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1.
To design a reliable 3D QSAR model of the intestinal Na(+)/bile acid cotransporter, we have used a training set of 17 inhibitors of the rabbit ileal Na(+)/bile acid cotransporter. The IC(50) values of the training set of compounds covered a range of four orders of magnitude for inhibition of [(3)H]cholyltaurine uptake by CHO cells expressing the rabbit ileal Na(+)/bile acid cotransporter allowing the generation of a pharmacophore using the CATALYST algorithm. After thorough conformational analysis of each molecule, CATALYST generated a pharmacophore model characterized by five chemical features: one hydrogen bond donor, one hydrogen bond acceptor, and three hydrophobic features. The 3D pharmacophore was enantiospecific and correctly estimated the activities of the members of the training set. The predicted interactions of natural bile acids with the pharmacophore model of the ileal Na(+)/bile acid cotransporter explain exactly the experimentally found structure;-activity relationships for the interaction of bile acids with the ileal Na(+)/bile acid cotransporter (Kramer et al. 1999. J. Lipid. Res. 40: 1604;-1617). The natural bile acid analogues cholyltaurine, chenodeoxycholyltaurine, or deoxycholyltaurine were able to map four of the five features of the pharmacophore model: a) the five-membered ring D and the methyl group at position 18 map one hydrophobic site and the 21-methyl group of the side chain maps a second hydrophobic site; b) one of the alpha-oriented hydroxyl groups at position 7 or 12 fits the hydrogen bond donor feature; c) the negatively charged side chain acts as hydrogen bond acceptor; and d) the hydroxy group at position 3 does not specifically map any of the five binding features of the pharmacophore model. The 3-hydroxy group of natural bile acids is not essential for interactions with ileal or hepatic Na(+)/bile acid cotransporters. A modification of the 3-position of a natural bile acid molecule is therefore the preferred position for drug targeting strategies using bile acid transport pathways. 相似文献
2.
Functional studies of a chimeric protein containing portions of the Na(+)/glucose and Na(+)/myo-inositol cotransporters 总被引:1,自引:0,他引:1
Coady MJ Jalal F Bissonnette P Cartier M Wallendorff B Lemay G Lapointe J 《Biochimica et biophysica acta》2000,1466(1-2):139-150
We obtained cDNA chimeras between Na/glucose cotransporter (SGLT1) and the homologous Na(+)/myo-inositol cotransporter (SMIT) by creating random chimeras in plasmids. Of 12 chimeras, two were functional when expressed in Xenopus laevis oocytes but, upon sequencing, only one of them (C1) produced an actual chimeric protein. In C1, the first 69 amino acids of SGLT1 were replaced by the corresponding 50 amino acids of SMIT. C1 transports the same sugars as does SGLT1. C1's affinity for all sugar substrates was systematically increased by a factor of 3.3+/-0.4 but the V(max) was diminished by a factor of 15-40. In contrast, the cotransport affinity for Na(+) was unchanged. The surface expression of C1 was one seventh that of SGLT1, which explains part of the reduced V(max) and implies a significant reduction in turnover rate. N-terminal truncated constructs of SGLT1 cDNA showed that deleting amino acids 2-14 does not affect cotransporter activity, but that the pentapeptide T(14)RPVET(19) is important for normal levels of SGLT1 current. The main result of a kinetic analysis of the systematic increase in apparent affinity for sugars, together with the intact Na apparent affinity, suggests enhanced access to the sugar binding site in C1. 相似文献
3.
Saeki T Munetaka Y Ueda K Iwami K Kanamoto R 《Bioscience, biotechnology, and biochemistry》2007,71(8):1865-1872
Although ileal and hepatic Na(+)-dependent bile acid transporters (SLC10A2 and SLC10A1 respectively) share structural similarities, the mutation of conserved amino acids often has distinct effects on them. We have identified two Cys residues in mouse Slc10a2 (Cys(51) and Cys(106)) the replacement of which by Ala remarkably reduces taurocholic acid (TCA) transport. Although Cys(51) is conserved in Slc10a1 as Cys(44), Ala substitution gave no apparent difference in TCA uptake. Here, we further analyzed the kinetics of TCA uptake and cell surface localization of these mutants. The C51A and C106A mutants of Slc10a2 showed significantly reduced TCA uptake, while no apparent difference in TCA uptake was observed for the Slc10a1-C44A mutant. The K(m) values for TCA uptake by these mutants were comparable, suggesting that these residues are not involved in the interaction with TCA. 相似文献
4.
Saeki T Kuroda T Matsumoto M Kanamoto R Iwami K 《Bioscience, biotechnology, and biochemistry》2002,66(2):467-470
All cysteines of mouse ileal and hepatic sodium-dependent bile acid transporters (Isbt and Ntcp, respectively) were individually replaced by alanine. Replacement of Cys106 in Isbt and Cys96 in Ntcp, which are located closely in alignment, decreased taurocholate uptake. Although Cys51 in Isbt is conserved in Ntcp, the replacement spoiled Isbt only. Both similarity and difference in the arrangement of functional sites are suggested. 相似文献
5.
Sun AQ Arrese MA Zeng L Swaby I Zhou MM Suchy FJ 《The Journal of biological chemistry》2001,276(9):6825-6833
To understand the potential functions of the cytoplasmic tail of Na(+)/taurocholate cotransporter (Ntcp) and to determine the basolateral sorting mechanisms for this transporter, green fluorescent protein-fused wild type and mutant rat Ntcps were constructed and the transport properties and cellular localization were assessed in transfected COS 7 and Madin-Darby canine kidney (MDCK) cells. Truncation of the 56-amino acid cytoplasmic tail demonstrates that the cytoplasmic tail of rat Ntcp is involved membrane delivery of this protein in nonpolarized and polarized cells and removal of the tail does not affect the bile acid transport function of Ntcp. Using site-directed mutagenesis, two tyrosine residues, Tyr-321 and Tyr-307, in the cytoplasmic tail of Ntcp have been identified as important for the basolateral sorting of rat Ntcp in transfected MDCK cells. Tyr-321 appears to be the major basolateral-sorting determinant, and Tyr-307 acts as a supporting determinant to ensure delivery of the transporter to the basolateral surface, especially at high levels of protein expression. When the two Tyr-based basolateral sorting motifs have been removed, the N-linked carbohydrate groups direct the tyrosine to alanine mutants to the apical surface of transfected MDCK cells. The major basolateral sorting determinant Tyr-321 is within a novel beta-turn unfavorable tetrapeptide Y(321)KAA, which has not been found in any naturally occurring basolateral sorting motifs. Two-dimensional NMR spectroscopy of a 24-mer peptide corresponding to the sequence from Tyr-307 to Thr-330 on the cytoplasmic tail of Ntcp confirms that both the Tyr-321 and Tyr-307 regions do not adopt any turn structure. Since the major motif YKAA contains a beta-turn unfavorable structure, the Ntcp basolateral sorting may not be related to the clathrin-adaptor complex pathway, as is the case for many basolateral proteins. 相似文献
6.
This study was designed to examine the activity of the Na(+)-H+ exchanger across the basolateral membranes of the ileal enterocyte and its developmental pattern. The function of the Na(+)-H+ exchanger was studied using a well validated basolateral membrane vesicle technique. Na+ uptake represented transport into the vesicle rather than binding as validated by initial rate studies. Na+ uptake represented an electroneutral process as shown by studies in which negative membrane potential was induced by the ionophore valinomycin. Various outwardly directed pH gradients significantly stimulated Na+ uptake compared with no pH gradient conditions at all age groups. However, the magnitude of stimulation was significantly different between the age groups with more marked stimulation of amiloride-sensitive Na+ uptake occurring in adolescent rats as compared to weanling or suckling rats. The amiloride sensitivity of the pH stimulated Na+ uptake was investigated using [Amiloride] = 10(-2)-10(-5) M at pHi/pHo = 5.2/7.5. At 10(-2) M amiloride concentration, Na+ uptake was inhibited by 80%, 70%, 77%, in the basolateral membranes of adolescent, weanling and suckling rats, respectively. Dixon plot analysis in both adolescent and weanling rats was consistent with two amiloride binding sites, a low affinity system and a high affinity system. In the suckling rat, on the other hand, the data supported a single high affinity binding site. Kinetic studies revealed a Km for amiloride-sensitive Na+ uptake of 12.6 +/- 6.6, 10.2 +/- 1.77, 9.46 and Vmax of 4.83 +/- 1.22, 4.47 +/- 0.36 and 8.08 +/- 1.92 n.mol.mg.protein-1.7 s-1 in suckling, weanling and adolescent rats, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
7.
Molecular cloning and expression of a cDNA encoding the rabbit ileal villus cell basolateral membrane Na+/H+ exchanger. 总被引:2,自引:0,他引:2
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C M Tse A I Ma V W Yang A J Watson S Levine M H Montrose J Potter C Sardet J Pouyssegur M Donowitz 《The EMBO journal》1991,10(8):1957-1967
A cDNA clone encoding a rabbit ileal villus cell Na+/H+ exchanger was isolated and its complete nucleotide sequence was determined. The cDNA is 4 kb long and contains 322 bp of 5'-untranslated region, 2451 bp of open reading frame and 1163 bp of 3'-untranslated area, with 70%, 91% and 40% identity to the human sequence, respectively. Amino acid sequence deduced from the longest open reading frame indicated a protein of 816 residues (predicted Mr 90,716) which exhibits 95% amino acid identity to the human Na+/H+ exchanger. The two putative glycosylation sites in the human Na+/H+ exchanger are conserved in this protein, suggesting that it is a glycoprotein. Stable transfection of the cDNA into an Na+/H+ exchanger deficient fibroblast cell line, established Na+/H+ exchange. The Na+/H+ exchanger was stimulated by serum and a phorbol ester but not by 8-Br-cAMP. In Northern blot analysis, the cDNA hybridized to a 4.8 kb message in rabbit ileal villus cells, kidney cortex, kidney medulla, adrenal gland, brain and descending colon and to a 5.2 kb message in cultured human colonic cancer cell lines, HT29-18 and Caco-2. In immunoblotting, a polyclonal antibody raised against a fusion protein of beta-galactosidase and the C-terminal 158 amino acids of the human Na+/H+ exchanger identified a rabbit ileal basolateral membrane protein of 94 kd and only weakly interacted with the ileal brush border membrane. In immunocytochemical studies using ileal villus and crypt epithelial cells, the same antibody identified basolateral and not brush border epitopes. Restriction analysis of genomic DNA with a 462 bp PstI-AccI fragment of the rabbit Na+/H+ exchanger strongly suggests the existence of closely related Na+/H+ exchanger genes. The near identity of the basolateral Na+/H+ exchanger and the human Na+/H+ exchanger plus the ubiquitous expression of this message suggests that the ileal basolateral Na+/H+ exchanger is the 'housekeeping' Na+/H+ exchanger. 相似文献
8.
Lucie Parent Stéphane Supplisson Donald D. F. Loo Ernest M. Wright 《The Journal of membrane biology》1992,125(1):49-62
Summary Cystic fibrosis (CF) is characterized by abnormal epithelial Cl– conductance (GCl). In vitro studies that have shown that cAMP regulation is an intrinsic property of the CF-affected GCl(CF-GCl) have been carried out previously on cultured secretory cells and on nonepithelial cells. Even though GCl in absorption is defective in CF, a clear demonstration of cAMP regulation of CF-GCl in a purely absorptive tissue is lacking. We studied the cAMP regulation of CF-GCl in the microperfused intact human reabsorptive sweat duct. About 40% of the ducts responded to cAMP (responsive) while the remainder of the ducts did not. In responsive ducts, cAMP-elevating agents: -adrenergic agonist isoproterenol (IPR), CPT-cAMP, forskolin, theophylline or IBMX increased G
tby about 2.3-fold (n = no. of ducts = 8). Removal of media Cl–, but not amiloride pretreatment (in the lumen), abolished the cAMP response, indicating exclusive activation of GCl. cAMP activated both apical and basolateral GCl. cAMP hyperpolarized gluconate: Cl– (lumen: bath) transepithelial bionic potentials (V
t=–20.3±5.2 mV, mean ±se, n=9) and transepithelial 3 1 luminal NaCl dilution diffusion potentials (V
t=–8.8±2.9 mV, n=5). cAMP activated basolateral GCl as indicated by increased bi-ionic (gluconate: Cl–, bath: lumen) diffusion potentials (by about 12 mV). The voltage divider ratio in symmetric NaCl solutions increased by 60%. Compared to responsive ducts, nonresponsive ducts were characterized by smaller spontaneous transepithelial potentials in symmetrical Ringer's solution (V
t=–6.9±0.8 mV, n=24, nonresponsive vs. –19.4±1.8 mV, n=22, responsive ducts) but larger bi-ionic potentials (–94±6 mV, n=35, nonresponsive vs. –65±5 mV, n=17, responsive ducts) and dilution diffusion potentials (–40±5 mV, n=11, nonresponsive vs. –29±3 mV, n=7, responsive ducts). These results are consistent with an inherently (prestimulus) maximal activation of GCl in nonresponsive ducts and submaximal activation of GCl in responsive ducts. We conclude that cAMP activates CF-G
Cl which is expressed and abnormal in both apical and basal membranes of this absorptive epithelium in CF.Abbreviations CF
cystic fibrosis
-
G
t
transepithelial conductance
-
V
b
electrical potential across the basolateral membrane
-
V
a
electrical potential across the apical membrane
-
V
t
transepithelial potential
- V
b
transepithelial currentinduced voltage deflections across the basolateral membrane
- V
a
transepithelial current-induced voltage deflections across the apical membrane
- V
t
transepithelial current-induced voltage deflection across the epithelium
- VDR
voltage divider ratio
- GCl
transepithelial Cl– conductance
- CF-GCl
cystic fibrosis-affected Cl– conductance
- EMF
electromotive force
- IPR
isoproterenol
- IBMX
3-isobutyl-1-methylxanthine
- CPT-cAMP
chlorophenylthio-adenosine 3-5 cyclic monophosphate
- PGE2
prostaglandin E2 相似文献
9.
Hollenback D Bonham L Law L Rossnagle E Romero L Carew H Tompkins CK Leung DW Singer JW White T 《Journal of lipid research》2006,47(3):593-604
Membranes of mammalian cells contain lysophosphatidic acid acyltransferase (LPAAT) activities that catalyze the acylation of sn-1-acyl lysophosphatidic acid (lysoPA) to form phosphatidic acid. As the biological roles and biochemical properties of the six known LPAAT isoforms have yet to be fully elucidated, we have characterized human LPAAT-beta activity using two different assays. In a membrane-based assay, LPAAT-beta used lysoPA and lysophosphatidylmethanol (lysoPM) but not other lysophosphoglycerides as an acyl acceptor, and it preferentially transferred 18:1, 18:0, and 16:0 acyl groups over 12:0, 14:0, 20:0, and 20:4 acyl groups. The fact that lysoPM could traverse cell membranes permitted additional characterization of LPAAT-beta activity in cells: PC-3 and DU145 cells converted exogenously added lysoPM and (14)C-labeled 18:1 into (14)C-labeled phosphatidylmethanol (PM). The rate of PM formation was higher in cells that overexpressed LPAAT-beta and was inhibited by the LPAAT-beta inhibitor CT-32501. In contrast, if lysoPM and (14)C-labeled 20:4 were added to PC-3 or DU145 cells, (14)C-labeled PM was also formed, but the rate was neither higher in cells that overexpressed LPAAT-beta nor inhibited by CT-32501. We propose that LPAAT-beta catalyzes the intracellular transfer of 18:1, 18:0, and 16:0 acyl groups but not 20:4 groups to lysoPA. 相似文献
10.
Substrate and inhibitor specificity of anion exchangers on the brush border membrane of rabbit ileum
Roy G. Knickelbein Peter S. Aronson John W. Dobbins 《The Journal of membrane biology》1985,88(2):199-204
Summary In previous studies we have found that several anions can be transported by an exchange process in rabbit ileal brush border membranes. We demonstrated exchanges of Cl for OH or HCO3, SO4 for OH, oxalate for OH, and oxalate for Cl. The purpose of these studies was to determine the number of distinct carriers mediating these exchanges. We utilized substrate and inhibitor specificity studies to distinguish between different anion exchange transporters. We conclude that SO4OH and oxalate: OH exchange occur on the same carrier because: (i) pH-gradient stimulated transport of both14C-oxalate and35SO4 were equally sensitive tocis-inhibition by unlabeled SO4 or oxalate; and (ii) both were inhibited equally by K. We conclude that oxalate: OH and oxalate: Cl exchanges occur on different carriers because: (i) Cl or SO4 caused unequalcis-inhibition of these two exchanges; and (ii) as compared to oxalate: Cl exchange, oxalate: OH exchange was more sensitive to inhibition by probenecid and K and less sensitive to inhibition by bumetanide. Finally, we conclude that oxalate: Cl exchange and ClHCO3 exchange occur on different carriers because: (i) ClHCO3 exchange was almost completely insensitive tocis-inhibition by oxalate; and (ii) oxalate: Cl exchange was more sensitive to inhibition by DIDS and bumetanide than ClHCO3 exchange. Thus we have found that there are at least three separate anion exchangers on rabbit ileal brush border: (i) a ClHCO3 exchanger; (ii) a SO4OH exchanger, which also transports oxalate; and (iii) an oxalate: Cl exchanger. 相似文献
11.
12.
13.
Eugene Emmer Richard P. Rood John H. Wesolek Michael E. Cohen R. Scott Braithwaite Geoffrey W. G. Sharp Heini Murer Mark Donowitz 《The Journal of membrane biology》1989,108(3):207-215
Summary In rabbit ileum, Ca2+/calmodulin (CaM) appears to be involved in physiologically inhibiting the linked NaCl absorptive process, since inhibitors of Ca2+/CaM stimulate linked Na+ and Cl– absorption. The role of Ca2+/CaM-dependent phosphorylation in regulation of the brush-border Na+/H+ antiporter, which is believed to be part of the neutral linked NaCl absorptive process, was studied using purified brush-border membrane vesicles, which contain both the Na+/H+ antiporter and Ca2+/CaM-dependent protein kinase(s) and its phosphoprotein substrates. Rabbit ileal villus cell brush-border membrane vesicles were prepared by Mg precipitation and depleted of ATP. Using a freezethaw technique, the ATP-depleted vesicles were loaded with Ca2+, CaM, ATP and an ATP-regenerating system consisting of creatine kinase and creatine phosphate. The combination of Ca2+/CaM and ATP inhibited Na+/H+ exchange by 45±13%. This effect was specific since Ca2+/CaM and ATP did not alter diffusive Na+ uptake, Na+-dependent glucose entry, or Na+ or glucose equilibrium volumes. The inhibition of the Na+/H+ exchanger by Ca2+/CaM/ATP was due to an effect on theV
max and not on theK
m
for Na+. In the presence of CaM and ATP, Ca2+ caused a concentration-dependent inhibition of Na+ uptake, with an effect 50% of maximum occurring at 120nm. This Ca2+ concentration dependence was similar to the Ca2+ concentration dependence of Ca2+/CaM-dependent phosphorylation of specific proteins in the vesicles. The Ca2+/CaM/ATP-inhibition of Na+/H+ exchange was reversed by W13, a Ca2+/CaM antagonist, but not by a hydrophobic control, W12, or by H-7, a protein kinase C antagonist. we conclude that Ca2+, acting through CaM, regulates ileal brush-border Na+/H+ exchange, and that this may be involved in the regulation of neutral linked NaCl absorption. 相似文献
14.
W Kramer S B Nicol F Girbig U Gutjahr S Kowalewski H Fasold 《Biochimica et biophysica acta》1992,1111(1):93-102
The Na(+)-dependent uptake system for bile acids in the ileum from rabbit small intestine was characterized using brush-border membrane vesicles. The uptake of [3H]taurocholate into vesicles prepared from the terminal ileum showed an overshoot uptake in the presence of an inwardly-directed Na(+)-gradient ([Na+]out > [Na+]in), in contrast to vesicles prepared from the jejunum. The Na(+)-dependent [3H]taurocholate uptake was cis-inhibited by natural bile acid derivatives, whereas cholephilic organic compounds, such as phalloidin, bromosulphophthalein, bilirubin, indocyanine green or DIDS - all interfering with hepatic bile-acid uptake - did not show a significant inhibitory effect. Photoaffinity labeling of ileal membrane vesicles with 3,3-azo- and 7,7-azo-derivatives of taurocholate resulted in specific labeling of a membrane polypeptide with apparent molecular mass 90 kDa. Bile-acid derivatives inhibiting [3H]taurocholate uptake by ileal vesicles also inhibited labeling of the 90 kDa polypeptide, whereas compounds with no inhibitory effect on ileal bile-acid transport failed to show a significant effect on the labeling of the 90 kDa polypeptide. The involvement of functional amino-acid side-chains in Na(+)-dependent taurocholate uptake was investigated by chemical modification of ileal brush-border membrane vesicles with a variety of group-specific agents. It was found that (vicinal) thiol groups and amino groups are involved in active ileal bile-acid uptake, whereas carboxyl- and hydroxyl-containing amino acids, as well as tyrosine, histidine or arginine are not essential for Na(+)-dependent bile-acid transport activity. The irreversible inhibition of [3H]taurocholate transport by DTNB or NBD-chloride could be partially reversed by thiols like 2-mercaptoethanol or DTT. Furthermore, increasing concentrations of taurocholate during chemical modification with NBD-chloride were able to protect the ileal bile-acid transporter from inactivation. These findings suggest that a membrane polypeptide of apparent M(r) 90,000 is a component of the active Na(+)-dependent bile-acid reabsorption system in the terminal ileum from rabbit small intestine. Vicinal thiol groups and amino groups of the transport system are involved in Na(+)-dependent transport activity, whereas other functional amino acids are not essential for transport activity. 相似文献
15.
Identification of a ligand-binding site in the Na+/bile acid cotransporting protein from rabbit ileum. 总被引:3,自引:0,他引:3
W Kramer F Girbig H Glombik D Corsiero S Stengelin C Weyland 《The Journal of biological chemistry》2001,276(38):36020-36027
Reabsorption of bile acids occurs in the terminal ileum by a Na(+)-dependent transport system composed of several subunits of the ileal bile acid transporter (IBAT) and the ileal lipid-binding protein. To identify the bile acid-binding site of the transporter protein IBAT, ileal brush border membrane vesicles from rabbit ileum were photoaffinity labeled with a radioactive 7-azi-derivative of cholyltaurine followed by enrichment of IBAT protein by preparative SDS gel electrophoresis. Enzymatic fragmentation with chymotrypsin yielded IBAT peptide fragments in the molecular range of 20.4-4 kDa. With epitope-specific antibodies generated against the C terminus a peptide of molecular mass of 6.6-7 kDa was identified as the smallest peptide fragment carrying both the C terminus and the covalently attached radiolabeled bile acid derivative. This clearly indicates that the ileal Na(+)/bile acid cotransporting protein IBAT contains a bile acid-binding site within the C-terminal 56-67 amino acids. Based on the seven-transmembrane domain model for IBAT, the bile acid-binding site is localized to a region containing the seventh transmembrane domain and the cytoplasmic C terminus. Alternatively, assuming the nine-transmembrane domain model, this bile acid-binding site is localized to the ninth transmembrane domain and the C terminus. 相似文献
16.
Characteristics of glutamic acid transport by rabbit intestinal brush-border membrane vesicles. Effects of Na+-, K+- and H+-gradients 总被引:1,自引:0,他引:1
A Berteloot 《Biochimica et biophysica acta》1984,775(2):129-140
In the presence of a Na+-gradient (out greater than in), L-glutamic acid and L-and D-aspartic acids were equally well concentrated inside the vesicles, while no transport above simple diffusion levels was seen by replacement of Na+ by K+. Equilibrium uptake values were found inversely proportional to the medium osmolarity, thus demonstrating uptake into an osmotically sensitive intravesicular space. The extrapolation of these lines to infinite medium osmolarity (zero space) showed only a small binding component in acidic amino-acid transport. When the same experiment was performed at saturating substrate concentrations, linear relationships extrapolating through the origin but showing smaller slope values were recorded, thus indicating that the binding component could be more important than suspected above. However, binding to the membrane was neglected in our studies as it was absent from initial rate measurements. Na+-dependent uphill transport of L-glutamic acid was stimulated by K+ present on the intravesicular side only but maximal stimulation was recorded under conditions of an outward K+-gradient (in greater than out). Quantitative and qualitative differences in the K+ effect were noted between pH 6.0 and 8.0. Initial uptake rates showed pH dependency in Na+-(out greater than in) + K+-(in greater than out) gradient conditions only with a physiological pH optimum between 7.0 and 7.5. It was also found that a pH-gradient (acidic outside) could stimulate both the Na+-gradient and the Na+ + K+-gradient-dependent transport of L-glutamic acid. However, pH- or K+-gradient alone were ineffective in stimulating uptake above simple diffusion level. Finally, it was found that increased rates of efflux were always observed with an acidic pH outside, whatever the conditions inside the vesicles. From these results, we propose a channel-type mechanism of L-glutamic acid transport in which Na+ and K+ effects are modulated by the surrounding pH. The model proposes a carrier with high or low affinity for Na+ in the protonated or unprotonated forms, respectively. We also propose that K+ binding occurs only to the unprotonated carrier and allows its fast recycling as compared to the free form of the carrier. Such a model would be maximally active and effective in the intestine in the in vivo physiological situations. 相似文献
17.
Basolateral plasma membrane vesicles were prepared from rat liver by a new technique using self-generating Percoll gradients. The method is rapid (total spin time of 2.5 h) and protein yields were high (0.64 mg/g of liver). Transmission electron microscopy studies and measurements of marker enzyme activities indicated that the preparation was highly enriched in basolateral membranes and substantially free of contamination by canalicular membranes or subcellular organelles. High total recoveries for protein yield and marker enzyme activities during the fractionation procedure indicated that enzymatic activity was neither lost (inactivation) nor increased (activation). Thus, the pattern of marker enzyme activities found in the membrane preparation truly reflected substantial enrichment in membranes from the basolateral surface. Analysis of freeze-fracture electron micrographs suggested that approximately 75% of the vesicles were oriented "right-side-out." In order to assess the functional properties of the vesicles, the uptake of [3H]taurocholate was studied. In the presence of a Na+ gradient, taurocholate uptake was markedly stimulated and the bile acid was transiently accumulated at a concentration 1.5- to 2-fold higher than that at equilibrium ("overshoot"). In the absence of a gradient but in the presence of equimolar Na+ inside and outside of the vesicle, taurocholate uptake was faster than in the absence of Na+. These findings support a direct co-transport mechanism for the uptake of taurocholate and Na+. Kinetic studies demonstrated that Na+-dependent taurocholate uptake was saturable with a Km of 36.5 microM and a Vmax of 5.36 nmol mg-1 protein min-1. The high yield, enzymatic profile and retention of transport properties suggest that this membrane preparation is well suited for studies of basolateral transport. 相似文献
18.
Alfred Berteloot 《生物化学与生物物理学报:生物膜》1984,775(2):129-140
In the presence of a Na+-gradient (out > in), l-glutamic acid and l-and d-aspartic acids were equally well concentrated inside the vesicles, while no transport above simple diffusion levels was seen by replacement of Na+ by K+. Equilibrium uptake values were found inversely proportional to the medium osmolarity, thus demonstrating uptake into an osmotically sensitive intravesicular space. The extrapolation of these lines to infinite medium osmolarity (zero space) showed only a small binding component in acidic amino-acid transport. When the same experiment was performed at saturating substrate concentrations, linear relationships extrapolating through the origin but showing smaller slope values were recorded, thus indicating that the binding component could be more important than suspected above. However, binding to the membrane was neglected in our studies as it was absent from initial rate measurements. Na+-dependent uphill transport of l-glutamic acid was stimulated by K+ present on the intravesicular side only but maximal stimulation was recorded under conditions of an outward K+-gradient (in > out). Quantitative and qualitative differences in the K+ effect were noted between pH 6.0 and 8.0. Initial uptake rates showed pH dependency in gradient conditions only with a physiological pH optimum between 7.0 and 7.5. It was also found that a pH-gradient (acidic outside) could stimulate both the Na+-gradient and the Na+ + K+-gradient-dependent transport of l-glutamic acid. However, pH- or K+-gradient alone were ineffective in stimulating uptake above simple diffusion level. Finally, it was found that increased rates of efflux were always observed with an acidic pH outside, whatever the conditions inside the vesicles. From these results, we propose a channel-type mechanism of l-glutamic acid transport in which Na+ and K+ effects are modulated by the surrounding pH. The model proposes a carrier with high or low affinity for Na+ in the protonated or unprotonated forms, respectively. We also propose that K+ binding occurs only to the unprotonated carrier and allows its fast recycling as compared to the free form of the carrier. Such a model would be maximally active and effective in the intestine in the in vivo physiological situations. 相似文献
19.