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1.
Phosphate transport across plasma membranes has been described in a wide variety of organisms and cell types including gastrointestinal epithelia. Phosphate transport across apical membranes of vertebrate gastrointestinal epithelia requires sodium; whereas, its transport across the basolateral membrane requires antiport processes involving primarily chloride or bicarbonate. To decipher the phosphate transport mechanism in the foregut apical membrane of the mollusc, Aplysia californica, in vitro short-circuited Aplysia californica gut was used. Bidirectional transepithelial fluxes of both sodium and phosphate were measured to see whether there was interaction between the fluxes. The net mucosal-to-serosal flux of Na+ was enhanced by the presence of phosphate and it was abolished by the presence of serosal ouabain. Similarly, the net mucosal-to-serosal flux of phosphate was dependent upon the presence of Na+ and was abolished by the presence of serosal ouabain. Theophylline, DIDS and bumetande, added to either side, had no effect on transepithelial difference or short-circuit current in the Aplysia gut bathed in a Na2HPO4 seawater medium. However, mucosal arsenate inhibited the net mucosal-to-serosal fluxes of both phosphate and Na+ and the arsenate-sensitive Na+ flux to that of phosphate was 2:1. These results suggest the presence of a Na-PO4 symporter in the mucosal membrane of the Aplysia californica foregut absorptive cell. 相似文献
2.
J E Lever 《The Journal of biological chemistry》1977,252(6):1990-1997
Selectively permeable membrane vesicles isolated from Simian virus 40-transformed mouse fibroblasts catalyzed Na+ gradient-coupled active transport of several neutral amino acids dissociated from intracellular metabolism. Na+-stimulated alanine transport activity accompanied plasma membrane material during centrifugation in discontinuous dextran 110 gradients. Carrier-mediated transport into the vesicle was demonstrated. When Na+ was equilibrated across the membrane, countertransport stimulation of L-[3H]alanine uptake occurred in the presence of accumulated unlabeled L-alanine, 2-aminoisobutyric acid, or L-methionine. Competitive interactions among neutral amino acids, pH profiles, and apparent Km values for Na+ gradient-stimulated transport into vesicles were similar to those previously described for amino acid uptake in Ehrlich ascites cells, which suggests that the transport activity assayed in vesicles is a component of the corresponding cellular uptake process. Both the initial rate and quasi-steady state of uptake were stimulated as a function of a Na+ gradient (external Na+ greater than internal Na+) applied artificially across the membrane and were independent of endogenous (Na+ + K+)-ATPase activity. Stimulation by Na+ was decreased when the Na+ gradient was dissipated by monensin, gramicidin D or Na+ preincubation. Na+ decreased the apparent Km for alanine, 2-aminoisobutyric acid, and glutamine transport. Na+ gradient-stimulated amino acid transport was electrogenic, stimulated by conditions expected to generate an interior-negative membrane potential, such as the presence of the permeant anions NO3- and SCN-. Na+-stimulated L-alanine transport was also stimulated by an electrogenic potassium diffusion potential (K+ internal greater than K+ external) catalyzed by valinomycin; this stimulation was blocked by nigericin. These observations provide support for a mechanism of active neutral amino acid transport via the "A system" of the plasma membrane in which both a Na+ gradient and membrane potential contribute to the total driving force. 相似文献
3.
Phosphate transport into brush-border membrane vesicles isolated from rat small intestine. 总被引:10,自引:0,他引:10
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Uptake of Pi into brush-border membrane vesicles isolated from rat small intestine was investigated by a rapid filtration technique. The following results were obtained. 1. At pH 7.4 in the presence of a NaCl gradient across the membrane (sodium concentration in the medium higher than sodium concentration in the vesicles), phosphate was taken up by a saturable transport system, which was competitively inhibited by arsenate. Phosphate entered the same osmotically reactive space as D-glucose, which indicates that transport into the vesicles rather than binding to the membranes was determined. 2. The amount of phosphate taken up initially was increased about fourfold by lowering the pH from 7.4 to 6.0.3. When Na+ was replaced by K+, Rb+ or Cs+, the initial rate of uptake decreased at pH 7.4 but was not altered at pH 6.0.4. Experiments with different anions (SCN-,Cl-, SO42-) and with ionophores (valinomycin, monactin) showed that at pH 7.4 phosphate transport in the presence of a Na+ gradient is almost independent of the electrical potential across the vesicle membrane, whereas at pH 6.0 phosphate transport involves the transfer of negative charge. It is concluded that intestinal brush-border membranes contain a Na+/phosphate co-transport system, which catalyses under physiological conditions an electroneutral entry of Pi and Na+ into the intestinal epithelial cell. In contrast with the kidney, probably univalent phosphate and one Na+ ion instead of bivalent phosphate and two Na+ ions are transported together. 相似文献
4.
The Na+-dependent phosphate transport system in the brush border of rabbit kidney exhibits a positive requirement for a divalent metal ion. Treatment of the brush-border membrane vesicles (BBMV) with a divalent metal chelator in combination with the divalent metal ionophore A23187 dramatically and selectively decreased the Na+-dependent uptake of phosphate; Na+-independent uptake of phosphate was not affected. The combination of chelator plus A23187 also inhibited uptake of phosphate in the presence of Na+ but in the absence of a gradient for sodium across the BBMV. This indicates that the inhibitor is not a result of an alteration in the Na+ gradient by chelator plus ionophore. The inhibited Na+ gradient-dependent transport of phosphate was restored by removing the chelator and adding Mn2+ to the BBMV. The phosphate-binding proteolipid (phosphorin) isolated from rabbit kidney BBMV binds inorganic phosphate with high affinity and specificity. Binding of phosphate to phosphorin is also inhibited by divalent metal chelators and can be restored by addition of a divalent metal. We conclude that a divalent metal ion is required both for the Na+-dependent phosphate transport in BBMV and for the binding of phosphate to the proteolipid phosphorin. These findings are consistent with our suggestion that phosphorin is a component of the Na+-dependent phosphate transport system in renal brush-border membranes. 相似文献
5.
Membrane vesicles were prepared from mouse fibroblasts transformed by SV40 virus (SV3T3). Following disruption of the cells by nitrogen cavitation, the membrane vesicles were obtained by differential centrifugation. As measured by enzyme markers, they consist mainly of membrane from the plasma membrane and smooth and rough endoplasmic reticulum. The vesicles transport Pi by two separate, mediated systems: one is independent of Na+, and the other is secondary active transport driven by a Na+ gradient. Electrical and chemical energy can be provided by a Na+ gradient to drive the concentrative uptake of Pi by the vesicles, one or both forces being used to energize transport. Evidence is provided that both the electrical and chemical potentials produced by the asymmetric distribution of Na+ across the membrane of SV3T3 membrane vesicles are utilized to concentrate phosphate in the vesicles. Phosphate transport by the vesicles cannot be accounted for by a small contamination of this fraction with mitochondria (1 to 4%). The Pi transport properties of the membrane vesicles differ from those of the fraction enriched in mitochondria in the following respects: their kinetic properties, and their responses to a Na+ gradient, N-ethylmaleimide, mersalyl, and succinate/acetate. However, the membrane vesicles share some properties of Pi transport with mitochondria. Cyanide, azide, oligomycin, 2,4-dinitrophenol, and carbonyl cyanide m-cholophenylhydrazone, inhibitors of Pi transport by mitochondria, also inhibit membrane vesicle, Pi transport. The vesicles retain all the features of Pi transport by SV3T3 cells that have been examined. They provide a simplified system for a determination of the details of the mechanism of Pi transport under conditions where transport is dissociated from intracellular reactions and in the presence of a defined electrochemical driving force. 相似文献
6.
The characteristics of phosphate transport across intestinal basolateral membranes of the rat were determined by using enriched preparations in which uphill Na+-dependent D-glucose transport could not be demonstrated, but ATP-dependent Ca2+ transport was present. Phosphate transport was saturable, Na+-dependent and exhibited Michaelis-Menten kinetics. Vmax. was 51.1 +/- 4.2 pmol/10 s per mg of protein and Km was 14 +/- 3.9 microM. The transport process was electroneutral. Tracer-exchange experiments and counter-transport studies confirmed the presence of a Na+-Pi carrier at the basolateral membrane. The presence of inside-positive membrane potential did not enhance phosphate uptake, indicating that the Na+ effect is secondary to the presence of the Na+-Pi carrier rather than an induction of positive membrane potential. The stoichiometry of this carrier at pH 7.4 was 2 Na+:1 phosphate, as shown by direct studies utilizing the static-head method. These studies are the first to determine the presence of a phosphate carrier at the basolateral membrane. 相似文献
7.
Rat liver basolateral plasma membrane (blLPM) vesicles resuspended in 5 mM Mg2(+)-, Ca2(+)-, Mn2(+)- or Co2(+)-containing media exhibited a markedly lower rate of Na(+)-stimulated L-alanine transport. Divalent cation inhibition of L-alanine uptake was dose dependent, and was observed only when the vesicles were pre-loaded with the divalent cations. The presence or absence of the metal ions in the extravesicular incubation media had no effect on L-alanine transport. Conversely, pretreatment of the vesicles with 0.2 mM of either EGTA or EDTA resulted in higher initial rates of L-alanine transport. This stimulation was overcome by addition of excess divalent cation to the vesicle suspension solution. Since these blLPM vesicles are primarily oriented right-side-out, the divalent cation inhibition of L-alanine transport appears to be a result of their interaction with cytosolic components of the cell membrane. Total Na+ flux as measured with 22Na+ was not affected by intravesicular 5 mM Mg2+ or Ca2+, indicating that the inhibition was not due to dissipation of the Na+ gradient. These observations suggest that intracellular divalent cations may serve to modulate L-alanine transport across the liver cell plasma membrane. 相似文献
8.
Sulphate and phosphate transport in the renal proximal tubule 总被引:2,自引:0,他引:2
K J Ullrich H Murer 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》1982,299(1097):549-558
Experiments performed on microperfused proximal tubules and brush-border membrane vesicles revealed that inorganic phosphate is actively reabsorbed in the proximal tubule involving a 2 Na+-HPO2-4 or H2PO-4 co-transport step in the brush-border membrane and a sodium-independent exit step in the basolateral cell membrane. Na+-phosphate co-transport is competitively inhibited by arsenate. The transtubular transport regulation is mirrored by the brush-border transport step: it is inhibited by parathyroid hormone intracellularly mediated by cyclic AMP. Transepithelial inorganic phosphate (Pi) transport and Na+-dependent Pi transport across the brush-border membrane correlates inversely with the Pi content of the diet. Intraluminal acidification as well as intracellular alkalinization led to a reduction of transepithelial Pi transport. Data from brush-border membrane vesicles indicate that high luminal H+ concentrations reduce the affinity for Na+ of the Na+-phosphate co-transport system, and that this mechanism might be responsible for the pH dependence of phosphate reabsorption. Contraluminal influx of Pi from the interstitium into the cell could be partly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS). It is not, however, changed when dicarboxylic acids are present or when the pH of the perfusate is reduced to pH 6. Sulphate is actively reabsorbed, involving electroneutral 2 Na+-SO2-4 co-transport through the brush-border membrane. This transport step is inhibited by thiosulphate and molybdate, but not by phosphate or tungstate. The transtubular active sulphate reabsorption is not pH dependent, but is diminished by the absence of bicarbonate. The transport of sulphate through the contraluminal cell side is inhibited by DIDS and diminished when the capillary perfusate contains no bicarbonate or chloride. The latter data indicate the presence of an anion exchange system in the contraluminal cell membrane like that in the erythrocyte membrane. 相似文献
9.
The steroid hormone aldosterone regulates reabsorptive Na+ transport across specific high resistance epithelia. The increase in Na+ transport induced by aldosterone is dependent on protein synthesis and is due, in part, to an increase in Na+ conductance of the apical membrane mediated by amiloride-sensitive Na+ channels. To examine whether an increment in the biochemical pool of Na+ channels expressed at the apical cell surface is a mechanism by which aldosterone increases apical membrane Na+ conductance, apical cell-surface proteins from the epithelial cell line A6 were specifically labeled by an enzyme-catalyzed radioiodination procedure following exposure of cells to aldosterone. Labeled Na+ channels were immunoprecipitated to quantify the biochemical pool of Na+ channels at the apical cell surface. The activation of Na+ transport across A6 cells by aldosterone was not accompanied by alterations in the biochemical pool of Na+ channels at the apical plasma membrane, despite a 3.7-4.2-fold increase in transepithelial Na+ transport. Similarly, no change in the distribution of immunoreactive protein was resolved by immunofluorescence microscopy. The oligomeric subunit composition of the channel remained unaltered, with one exception. A 75,000-Da polypeptide and a broad 70,000-Da polypeptide were observed in controls. Following addition of aldosterone, the 75,000-Da polypeptide was not resolved, and the 70,000-Da polypeptide was the major polypeptide found in this molecular mass region. Aldosterone did not alter rates of Na+ channel biosynthesis. These data suggest that neither changes in rates of Na+ channel biosynthesis nor changes in its apical cell-surface expression are required for activation of transepithelial Na+ transport by aldosterone. Post-translational modification of the Na+ channel, possibly the 75,000 or 70,000-Da polypeptide, may be one of the cellular events required for Na+ channel activation by aldosterone. 相似文献
10.
The use of membrane vesicles in transport studies 总被引:3,自引:0,他引:3
J E Lever 《CRC critical reviews in biochemistry》1980,7(3):187-246
Transport-competent plasma membrane vesicles isolated from mammalian cells provide a system to investigate mechanisms and regulation of nutrient and ion transport systems. The characteristics of membrane vesicle systems to study transport in erythrocytes, renal and epithelial membranes, Ehrlich ascites cells, and mouse fibroblasts are discussed. Studies of Na+-stimulated and Na+-independent amino acid and glucose transport in these systems are evaluated, with emphasis on experimental verification of concepts stated in the Na+ gradient hypothesis. Nucleoside, phosphate, and calcium transport systems in plasma membrane vesicles from mouse fibroblast cultures are discussed. Also, current biochemical approaches to investigate mechanisms of regulation of nutrient transport systems by hormones or cellular proliferative state are described. 相似文献
11.
Mechanism of the stimulation of serine and alanine transport into isolated rat liver cells by bicarbonate ions. 总被引:1,自引:0,他引:1
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J D McGivan 《The Biochemical journal》1979,182(3):697-705
1. Bicarbonate ions stimulate the transport of serine and alanine into isolated hepatocytes. 2. The effect of bicarbonate is to increase the Vmax. of the transport process without changing the apparent Km. 3. The intracellular pH was estimated from the distribution of the weak base methylamine and the weak acid 5,5'-dimethyloxazolidine-2,4-dione (DMO) across the plasma membrane. 4. The addition of bicarbonate to a cell suspension caused the internal pH to become more acid. 5. The initial rate of serine, alanine and glycine transport was a linear function of the initial difference in pH across the membrane. 6. It is concluded that bicarbonate activates the transport of these amino acids primarily by increasing the pH difference across the plasma membrane. 7. It is suggested that the uptake of serine together with Na+ ions occurs in exchange for H+ ions, which are translocated outwards on the same carrier system. Some preliminary evidence consistent with this model is presented. 相似文献
12.
Studies on the transport of carnitine in the brain using synaptosomes isolated from guinea-pig cerebral cortex 总被引:1,自引:0,他引:1
Synaptosomes isolated from guinea pig cerebral cortex accumulate L-carnitine from the medium in an active process, dependent on the sodium gradient across the plasma membrane and on (Na+ + K+)-ATPase activity. L-Carnitine uptake is inhibited by oxidative phosphorylation uncouplers and by ouabain, a known inhibitor of (Na+ + K+)-ATPase. In addition, the omission of Na+ or its replacement by Li+ inhibited the transport, which was also competitively inhibited by gamma-aminobutyrate. The kinetics of carnitine uptake show that the overall process would consist of two components: a passive diffusion and a carrier-mediated transport which is saturated at 1-2 mM carnitine concentration. 相似文献
13.
The regulation by thyroid hormone of phosphate transport in primary cultured chick renal cells was examined. The more physiologically active L-analogs of triiodothyronine and thyroxine, but not the D-analogs of the hormones, stimulated the Na+-dependent phosphate uptake system. Na+-independent phosphate uptake and Na+-dependent uptakes of alpha-methylglucoside and L-proline were unaffected. The increase in Na+-dependent phosphate uptake was concentration dependent, exhibited an induction period, and was blocked by inhibitors of RNA and protein synthesis. The stimulation of phosphate uptake by triiodothyronine was due to an increased Vmax rather than to an altered affinity for phosphate. These findings demonstrate that thyroid hormone acts directly on renal cells to modulate phosphate transport and suggest that the renal cell system may serve as a model to examine the mechanism by which thyroid hormone controls gene expression and regulates plasma membrane transport function. 相似文献
14.
Type-I diabetes is associated with a decrease in magnesium content in various tissues, including liver. We have reported that hepatocytes from streptozotocin-injected rats have lost the ability to accumulate Mg2+ following hormonal stimulation. To assess whether the defect is inherent to the Mg2+ transport mechanism located in the hepatocyte cell membrane, plasma membrane vesicles were purified from diabetic livers. Diabetic plasma membranes do not retain intravesicular Mg2+ as tightly as vesicles purified from livers of age-matched non-diabetic rats. In addition, the amount of intravesicular Mg2+ these vesicles exchange for extravesicular Na+ or Ca2+ is 2-3-fold larger than in non-diabetic vesicles. The partition of Ca2+/Mg2+ and Na+/Mg2+ exchange mechanisms in the apical and basolateral domains of liver plasma membrane is maintained under diabetic conditions, although the Na+/Mg2+ exchanger in diabetic basolateral membranes has lost the ability to operate in reverse and favor an accumulation of extravesicular Mg2+ within the vesicles in exchange for entrapped Na+. These data indicate the occurrence of a major alteration in Mg2+ transport across the hepatocyte membrane, which can explain, at least in part, the decrease in liver magnesium content observed in diabetic animals and patients. 相似文献
15.
The uptake of L-phenylalanine into brush border microvilli vesicles and basolateral plasma membrane vesicles isolated from rat kidney cortex by differential centrifugation and free flow electrophoresis was investigated using filtration techniques. Brush border microvilli but not basolateral plasma membrane vesicles take up L-phenylalanine by an Na+-dependent, saturable transport system. The apparent affinity of the transport system for L-phenylalanine is 6.1 mM at 100 mM Na+ and for Na+ 13mM at 1 mM L-phenylalanine. Reduction of the Na+ concentration reduces the apparent affinity of the transport system for L-phenylalanine but does not alter the maximum velocity. In the presence of an electrochemical potential difference of Na+ across the membrane (etaNao greater than etaNai) the brush border microvilli accumulate transiently L-phenylalanine over the concentration in the incubation medium (overshoot pheomenon). This overshoot and the initial rate of uptake are markedly increased when the intravesicular space is rendered electrically more negative by membrane diffusion potentials induced by the use of highly permeant anions, of valinomycin in the presence of an outwardly directed K+ gradient and of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence of an outward-directed proton gradient. These results indicate that the entry of L-phenylalanine across the brush border membrane into the proximal tubular epithelial cells involves cotransport with Na+ and is dependent on the concentration difference of the amino acid, on the concentration difference of Na+ and on the electrical potential difference. The exit of L-phenylalanine across the basolateral plasma membranes is Na+-independent and probably involves facilitated diffusion. 相似文献
16.
The Gibbs-Donnan near-equilibrium system of heart 总被引:3,自引:0,他引:3
The gradients of the major inorganic ions across the plasma membrane of heart were examined to determine the factors controlling the extent and direction of the changes induced during injury, certain diseases, and electrolyte disturbances. The ionic environment was altered by changing only the concentration of inorganic phosphate, [sigma Pi]o, from 0 to 1.2 to 5 mM in the Krebs-Henseleit buffer perfusing working rat hearts. Raising [sigma Pi]o from 1.2 to 5 mM resulted in a decrease in total Mg2+ content and calculated free cytosolic [Mg2+] from 0.44 to 0.04 mM, conversion of 4 mmol of MgATP2- to ATP4- and a decrease in measured intracellular [Cl-]i from 41 to 16 mM. At all levels of [sigma Pi]o, both the [Na+]i and [K+]i were invariant at about 3 mM and 130 mM, respectively, as was the energy of hydrolysis of the terminal phosphate bond of sigma ATP, delta GATP Hydr, of -13.2 kcal/mol. The relationship maintained between the ions on both sides of the plasma membrane by the 3Na+/2K(+)transporting ATPase (EC 3.6.1.37) and an open K+ channel was: (formula; see text) The energy of the gradients of the other inorganic ions across the plasma membrane, delta G[ion]o/i, exhibited three distinct quanta of energy derived from the prime quantum of delta GATP Hydr of -13.2 kcal/mol. The second quantum was about one-third of delta GATP Hydr or +/- 4.4 kcal/mol and comprised the delta G[Na+]o/i, delta G[Mg2+]o/i, and delta G[HPO42-]o/i. These results indicated near-equilibrium was achieved by the reactants of the 3Na+/2K(+)-ATPase, the K+ channel, the Na(+)-Pi co-transporter, and a postulated net Mg2+/H2PO4- exchanger. The third quantum was one-third of delta G[Na+]o/i or about +/- 1.5 kcal/mol and comprised delta G[H+]o/i, delta G[HCO3-]o/i, and delta G[Cl-]o/i. The delta G[K+]o/i was 0, indicating near-equilibrium between the chemical energy of [K+]o/i and the E across the plasma membrane of -83 mV. It is concluded that the gradients of the major inorganic ions across the plasma membrane and the potential across that membrane constitute a Gibbs-Donnan equilibrium system catalyzed by transport enzymes sharing common substrates. The chemical and electrical energies of those gradients are equal in magnitude and opposite in sign to the chemical energy of ATP hydrolysis. 相似文献
17.
The freshwater cyanobacterium Synechococcus PCC 6311 is able to adapt to grow after sudden exposure to salt (NaCl) stress. We have investigated the mechanism of Na+ transport in these cells during adaptation to high salinity. Na+ influx under dark aerobic conditions occurred independently of delta pH or delta psi across the cytoplasmic membrane, ATPase activity, and respiratory electron transport. These findings are consistent with the existence of Na+/monovalent anion cotransport or simultaneous Na+/H+ +anion/OH- exchange. Na+ influx was dependent on Cl-, Br-, NO3-, or NO2-. No Na+ uptake occurred after addition of NaI, NaHCO3, or Na2SO4. Na+ extrusion was absolutely dependent on delta pH and on an ATPase activity and/or on respiratory electron transport. This indicates that Na+ extrusion via Na+/H+ exchange is driven by primary H+ pumps in the cytoplasmic membrane. Cells grown for 4 days in 0.5 m NaCl medium, "salt-grown cells," differ from control cells by a lower maximum velocity of Na+ influx and by lower steady-state ratios of [Na+]in/[Na+]out. These results indicate that cells grown in high-salt medium increase their capacity to extrude Na+. During salt adaptation Na+ extrusion driven by respiratory electron transport increased from about 15 to 50%. 相似文献
18.
Inhibition by glucocorticoids of phosphate transport in primary cultured renal cells 总被引:4,自引:0,他引:4
The regulation by glucocorticoids of phosphate transport in primary cultured chick renal cells was examined. Dexamethasone inhibited the Na+-dependent phosphate uptake system. Na+-independent phosphate uptake and Na+-dependent uptakes of alpha-methylglucoside and L-proline were unaffected. The mineralocorticoid aldosterone did not alter phosphate uptake. The inhibition of Na+-dependent phosphate uptake by dexamethasone was concentration-dependent, exhibited an induction period, was blocked by inhibitors of RNA and protein synthesis, and was rapidly reversed when the steroid was removed. Following reversal, the cells could respond a second time to the glucocorticoid. However, this time the response was rapid, could be evoked at least for 24 h after glucocorticoid withdrawal, and might be prevented by actinomycin D and cycloheximide. These findings demonstrate that glucocorticoids act on renal cells to modulate phosphate transport and suggest that the renal cell system provides an attractive model to examine the mechanism by which glucocorticoids control gene expression and regulate plasma membrane transport function. 相似文献
19.
Purified plasma membrane vesicles from GH3 rat anterior pituitary cells exhibit a Mg2+-ATP-dependent Ca2+ transport activity. Concentrative uptake of Ca2+ is abolished by exclusion of either Mg2+ or ATP or by inclusion of the Ca2+ ionophore A23187. Furthermore, addition of A23187 to vesicles which have reached a steady state of ATP-supported Ca2+ accumulation rapidly and completely discharges accumulated cation. Ca2+ uptake is unaffected by treatment of vesicles with oligomycin, the uncoupler CCCP, or valinomycin and is greatly reduced in non-plasma membrane fractions. Likewise, Ca2+ accumulation is not stimulated by oxalate, consistent with the plasma membrane origin of this transport system. (Na+, K+)-ATPase participation in the Ca2+ transport process (i.e. via coupled Na+/Ca2+ exchange) was eliminated by omitting Na+ and including ouabain in the reaction medium. Ca2+ transport activity in GH3 vesicles has a similar pH dependence as that seen in a number of other plasma membrane systems and is inhibited by orthovanadate in the micromolar range. Inhibition is enhanced if the membranes are preincubated with vanadate for a short time. A kinetic analysis of transport indicates that the apparent Km for free Ca2+ and ATP are 0.7 and 125 microM, respectively. The average Vmax is 3.6 nmol of Ca2+/min/mg of protein at 37 degrees C. Addition of exogenous calmodulin or calmodulin antagonists had no significant effect on these kinetic properties. GH3 plasma membranes also contain a Na+/Ca2+ exchange system. The apparent Km for Ca2+ is almost 10-fold higher in this system than that for ATP-driven Ca2+ uptake. When both processes are compared under similar conditions, the Vmax of the exchanger is approximately 2-3 times that of ATP-dependent Ca2+ accumulation. Similar results are obtained when purified plasma membranes from bovine anterior pituitary glands were investigated. It is suggested that both Na+/Ca2+ exchange and the (Ca2+ + Mg2+)-ATPase are important in controlling intracellular levels of Ca2+ in anterior pituitary cells. 相似文献
20.
Amiloride-sensitive Na+ channels are expressed at the apical membrane of high resistance, Na+-transporting epithelial. The specific interaction of amiloride with this transport protein suggested the feasibility of raising anti-Na+ channel antibodies by an antiidiotypic approach designed to generate antibodies directed against the amiloride-binding domain on the channel. Antiidiotypic monoclonal antibody RA6.3 mimicked the effect of amiloride by inhibiting Na+ transport across A6 cell monolayers when applied to the apical cell surface. Inhibition of transport required pretreatment of the apical cell surface with trypsin in the presence of amiloride in order to enhance accessibility of the antibody to the amiloride-binding site. This antibody specifically immunoprecipitated a large 750,000-700,000 Da protein from [35S]methionine-labeled A6 cell cultures, which was resolved further under reducing conditions as a set of polypeptides with apparent molecular masses of 260,000-230,000, 180,000, 140,000-110,000, and 70,000 Da. The antibody recognized the 140,000-Da subunit, known to contain the amiloride-binding domain, on immunoblots of purified A6 cell Na+ channel. Immunoprecipitation of apical or basolateral plasma membrane proteins selectively labeled with 125I demonstrated that expression of the oligomeric Na+ channel was restricted to the apical plasma membrane. Immunocytochemical localization in A6 cultures revealed apical membrane as well as cytosolic immunoreactive sites. Immunostaining was also observed at or near the basolateral plasma membrane. 相似文献