phospholipids.Abbreviations Cai
Ca2+ inside vesicles
- Ca0
Ca2+ outside vesicles
- SR
sarcoplasmic reticulum
- PC
phosphatidylcholine
- PS
phosphatidylserine
- PG
phosphatidylglycerol
- PE
phosphatidylethanolamine
- DPH
1,6-diphenyl-1,3,5-hexatriene
- n-AS
n-(9-anthroyloxy) fatty acids
- TMA-DPH
1-(4-trimethylammoniumphenyl)-6)-phenyl-1,3,5-hexatriene
- FCCP
carbonylcyanide-p-trifluoromethoxyphenylhydrazone
- -AR
-adrenergic receptors
- DHA
dihydroalprenolol
- AC
adenylate cyclase
- AC·Lca+–
higher Ca2+ inside vesicles
- AC·Lca– –
lower Ca2+ on both side of vesicles
- AC·Lca++
higher Ca2+ on both side of vesicles
- AC·Lca– +
higher Ca2+ outside vesicles
- cAMP
cyclic adenosine monophosphate
- Gs
stimulatory GTP-binding protein
- GTP
guanosine triposphate
- GTPS
guanosine 50-(3-thiotriphosphate) 相似文献
5.
Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves 总被引:3,自引:0,他引:3
Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca2+ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment.45Ca2+ transport was assayed by membrane filtration technique. Results showed that Ca2+ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca2+ transport system had a high affinity for Ca2+(K
m
(Ca2+)=0.4 m) and ATP(K
m
(ATP)=3.9 m), and showed pH optimum at 7.5. ATP-dependent Ca2+ uptake in the plasma membrane vesicles was stimulated in the presence of Cl– or NO
3
–
. Quenching of quinacrine fluorescence showed that these anions also induced H+ transport into the vesicles. The Ca2+ uptake stimulated by Cl– was dependent on the activity of H+ transport into the vesicles. However, carbonylcyanidem-chlorophenylhydrazone (CCCP) and VO
4
3–
which is known to inhibit the H+ pump associated with the plasma membrane, canceled almost all of the Cl–-stimulated Ca2+ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca2+ uptake into the vesicles. These results suggest that the Cl–-stimulated Ca2+ uptake is caused by the efflux of H+ from the vesicles by the operation of Ca2+/H+ antiport system in the plasma membrane. In Cl–-free medium, H+ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca2+ uptake into the vesicles. These results suggest that two Ca2+ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca2+ transport system (Ca2+ pump) and the other is a Ca2+/H+ antiport system. Little difference in characteristics of Ca2+ transport was observed between the plasma membranes isolated from etiolated and green corn leaves. 相似文献
6.
Varda Shoshan-Barmatz 《The Journal of membrane biology》1988,103(1):67-77
Summary The relationship between Ca2+ release from sarcoplasmic reticulum, induced by elevated pH, tetraphenylboron (TPB–) or chemical modification, and the change in the surface charge of the membranes as measured by the fluorescence intensity of anilinonaphthalene sulfonate (ANS) is examined. The stimulated Ca2+ release is inhibited by dicyclohexylcarbodiimide and external Ca2+. TPB–, but not tetraphenylarsonium (TPA+), causes a decrease in ANS– fluorescence, with 50% decrease occurring at about 5 m TPB–. The decrease in ANS– fluorescence as well as the inhibition of Ca2+ accumulation induced by TPB– are prevented by TPA+. A linear relationship between the decrease in membrane surface potential and the extent of the Ca2+ released by TPB– is obtained. Similar levels of [3H]TPB– bound to sarcoplasmic reticulum membranes were obtained regardless of whether or not the vesicles have taken up Ca2+. The inhibition of Ca2+ accumulation and the [3H]TPB– incorporation into the membranes were correlated. Ca2+ release from sarcoplasmic reticulum, by pH elevation, chemical modification or by addition of NaSCN (0.2 to 0.5m) or the Ca2+ ionophore ionomycin, is also accompanied by a decrease in ANS– fluorescence intensity. However, chemical modification and elevated pH affects the surface potential much less than SCN– or TPB– do. These results suggest that the enhancement of Ca2+ release by these treatments is not due to a general effect on the membrane surface potential, but rather through the modification of a specific protein. They also suggest that membrane surface charges might play an important role in the control mechanism of Ca2+ release. 相似文献
7.
The characteristics of Cl– transport in isolated tonoplast vesicles from red-beet (Beta vulgaris L.) storage tissue have been investigated using the Cl–-sensitive fluorescent probe, 6-methoxy-1-(3-sulfonatopropyl)-quinolinium (SPQ). The imposition of (inside) positive diffusion potentials, generated with K+ and valinomycin, increased the initial rate of Cl– transport, demonstrating that Cl– could be electrically driven into the vesicles. Chloride influx was unaffected by SO
4
2-
, but was competitively blocked by NO
3
–
, indicating that both Cl– and NO
3
–
may be transported by the same porter. In some preparations, increases in free-Ca2+ concentration from 10–8 to 10–5 mol·dm–3 caused a significant decrease in Cl– influx, which may indicate that cytosolic Ca2+ concentration has a role in controlling Cl– fluxes at the tonoplast. However, this effect was only seen in about 50% of membrane preparations and some doubt remains over its physiological significance. A range of compounds known to block anion transport in other systems was tested, and some partially blocked Cl– transport. However, many of these inhibitors interfered with SPQ fluorescence and so only irreversible effects could be tested. The results are discussed in the context of recent advances made using the patch-clamp technique on isolated vacuoles.Abbreviations and Symbols BTP
1,3-bis[tris(hydroxymethyl)-methylamino]propane
- DTT
dithiothreitol
- EDTA
ethylenediaminetetraacetic acid
-
membrane potential
- pH
pH gradient
- SPQ
6-methoxy-1-(3-sulfonatopropyl)quinolinium
- Tricine
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl] glycine 相似文献
8.
The kinetics of CNProto- and CNDeutero-hemin binding to apohemoglobin A2 was investigated in a stopped-flow device in 0.05 M potassium phosphate buffer, pH 7, at 10°C. The overall kinetic profile exhibited multiple phases: Phases I–IV corresponding with heme insertion (8.5–13 × 107 M–1 s–1), local structural rearrangement (0.21–0.23 s–1), global structural event (0.071–0.098 s–1), and formation of the Fe–His bond (0.009–0.012 s–1), respectively. Kinetic differences observed between apohemoglobin A2 and apohemoglobin A (previously studied) prompted an analysis of the structures of and chains through molecular modeling. This revealed a structural repositioning of the residues not only at, but also distant from the site of the amino acid substitutions, specifically those involved in the heme contact and subunit interface. A significant global change was observed in the structure of the exon-coded 3 region and provided additional evidence for the designation of this as the subunit assembly domain. 相似文献
9.
1. In Lymnaea stagnalis L. (Pulmonata, Basommatophora) the neurons in the osphradium were visualized by staining through the inner right parietal nerve by 5,6-carboxyfluorescein (5,6-CF). Three types of neurons were identified: three large ganglionic cells (GC1-3; 80–100 m), the small putative sensory neurons (SC; 20 m) and very small sensory cells (3–5 m).2. The ganglionic and putative sensory neurons were investigated by whole cell patch-clamp method in current-clamp condition. The three giant ganglionic neurons (GC1-3) located closely to the root of osphradial nerve, had a membrane potential (MP) between –30 and –70 mV and showed tonic or bursting activities. The small putative sensory cells (SCs) scattered throughout the osphradial ganglion, possessed a MP between –25 and –55 mV and showed an irregular firing pattern with membrane oscillations. At resting MP the GC1-3 cells were depolarized and increased the frequency of their firing, while the SCs were hyperpolarized and inhibited by NaCl (10–2
M) and L-aspartate (10–5
M) applied to the osphradium.3. 5-Hydroxytryptamine (5HT, 10–6
M), -aminobutyric acid (GABA; 10–6
M) and the GABAB agonist baclofen (10–6
M) depolarized the neurons GC1-3 and increased their firing frequency. In contrast, on the GC1-3 neurons, acetylcholine (Ach; 10–6
M) and FMRFamide (10–6
M) caused hyperpolarization and cessation of the firing activity. The 5HT effect was blocked by mianserin (10–6
M) but picrotoxin (10–5
M) failed to block the GABA-induced effect on the GC1-3 cells.4. The small putative sensory neurons (SCs) were excited by Ach (10–6
M) and 5HT (10–6
M) but were inhibited by GABA (10–6
M). FMRFamide (10–6
M) had a biphasic response. The Ach effect was blocked by hexamethonium (10–6
M) and tetraethylammonium (10–6
M), indicating the involvement of nicotinic cholinergic receptors.5. The distinct responses of the two populations of osphradial neurons to chemical stimuli and neurotransmitters suggest that they can differently perceive signals from environment and hemolymph. 相似文献
10.
Walter Borzani 《Biotechnology letters》1995,17(12):1295-1298
Summary The following equations represent the influence of the ethanol concentration (E) on the specific growth rate of the yeast cells () and on the specific production rate of ethanol () during the reactor filling phase in fed-batch fermentation of sugar-cane blackstrap molasses: =
0 - k · E and v = v
0
· K/(K +E)
Nomenclature E
ethanol concentration in the aqueous phase of the fermenting medium (g.L–1)
- Em
value of E when = 0 or = 0 (g.L–1)
- F
medium feeding rate (L.h–1)
- k
empirical constant (L.g–1.h–1)
- K
empirical constant (g.L–1)
- Mas
mass of TRS added to the, reactor (g)
- Mcs
mass of consumed TRS (g)
- Me
mass of ethanol in the aqueous phase of the fermenting medium (g)
- Ms
mass of TRS in the aqueous phase of the fermenting medium (g)
- Mx
mass of yeast cells (dry matter) in the fermenting medium (g)
- r
correlation coefficient
- S
TRS concentration in the aqueous phase of the fermenting medium (g.L–1)
- Sm
TRS concentration of the feeding medium (g.L–1)
- t
time (h)
- T
temperature (° C)
- TRS
total reducing sugars calculated as glucose
- V
volume of the fermenting medium (L)
- V0
volume of the inoculum (L)
- X
yeast cells concentration (dry matter) in the fermenting medium (g.L–1)
-
filling-up time (h)
-
specific growth rate of the yeast cells (h–1)
- 0
value of when E=0
-
specific production rate of ethanol (h–1)
- 0
value of when E=0
-
density of the yeast cells (g.L–1)
-
dry matter content of the yeast cells 相似文献
11.
Pyruvate is a key metabolic intermediate and the substrate for diacetyl and acetoin synthesis. The mechanism of pyruvate transport was determined inLactobacillus plantarum by use of cells and membrane vesicles. In the cells, protonophores inhibited pyruvate transport, whereas valinomycin did not. Pyruvate was accumulated against a gradient in membrane vesicles. The transport rate and the degree of accumulation increased as the proton gradient increased, but an imposed K potential of –61mV did not drive pyruvate transport. The maximum transport rate (35 nmol/min/mg protein) and accumulation ratio (162-fold) were at pH 3.0, with an apparent Km value of 35 M. These results suggested that pyruvate was transported by a proton symport. 相似文献
12.
Summary Permeability properties and the effects of a changed membrane potential on Ca2+ release of sarcoplasmic reticulum vesicles of rabbit skeletal muscle were investigated by Millipore filtration. The relative permeability of sarcoplasmic reticulum to solutes determined under conditions of isotope exchange at equilibrium and/or under conditions of net flow of solute and water into the vesicles was as follows: sucrose, Ca2+, Mn2+–, choline+, Tris+–+, Na+, Li+, Cl–. Transient membrane potentials were induced by rapidly changing the ionic environment of the vesicles. Knowledge of the relative permeation rates of the above ions allowed prediction of the direction and extent of membrane polarization. Osmotic effects in the polarization measurements due to the rapid influx of solute and water into the vesicles were minimized by using media containing a fast (K+ or Cl–) and a relatively slow (gluconate– or choline+) penetrating ion.45Ca2+ efflux from vesicles derived from different parts of the sarcoplasmic reticulum structure was not appreciably changed when vesicles were made more positive inside (choline chloride potassium gluconate) or more negative inside (potassium gluconate choline chloride). These studies suggest that part or all of the ion-induced changes in sarcoplasmic reticulum membrane permeability, previously interpreted to indicate depolarization-induced Ca2+ release, may be due to osmotic effects. 相似文献
13.
Bonaventura Ruiz-Montasell F. Javier Casado Antonio Felipe Marçal Pastor-Anglada 《The Journal of membrane biology》1992,128(3):227-233
Summary The characteristics of uridine transport were studied in basolateral plasma membrane vesicles isolated from rat liver. Uridine was not metabolized under transport measurement conditions and was taken up into an osmotically active space with no significant binding of uridine to the membrane vesicles. Uridine uptake was sodium dependent, showing no significant stimulation by other monovalent cations. Kinetic analysis of the sodium-dependent component showed a single system with Michaelis-Menten kinetics. Parameter values were K
M 8.9
m and V
max 0.57 pmol/mg prot/sec. Uridine transport proved to be electrogenic, since, firstly, the Hill plot of the kinetic data suggested a 1 uridine: 1 Na+ stoichiometry, secondly, valinomycin enhanced basal uridine uptake rates and, thirdly, the permeant nature of the Na+ counterions determined uridine transport rates (SCN– > NO
3
–
> Cl– > SO
4
2–
). Other purines and pyrimidines cis-inhibited and trans-stimulated uridine uptake.This work has been partially supported by grant PM90-0162 from D.G.I.C.Y.T. (Ministerio de Educación y Ciencia, Spain). B.R.-M. is a research fellow supported by the Nestlé Nutrition Research Grant Programme. 相似文献
14.
Summary Loop diuretic-sensitive (Na+,K+,Cl–)-cotransport activity was found to be present in basolateral membrane vesicles of surface and crypt cells of rabbit distal colon epithelium. The presence of grandients of all three ions was essential for optimal transport activity (Na+,K+) gradien-driven36Cl fluxes weree half-maximally inhibited by 0.14 m bumetanide and 44 m furosimide. While86Rb uptake rates showed hyperbolic dependencies on Na+ and K+ concentrations with Hill coefficients of 0.8 and 0.9, respectively, uptakes were sigmoidally related to the Cl– concentration, Hill coefficient 1.8, indicating a 1 Na+: 1 K+:2 Cl– stoichiometry of ion transport.The interaction of putative (Na+, K+, Cl–)-cotransport proteins with loop diuretics was studied from equilibrium-binding experiments using [3H]-bumetanide. The requirement for the simulataneous presence of Na+,K+, and Cl–, saturability, reversibility, and specificity for diuretics suggest specific binding to the (Na+, K+, Cl–)-cotransporter. [3H]-bumetanide recognizes a minimum of two classes of diuretic receptors sites. high-affinity (K
D1=0.13 m;B
max1 =6.4 pmol/mg of protein) and low-affinity (K
D2=34 m;B
max2=153 pmol/mg of protein) sites. The specific binding to the high-affinity receptor was found to be linearly competitive with Cl– (K
1=60mm), whereas low-affinity sites seem to be unaffected by Cl–. We have shown that only high-affinity [3H]-bumetanide binding correlates with transport inhibition raising questions on the physiological significance of diuretic receptor site heterogeneity observed in rabbit distal colon epithelium. 相似文献
15.
Summary A method was developed for the study of divalent cation transport events on the time scale of 20 msec or longer. Passive Ca2+ equilibration across the membranes of the Ca2+-ATPase rich fraction of sarcoplasmic reticulum (SR) was studied. The method makes use of the divalent cation sensitivity of the surface binding of the fluorescent probe 1-anilino-8-naphthalenesulfonate (ANS–). Binding to the inside and outside surfaces is distinguished in fluorescent stopped-flow experiments. The surface binding reactions of the probe are faster than the time resolution of the instrument (ca. 3 msec), while binding reactions requiring transport across the membrane could be resolved. In Ca2+ influx experiments, the time course of fluorescent enhancement was monitored following a Ca2+ jump. The kinetics of Ca2+ efflux were studied by pre-equilibrating Ca2+ across the membrane, removing the external Ca2+ with an EGTA jump, and observing the time course of the fluorescence decrease. Rapid transport of ANS– (coupled to K+) was ensured by the addition of valinomycin. Two processes of Ca2+ influx were observed: (i) a rapid process with small fluorescent amplitudes and at
1/2 of 40–60 msec and (ii) a slow process with a large amplitude and at
1/2 of 70–100 sec. The rates and extents of the two phases were quantitated in terms of the rates and extents of change in the Ca2+ concentration in the SR lumen. The slow phase accounted for a larger change, in the internal free Ca2+ concentration than did the first phase. For the influx of 10 mM Ca2+, the rapid phase raises the internal Ca2+ concentration to ca. 1 mM within its apparentt
1/2 of 20 msec. The slow phase brings about an increase of the internal Ca2+ concentration to 4 mM within its apparentt
1/2 of 90 sec. The two phases have average rates of increase of internal free Ca2+ concentration, [Ca]
i
/sec of ca. 50 mM/sec and ca. 0.02 mM/sec, respectively. The Ca2+ influx rates increased with increasing KCl concentration and with increasing external Ca2+ concentration.Two phases of Ca2+ efflux were observed. The amplitudes and rates were analyzed and the fast phase was shown to account for more Ca2+ movement than the slow phase. The rate of the fast phase was greatly increased by increasing the K+ concentration. The rate of the slow phase efflux decreased with increasing Ca2+ concentration in the external medium. The concentration for half-maximal inhibition was 4 M, a value close to the dissociation constant of the high affinity site on the Ca2+-ATPase.The above constitutes a body of circumstantial evidence that the passive Ca2+ permeability observed is mediated by the Ca2+-ATPase, acting as a Ca2+ for 2 K+ exchanger. The fast phases are explained as a partial turnover of the pump in the steady state. The slow rate is explained by a preference of the ion binding translocator site of the carrier for an outward orientation.The ANS– technique was applied to the monovalent cation permeability of the Ca2+-ATPase rich SR and the results of other studies were corroborated and extended. The interaction of valinomycin with intrinsic permeability mechanisms of the SR was considered. 相似文献
16.
Charles M. Schron 《The Journal of membrane biology》1991,120(2):192-200
Summary In jejunal brush-border membrane vesicles, an outwardly directed OH– gradient (in>out) stimulates DIDS-sensitive, saturable folate (F) uptake (Schron, C.M. 1985.J. Clin. Invest.
76:2030–2033), suggesting carrier-mediated folate: OH– exchange (or phenomenologically indistinguishable H+: folate cotransport). In the present study, the precise role of pH in the transport process was elucidated by examining F uptake at varying pH. For pH gradients of identical magnitude, F uptake (0.1 M) was greater at lower (pHint/pHext: 5.5/4.5) compared with higher (6.5/5.5) pH ranges. In the absence of a pH gradient, internal Ftrans stimulated DIDS-sensitive3H-folate uptake only at pH6.0. Since stepwise increments ininternal pH (4.57.5; pHext=4.5) stimulated F uptake, an inhibitory effect of higherinternal pH was excluded. In contrast, with increasing external pH (4.356.5; pHint=7.8), a 50-fold decrement in F uptake was observed (H+
K
m
=12.8±1.2 M). Hill plots of these data suggest involvement of at least one H+ (OH–) at low pH (monovalent F– predominates) and at least 2 H+ (OH–) at high pH (divalent F–2 predominates). Since an inside-negative electrical potential did not affect F uptake at either pHext 4.55 or 5.8, transport of F– and F–2 is electroneutral. Kinetic parameters for F– and F–2 were calculated from uptake data at pHext 4.55 and 5.0. Comparison of predictedvs. experimentally determined kinetic parameters at pHext5.8 (K
m
=1.33vs. 1.70 M;V
max=123.8vs. 58.0 pmol/mg prot min) suggest that increasing external pH lowers theV
max, but does not affect theK
m
for carrier-mediated F transport. These data are consistent with similarK
i
'
s for sulfasalazine (competitive inhibitor) at pHext 5.35 and 5.8 (64.7 and 58.5 M, respectively). In summary, the jejunal F carrier mediates electroneutral transport of mono- and divalent F and is sensitive to external pH with a H+
K
m
(or OH– lC50) corresponding to pH 4.89. External pH effects theV
max, but not theK
m
for carriermediated F uptake suggesting a reaction mechanism involving a ternary complex between the outward-facing conformation of the carrier and the transported ions (F and either OH– or H+),rather than competitive binding that is mutually exclusive. 相似文献
17.
Acidification inside the vacuo-lysosome systems is ubiquitous in eukaryotic organisms and essential for organelle functions. The acidification of these organelles is accomplished by proton-translocating ATPase belonging to the V-type H+-ATPase superfamily. However, in terms of chemiosmotic energy transduction, electrogenic proton pumping alone is not sufficient to establish and maintain those compartments inside acidic. Current studies have shown that thein situ acidification depends upon the activity of V-ATPase and vacuolar anion conductance; the latter is required for shunting a membrane potential (interior positive) generated by the positively charged proton translocation. Yeast vacuoles possess two distinct Cl– transport systems both participating in the acidification inside the vacuole, a large acidic compartment with digestive and storage functions. These two transport systems have distinct characteristics for their kinetics of Cl– uptake or sensitivity to a stilbene derivative. One shows linear dependence on a Cl– concentration and is inhibited by 4,4-diisothiocyano-2,2-stilbenedisulfonic acid (DIDS). The other shows saturable kinetics with an apparentK
m
for Cl– of approximately 20 mM. Molecular mechanisms of the chemiosmotic coupling in the vacuolar ion transport and acidification inside are discussed in detail. 相似文献
18.
Plant vacuoles were isolated from cotyledons of germinatingAcacia mangium seeds, which had been treated with or withoutcolchicine, to measure vacuolar membrane pyrophosphate (PPi)- andATP-dependent H+ transport activities, and enzymaticactivities of H+-pyrophosphatase(H+-PPase) and H+-ATPase. Innon-colchicine-treated seeds, activities of the two enzymes increasedrapidly after seed germination to almost a maximal level on the seventhday. A linear function relationship exists in magnitude between PPi- orATP-dependent H+transport activity and its correspondingenzymatic activity. The former regression equation is: PPi-dependentH+ transport activity(%A.min–1.g–1) =–0.039 + H+-PPase activity(units.mg–1) × 1.574, the latter is:ATP-dependent H+ transport activity(%A.min–1.g–1) =–0.003 + H+-ATPase activity(units.mg–1) × 0.549. In colchicine-treatedseeds, activities of the two enzymes increased very slowly during 8 daysof germination and the relationship to their respectiveH+ transport activities was not in agreement with theabove-mentioned regression equations. PPi- and ATP-dependentH+ transport activities were lower than thecorresponding values calculated from H+-PPase activityand H+-ATPase activity according to the two regressionequations, respectively. However, when sucrose, indole butyric acid(IBA), or 6-benzyladenine (6-BA) were applied exogenously to the seedsfollowing colchicine treatment for 3 days, activities ofH+-PPase, H+-ATPase, PPi- andATP-dependent H+ transport in the 6-day-old seedlingsall increased. By statistical analysis, it was concluded that colchicineinhibits cotyledon vacuolar membrane H+-PPase,H+-ATPase activities, PPi- and ATP-dependentH+ transport activities during seed germination andearly seedling growth of Acacia mangium. The inhibitory effectsof colchicine could be overcome by IBA, 6-BA and sucrose to varyingdegrees. 相似文献
19.
Single-channel conductance fluctuations are analysed for gramicidin A incorporated into binary-mixed black lipid membranes of charged phosphatidic acid and neutral lecithin in different molar ratios. At very low Ca++ concentrations in the electrolyte (i.e. in the presence of EDTA) homogeneous lipid mixtures are identified through their conductance and life time probability distributions for integral gramicidin pores. As for the pure lipid components, the conductance histograms each show a single maximum with regular width and for all channels a single mean lifetime is found.For Ca++-levels (10-6–10-5
M) that are close to the critical demixing concentration (10-4
M) unusually broad conductance distributions and reduced lifetimes are found provided the PC content, x, of the membrane is close to the critical mixture (x
crit0.5). We interpret this as a first example of the coupling of a membrane function (the transport of ions) to a lipid matrix with locally fluctuating composition close to a critical demixing point.For
the conductance histogram of gramicidin A in an equimolar mixture of PA and PC shows two well-separated maxima. A correlation analysis between conductance and lifetime of the single pores shows that the two channel populations also differ significantly in their mean channel lifetime, *. This finding is interpreted as being direct evidence for Ca++-induced lateral phase separation in black lipid membranes, as has been postulated recently.Abbreviations used HEPES
N-2-hydroxyethyl-piperazine-N-2-ethane-sulfonic acid
- EDTA
ethylenediaminetetraacetic acid 相似文献
20.
Summary The effect of energy deprivation on the intracellular transport and secretion of thyroglobulin was studied in open follicles isolated from porcine thyroids. Follicles were pulse-labeled with 3H-leucine or 3H-galactose. Labeled thyroglobulin was secreted into the incubation medium where it was isolated by means of immunoprecipitation. Secretion was followed in chase incubations under various experimental conditions using CCCP (carbonyl-cyanide-mchlorophenylhydrazone) or DNP (dinitrophenol), both uncouplers of oxidative phosphorylation, or CN–, which inhibits respiration. CCCP (1 M) was shown to inhibit exocytosis by about 80%, DNP (0.1–5 mM) by 45–85%, and CN– (0.5–1.1 mM) by 5–55%. By combining CN– with the ionophore monensin, which blocks transport through the Golgi complex but does not essentially interfere with exocytosis, evidence was obtained that CN– also inhibits transport of thyroglobulin from the Golgi cisternae to the exocytic vesicles by 40%. Electron-miroscopic autoradiography of isolated thyroid lobes from the rat also indicated that transport of 3H-leucine label into the follicle lumen is inhibited in the presence of CCCP or CN–. Intracellular ATP content was found to be about 40% of the control level in follicles incubated with CCCP (1 uM) or CN– (0.9 mM). The results show that the transport of thyroglobulin from the Golgi complex to the exocytic vesicles as well as from the exocytic vesicles into the follicle lumen is dependent upon metabolic energy. The transport blocks are probably associated with inhibited membrane fusions and fissions.Abbreviations CCCP
carbonylcyanide-m-chlorophenylhydrazone
- FCCP
carbonylcyanide-p-trifluoromethoxyphenylhydrazone
- DNP
dinitrophenol 相似文献