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At least 55 records · Page 3Linked to original sources
PubMed2005

Delineation of human peptide transporter 1 (hPepT1)-mediated uptake and transport of substrates with varying transporter affinities utilizing stably transfected hPepT1/Madin-Darby canine kidney clones and Caco-2 cells.

In the present investigation, the uptake and transport kinetics of valacyclovir (VACV), 5-aminolevulinic acid (5-ALA), and benzylpenicillin (BENZ) were studied in stably transfected Madin-Darby canine kidney (MDCK)/human peptide transporter 1 (hPepT1)-V5&His clonal cell lines expressing varying levels of epitope-tagged hPepT1 protein (low, medium, and high expression) and in Caco-2 cells to delineate hPepT1-mediated transport kinetics. These compounds were selected due to the fact that they are known PepT1 substrates, yet also have affinity for other transporters. Caco-2 cells, traditionally used for studying peptide-based drug transport, were included for comparison purposes. The time, pH, sodium, and concentration dependence of cellular uptake and permeability were measured using mock, clonal hPepT1-MDCK, and Caco-2 cells. A pH-dependent effect was observed in the hPepT1-expressing clones and Caco-2 cells, with an increase of 1.96-, 1.84-, and 2.05-fold for VACV, 5-ALA, and BENZ uptake, respectively, at pH 6 versus 7.4 in the high-expressing hPepT1 cells. BENZ uptake was significantly decreased in Caco-2 and MDCK cells in Na(+)-depleted buffer, whereas VACV uptake only decreased in Caco-2 cells. Concentration-dependent uptake studies in the mock-corrected hPepT1-MDCK and Caco-2 cells demonstrated hPepT1 affinity ranking of VACV > 5-ALA > BENZ. The apical-to-basal apparent permeability coefficient (P(app)) values of VACV, 5-ALA, and BENZ in mock-corrected hPepT1-MDCK cells showed solely hPepT1-mediated transport in contrast to Caco-2 cells. Lower K(m) values and higher P(app) in Caco-2 cells compared with hPepT1-MDCK cells suggested the involvement of multiple transporters in Caco-2 cells. Thus, hPepT1-MDCK cells corrected for endogenous transporter expression may be a more appropriate model for screening compounds for their affinity to hPepT1.

Acyclovir↗
PubMed1999

Interaction of kyotorphin and brain peptide transporter in synaptosomes prepared from rat cerebellum: implication of high affinity type H+/peptide transporter PEPT2 mediated transport system.

High-affinity type H+/peptide cotransporter PEPT2 is preferentially expressed in the kidney, and is responsible for reabsorption of di- and tripeptides in epithelial tubules. Interestingly, PEPT2 has been recently cloned from rat brain. However, there is very little information available on the peptide transporter activity in the brain. In the present study, we investigated the interaction of kyotorphin (L-tyrosyl-L-arginine) with the peptide transporter using synaptosomes prepared from rat cerebellum. The activity of the peptide transporter was assessed by measuring the uptake of radiolabeled glycyl-sarcosine (Gly-Sar), which is a prototypical substrate for the peptide transporter, in the presence of H+-gradient. Kyotorphin competitively inhibited the uptake of Gly-Sar with an inhibitory constant (Ki) of 30 +/- 4 microM in rat cerebellum synaptosomes. This uptake property is very close to that of PEPT2. Carnosine (beta-alanyl-L-histidine) also inhibited the uptake of Gly-Sar, on the other hand, TRH did not interact with the peptide transporter. RT-PCR using specific primers revealed that PEPT2 mRNA exists in cerebellum in rat. Taken collectively, these results indicate that the functional peptide transport system in rat cerebellum might be the high affinity transporter PEPT2.

Animals↗
PubMed1998

Acute regulation of norepinephrine transport: I. protein kinase C-linked muscarinic receptors influence transport capacity and transporter density in SK-N-SH cells.

Using SK-N-SH cells, we observe that muscarinic acetylcholine receptor activation by methacholine (MCh) rapidly and selectively diminishes l-NE transport capacity (Vmax) with little or no change in norepinephrine (NE) Km and without apparent effects on membrane potential monitored directly under current clamp. Over the same time frame, MCh exposure reduces the density of [3H]nisoxetine binding sites (Bmax) in intact cells but not in total membrane fractions, consistent with a loss of transport capacity mediated by sequestration of transporters rather than changes in intrinsic transport activity or protein degradation. Similar changes in NE transport and [3H]nisoxetine binding capacity are observed after phorbol ester (beta-PMA) treatment. Inhibition of PKC by antagonists and downregulation of PKC by chronic treatment with phorbol esters abolishes beta-PMA-mediated effects but produce only a partial blockade of MCh-induced effects. Neither muscarinic acetylcholine receptor nor PKC activation require extracellular Ca++ to diminish NET activity. In contrast, treatment of cells with the Ca++/ATPase antagonist, thapsigargin in Ca++-free medium, eliminates the staurosporine-insensitive component of MCh regulation. These findings were further corroborated by the ability of [1, 2-bis(o-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester application in Ca++-free medium to abolish NET regulation by MCh. Although they may contribute to basal NET expression, we could not implicate CaMKII-, PKA- or nitric oxide-linked pathways in MCh regulation. Together, these findings 1) provide evidence in support of G-protein coupled receptor-mediated regulation of catecholamine transport, 2) reveal intracellular Ca++-sensitive, PKC-dependent and -independent pathways that serve to regulate NET expression and 3) indicate that the diminished capacity for NE transport evident after mAChR and PKC activation involves a redistribution of NET protein.

Biological Transport↗
PubMed2002

Human organic anion transporters and human organic cation transporters mediate renal transport of prostaglandins.

Prostaglandin E(2) (PGE(2)) and prostaglandin F(2 alpha) (PGF(2 alpha)) have been used for the induction of labor and the termination of pregnancy. Renal excretion is shown to be an important pathway for the elimination of PGE(2) and PGF(2 alpha). The purpose of this study was to elucidate the molecular mechanism of renal PGE(2) and PGF(2 alpha) transport using cells stably expressing human organic anion transporter (hOAT) 1, hOAT2, hOAT3, and hOAT4, and human organic cation transporter (hOCT) 1 and hOCT2. A time- and dose-dependent increase in PGE(2) and PGF(2 alpha) uptake was observed in cells expressing hOAT1, hOAT2, hOAT3, hOAT4, hOCT1, and hOCT2. The K(m) values of PGE(2) uptake by hOAT1, hOAT2, hOAT3, hOAT4, hOCT1, and hOCT2 were 970, 713, 345, 154, 657, and 28.9 nM, respectively, whereas those of PGF(2 alpha) uptake by hOAT1, hOAT3, hOAT4, hOCT1, and hOCT2 were 575, 1092, 692, 477, and 334 nM, respectively. PGE(2) and PGF(2 alpha) significantly inhibited organic anion uptake by hOATs and organic cation uptake by hOCTs. In conclusion, considering the localization of these transporters, the results suggest that PGE(2) and PGF(2 alpha) transport in the basolateral membrane of the proximal tubule is mediated by multiple pathways including hOAT1, hOAT2, hOAT3, and hOCT2, whereas that in the apical side is mediated by hOAT4.

Animals↗
PubMed1998

Replacement of Ala-166 with cysteine in the high affinity rabbit sodium/glucose transporter alters transport kinetics and allows methanethiosulfonate ethylamine to inhibit transporter function.

An alanine to cysteine mutation at position 166 has been introduced by site-directed mutagenesis into the rabbit sodium/glucose transporter (rSGLT1). When expressed in Xenopus laevis oocytes, this mutant transporter (A166C rSGLT1) demonstrates a significantly lower apparent affinity for alpha-methyl glucoside (alphaMG) compared with the wild-type transporter (apparent Km = 0.8 versus 0.15 mM). Using the two-electrode voltage clamp technique, transient currents have also been measured, and for the mutant transporter, the transients induced by large depolarizations exhibit longer time constants than those for wild type. Moreover, the substitution of Ala-166 with a cysteine allows the sulfydryl specific reagent, methanethiosulfonate ethylamine (MTSEA), to react with and alter the function of the transporter. Whereas the wild-type transporter is unaffected by reaction with MTSEA, A166C rSGLT1 has its steady-state currents induced by 1 mM alphaMG inhibited 83% within a minute of exposure to MTSEA. Furthermore, the pre-steady-state transients of the A166C mutant after MTSEA exposure demonstrate much shorter time constants than before while the total amount of charge transferred is only slightly diminished. These results together provide evidence that position 166 is situated in a region critical to the functioning of rSGLT1.

Alanine↗
PubMed2004

Homocysteine and the renal epithelial transport and toxicity of inorganic mercury: role of basolateral transporter organic anion transporter 1.

The epithelial cells that line the renal proximal tubule have been shown to be the primary cellular targets where mercuric ions gain entry, accumulate, and induce pathologic effects in vivo. Recent data have implicated at least one of the organic anion transport systems in the basolateral uptake of inorganic mercury (Hg). With the use of a line of type II MDCK cells transfected stably with the human organic anion transporter 1 (hOAT1), the hypothesis that hOAT1 can transport mercuric conjugates of homocysteine (Hcy) was tested. Indeed, MDCK II cells expressing a functional form of hOAT1 gained the ability to transport the mercuric conjugate 2-amino-4-(3-amino-3-carboxy-propylsulfanylmercuricsulfanyl) butyric acid (Hcy-S-Hg-S-Hcy). In addition, p-aminohippurate and the dicarboxylates adipate and glutarate (but not succinate or malonate) inhibited individually the uptake of Hcy-S-Hg-S-Hcy in a concentration-dependent manner. Furthermore, a direct relationship between the uptake of Hcy-S-Hg-S-Hcy and the induction of cellular injury and death was demonstrated in the hOAT1-expressing MDCK II cells only. These data represent the first line of direct evidence implicating one of the organic anion transporters in the uptake of a mercuric conjugate of Hcy in a mammalian cell. Thus, mercuric conjugates of Hcy are potential transportable substrates of OAT1. More important, the findings from the present study implicate the activity of OAT1 in the uptake and toxicity of Hg (when in the form of Hcy-S-Hg-S-Hcy in the extracellular compartment) in proximal tubular epithelial cells in vivo.

Adipates↗
PubMed1993

Regulation of the human serotonin transporter. Cholera toxin-induced stimulation of serotonin uptake in human placental choriocarcinoma cells is accompanied by increased serotonin transporter mRNA levels and serotonin transporter-specific ligand binding.

Treatment of confluent cultures of JAR human placental choriocarcinoma cells with cholera toxin or forskolin for 16 h markedly stimulated (2.4-fold) serotonin transport activity in these cells. Cycloheximide, an inhibitor of protein synthesis or actinomycin D, an inhibitor of mRNA synthesis effectively blocked this stimulation. Northern blot analysis revealed that treatment with cholera toxin resulted in severalfold increase in the concentrations of the three mRNA species (6.8, 4.9 and 3.0 kilobases in size) which hybridized to the human placental serotonin transporter cDNA. Under similar conditions, the concentrations of the mRNA species which hybridized to the human placental taurine transporter cDNA or to the human beta-actin cDNA were not affected. Analysis of paroxetine-sensitive binding of the cocaine analog 2 beta-carbomethoxy-3 beta-(4- [125I]iodophenyl)tropane to the membranes prepared from control and cholera toxin-treated cells indicated that the maximal binding capacity was increased 2.5-fold by cholera toxin, with no significant change in the binding affinity. Thus, stimulation of serotonin transporter activity in the placental choriocarcinoma cells following cholera toxin treatment is likely a result of an increase in cell surface density of the serotonin transporter protein as a consequence of increased steady state serotonin transporter mRNA levels.

Binding Sites↗
PubMed2005

Further structure-activity relationship studies of piperidine-based monoamine transporter inhibitors: effects of piperidine ring stereochemistry on potency. Identification of norepinephrine transporter selective ligands and broad-spectrum transporter inhibitors.

4-(4-Chlorophenyl)piperidine analogues each bearing a thioacetamide side chain appendage similar to that found in the wake-promoting drug modafinil have been synthesized. The transporter inhibitory activity of both the cis and trans isomers of these 3,4-disubstituted piperidines in both their (+)- and (-)-enantiomeric forms was determined. These studies reveal that the (-)-cis analogues exhibit dopamine transporter/norepinephrine transporter (DAT/NET) selectivity as was previously reported for the (+)-trans analogues. On the other hand, the (-)-trans and the (+)-cis isomers show serotonin transporter (SERT) or SERT/NET selectivity. Among them, (+)-cis-5b shows a low nanomolar Ki for the NET with 39-fold and 321-fold lower potency at the DAT and SERT, respectively, thus making it a useful pharmacological research tool for exploring NET-associated behavioral signatures. On the other hand, several of the compounds described herein, such as (+)-trans-5c, show comparable activity at all three transporters. Because broad-spectrum transporter inhibitors have been hypothesized to exhibit a more rapid onset of action and/or a greater efficacy as antidepressant agents than those selective for SERT or SERT + NET, some of the present compounds will be valuable to study in animal models of depression.

Animals↗
PubMed2000

Nucleoside transporter proteins of Saccharomyces cerevisiae. Demonstration of a transporter (FUI1) with high uridine selectivity in plasma membranes and a transporter (FUN26) with broad nucleoside selectivity in intracellular membranes.

FUI1 and function unknown now 26 (FUN26) are proteins of uncertain function with sequence similarities to members of the uracil/allantoin permease and equilibrative nucleoside transporter families of transporter proteins, respectively. [(3)H]Uridine influx was eliminated by disruption of the gene encoding FUI1 (fui1) and restored by expression of FUI1 cDNA, whereas influx in transport-competent and fui1-negative yeast were unaffected, respectively, by disruption of the FUN26 gene or overexpression of FUN26 cDNA. FUI1 transported uridine with high affinity (K(m), 22 +/- 3 micrometer) and was unaffected or inhibited only partially by high concentrations (1 mm) of a variety of ribo- and deoxyribonucleosides or nucleobases. When FUN26 cDNA was expressed in oocytes of Xenopus laevis, inward fluxes of [(3)H]uridine, [(3)H]adenosine, and [(3)H]cytidine were stimulated, and uridine influx was independent of pH and not inhibited by dilazep, dipyridamole, or nitrobenzylmercaptopurine ribonucleoside. Fractionation of yeast membranes containing immunotagged recombinant FUN26 (shown to be functional in oocytes) demonstrated that the protein was primarily in intracellular membranes. These results indicated that FUI1 has high selectivity for uracil-containing ribonucleosides and imports uridine across cell-surface membranes, whereas FUN26 has broad nucleoside selectivity and most likely functions to transport nucleosides across intracellular membranes.

Animals↗
PubMed2002

Human organic anion transporters and human organic cation transporters mediate renal antiviral transport.

Renal excretion is an important elimination pathway for antiviral agents, such as acyclovir (ACV), ganciclovir (GCV), and zidovudine (AZT). The purpose of this study was to elucidate the molecular mechanisms of renal ACV, GCV, and AZT transport using cells stably expressing human organic anion transporter 1 (hOAT1), hOAT2, hOAT3, and hOAT4, and human organic cation transporter 1 (hOCT1) and hOCT2. Time- and concentration-dependent uptake of ACV and GCV was observed in hOAT1- and hOCT1-expressing cells. In contrast, uptake of valacyclovir, L-valyl ester of ACV, was observed only in hOAT3-expressing cells. On the other hand, AZT uptake was observed in hOAT1-, hOAT2-, hOAT3-, and hOAT4-expressing cells. The Km values of ACV uptake by hOAT1 and hOCT1 were 342.3 and 151.2 microM, respectively, whereas those of GCV uptake by hOAT1 and hOCT1 were 895.5 and 516.2 microM, respectively. On the other hand, the Km values of AZT uptake by hOAT1, hOAT2, hOAT3, and hOAT4 were 45.9, 26.8, 145.1, and 151.8 microM, respectively. In addition, probenecid weakly inhibited the hOAT1-mediated ACV uptake. In conclusion, these results suggest that hOAT1 and hOCT1 mediate renal ACV and GCV transport, whereas hOAT1, hOAT2, hOAT3, and hOAT4 mediate renal AZT transport. In addition, L-valyl ester appears to be important in differential substrate recognition between hOAT1 and hOAT3. hOAT1 may not be the molecule responsible for the drug interaction between ACV and probenecid.

Acyclovir↗
PubMed1981

A Fickian diffusion transport process with features of transport catalysis. Doxorubicin transport in human red blood cells.

The transport of the antineoplastic drug doxorubicin (Adriamycin) in human red blood cells was investigated by measuring the net efflux from loaded cells. Previous data indicated that doxorubicin transport was a Fickian diffusion transport process of the electrically neutral molecule through the lipid domain of the cell membrane (Dalmark, 1981 [In press]). However, doxorubicin transport showed saturation kinetics and a concentration-dependent temperature dependence with nonlinear Arrhenius plots. The two phenomena were related to the doxorubicin partition coefficient between 1-octanol and a water phase. This relationship indicated that the two phenomena were caused by changes in the physiochemical properties of doxorubicin in the aqueous phase and were not caused by interaction of doxorubicin with cell membrane components. The physicochemical properties of doxorubicin varied with concentration and temperature because of the ability of doxorubicin to form polymers by self-association in aqueous solution like other planar aromatic molecules through pi-electron orbital interaction. The hypothesis is proposed that doxorubicin transport across cell membranes takes place by simple Fickian diffusion.

Biological Transport↗
PubMed1990

Glucose transport in fish erythrocytes: variable cytochalasin-B-sensitive hexose transport activity in the common eel (Anguilla japonica) and transport deficiency in the paddyfield eel (Monopterus albus) and rainbow trout (Salmo gairdneri).

Erythrocytes from individual common eels (Anguilla japonica Temminck and Schlegel) exhibited widely variable initial rates of cytochalasin-B-sensitive 3-O-methyl-D-glucose (3-OMG) zero-trans influx, in the range of 0-19.5 mmol l-cells-1 h-1 (5 mmol l-1 extracellular concentration at 20 degrees C, 50 animals tested). Storage of cells at 4 degrees C in a glucose-containing medium for up to 72 h had no effect on 3-OMG uptake, and there was no correlation between the sugar permeabilities of erythrocytes from different fish and intracellular ATP levels. Adrenaline and noradrenaline increased cytochalasin-B-sensitive 3-OMG transport activity; half-maximal stimulation occurred at catecholamine concentrations in the region of 1 mumol l-1. This catecholamine-induced stimulation of sugar transport appeared to be independent of the basal cytochalasin-B-sensitive 3-OMG permeability of the cells. Kinetically, catecholamines increased the Vm of transport without changing the apparent Km (approx. 1.4 mmol l-1). Saturable 3-OMG influx was inhibited by phloretin, D-glucose, D-deoxyglucose and D-galactose, but not by D-fructose and L-glucose. Transporter stereoselectivity was confirmed by direct measurements of D- and L-glucose uptake. Erythrocytes from two other fish species, Monopterus albus Richardson (paddyfield eel) and Salmo gairdneri Richardson (rainbow trout), unlike those from the common eel, were uniformly deficient with respect to cytochalasin-B-sensitive 3-OMG and D-glucose transport activity. Catecholamines had no effect on sugar uptake in these species.

3-O-Methylglucose↗
PubMed1991

Primaquine blocks transport by inhibiting the formation of functional transport vesicles. Studies in a cell-free assay of protein transport through the Golgi apparatus.

The lysosomotropic amine primaquine has previously been shown to inhibit both secretory and recycling processes of cells in culture. We have used a cell-free assay that reconstitutes glycoprotein transport through the Golgi apparatus to investigate the mechanism of action of primaquine. In this assay, primaquine inhibits protein transport at a half-maximal concentration of 50 microM, similar to the concentration previously reported to disrupt protein secretion in cultured cells. Kinetic analysis of primaquine inhibition indicates that its point of action is at an early step in the vesicular transport mechanism. Primaquine does not inhibit the fusion of vesicles already attached to their target membranes. Primaquine irreversibly inactivates the membranes that form transport vesicles (donor), but not the membranes that are the destination of those vesicles (acceptor). Morphological data indicate that primaquine inhibits the budding of vesicles from the donor membranes. Once formed, the vesicles are refractile to primaquine action, and their attachment to and fusion with acceptor membranes proceeds unimpeded. In addition to illuminating the mechanism of action of primaquine, this study suggests that the selective action of this agent will make it a useful tool in the study of the formation of transport vesicles.

Animals↗
PubMed1987

[Physico-chemical mechanisms of organic acid transport in the apical membrane cells of the proximal kidney tubules in rats. I. Kinetic transport parameters and effect of the lipid phasic state on transport].

The kinetics of the transport of 3H-para-aminohippuric acid (PAH) and the influence of the temperature on the initial rate of transport were studied on the vesicles of a purified fraction of the apical membrane isolated from cells of kidney proximal tubules. The PAH transport is accomplished owing to the facilitate diffusion mechanism. The apparent Michaelis constant at 36 degrees C was equal to 7.0 + 1.0 mM, the maximum rate was 15 nmol/min on 1 mg of protein, the inhibition constant for the PAH transport by probenecid being 0.5 mM. At 22 degrees C the apparent Michaelis constant was drastically increased. When the temperature dependence of the initial rate of PAH transport into vesicles was replotted in the form of the Arrhenius plot, there was a turning-point of the line at 28-30 degrees C. The same turning-point is shown on the Arrhenius plot for temperature dependence of alkaline phosphatase activity (a marker enzyme for the apical membrane). The electron paramagnetic resonance spectra analysis of 5-doxylstearate-labeled apical membrane preparation reveals a thermotropic transition near 21-29 degrees C. It is concluded that the function of the carrier and the activity of alkaline phosphatase depend on the phasic state of membrane lipids; the normal function of membrane proteins is possible under the liquid-crystalline state of the lipid bilayer.

Aminohippuric Acids↗
PubMed1980

Effects of metabolic and transport inhibitors on sodium transport and CO2 production of toad bladders: use of an uncoupling agent to distinguish between effects on transport and metabolism.

Agents that directly inhibit sodium transport secondary reduce the rat of metabolism. Conversely, agents that directly inhibit energy metabolism cause a secondary inhibition of active transport. The aim of the present investigation was to distinguish between these two effects with an uncoupling agent, 2,4-dinitrophenol (DNP). The rates of sodium transport (TNa) and CO2 production (QCO2) by paired urinary hemibladders from Dominican toads were simultaneously measured. DNP alone inhibited TNa, but stimulated QCO2 of the hemibladders, consistent with its action as an uncoupling agent. Ouabain, which directly inhibits sodium transport, inhibited TNa and QCO2 of toad hemibladders; the subsequent addition of DNP stimulated QCO2 to a rate similar to that of the control hemibladders. The metabolic inhibitors antimycin A and rotenone also inhibited TNa and QCO2; in contrast to the results with ouabain, the subsequent addition of DNP did not stimulate QCO2 in the presence of antimycin A or rotenone. Therefore, the respiratory response to DNP clearly distinguishes between direct inhibition of metabolism and direct inhibition of active transport. This approach may be useful for the investigation of the mechanism of action of drugs, hormones and other biological phenomena.

Animals↗
PubMed1991

Glucose transport of Haloferax volcanii requires the Na(+)-electrochemical potential gradient and inhibitors for the mammalian glucose transporter inhibit the transport.

The uptake of glucose and its non-metabolizing analogues by Haloferax volcanii, one of the glucose-utilizing Halobacteria, was examined using intact cells and envelope vesicles. Results obtained were: (1) The transport system is inducible. (2) The uptake requires the gradient of Na(+)-electrochemical potential. (3) Inhibitors for mammalian glucose transport also have an effect on this system, implying that the transporters resemble each other. (4) It is suggested that the mobility of the transporter is regulated by the membrane energization.

Biological Transport, Active↗
PubMed1991

Location of the Ca(2+)-transport site of Ca(2+)-transporting ATPase of the sarcoplasmic reticulum as determined by analysis of paramagnetic interaction between Gd3+ ions bound at the transport site and membrane-embedded nitroxide spin probes.

Gd3+ ions were bound to the Ca(2+)-transport site of Ca(2+)-transporting ATPase of the sarcoplasmic reticulum (SR-ATPase) and their effect on the ESR spectrum of spin-probes, which were attached to specific sites on SR-ATPase and embedded in the membranous lipid at various depths from the surface of the membrane, was studied. Spin-labeled reagents, 1-oxyl-2,2-dimethyl-oxazolidine derivatives of maleimidoethyl-keto stearate, collectively abbreviated as MSL(m,n) were mainly used for labeling SR-ATPase. They have Cm- and Cn-hydrocarbon chains, respectively, on both sides of the spin label, of which the Cm-hydrocarbon chain is located distal to the carboxyl group of the keto stearate moiety. Paramagnetic interaction between Gd3+ and a spin probe was detected by measuring the decrease in the intensity of the ESR signal of the probe. Displacement of Gd3+ from the Ca(2+)-transport site by Ca2+, which had been confirmed previously by using fluorescently labeled SR-ATPase (described in the preceding article), led to a significant reversal of the paramagnetic effect of Gd3+ on MSL(12,3) and MSL(10,5) attached to SR-ATPase. On the other hand, the effect of Gd3+ was not reversed by Ca2+ when SR-ATPase labeled with MSL(1,14) or a spin-label specific for the cytoplasmic domain was used. These results led us to conclude that the Ca(2+)-transport site of SR-ATPase is located in the membranous region of the molecule, but that the site is not very far from the surface of the membrane of the sarcoplasmic reticulum.

Animals↗