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The basolateral segment of the rat renal tubular plasma membrane possesses Ca2+-dependent ATPase activity which was independent of Mg2+. Two kinetic forms were found: one, was a high affinity (apparent Km for free Ca2+ of 172 nM) low capacity (Vmax of 144 nmol of Pi X min-1 mg-1 protein) type; the other, had low affinity (apparent Km of 25 microM) and high capacity (896 nmol of Pi X min-1 X mg-1 protein). Mg2+ inhibited both Ca2+-ATPases. The high affinity enzyme exhibited positive cooperativity with respect to ATP, with a n value of 1.6. Ca2+-ATPase activity was not affected by calmodulin and was not inhibited by vanadate. On the other hand, both high and low affinity Ca2+-ATPase activities were increased when 1,25-dihydroxycholecalciferol was given to vitamin D-deficient rats. Kinetically, the enhanced activities were due to an increase in the Vmax values; the apparent affinities for free Ca2+ were not changed. The physiological function of the vitamin D-sensitive, Mg+-independent, Ca2+-ATPase activities remains to be established. 相似文献
3.
Differential binding of folates by rat renal cortex brush border and basolateral membrane preparations 总被引:1,自引:0,他引:1
R Corrocher R G Abramson V F King C Schreiber S Dikman S Waxman 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1985,178(1):73-84
The binding of radioactive 5-methyltetrahydrofolate and folic acid was found to be greater in brush border than in basolateral membrane preparations of rat renal cortex. This appeared to be due to an increased amount of a specific folate binding protein in the brush border membrane preparations as compared to those of the basolateral membrane. The binding was saturable and inhibited by nonradioactive folic acid and, therefore, a specific, rather than nonspecific process. The Km's for folic acid binding in brush border and basolateral membrane preparations were similar and involved a single high-affinity binding site. In contrast, methotrexate was found to bind equally well to both brush border and basolateral membrane preparations. Moreover, folic acid binding was not inhibited by an equimolar amount of methotrexate. A folate binding protein could be extracted from either membrane preparation with 1% Triton X-100 and, to a lesser extent, with 0.6 M NaCl. These different extraction procedures resulted in different apparent molecular weights for folate binding protein (greater than 160,000 for Triton X-100-extracted samples and 40,000 for NaCl-extracted samples). The membrane preparation pellets remaining after NaCl extraction were able to rebind tritiated folic acid and also the 40,000-Da folate binding protein. On the other hand, membrane preparations extracted with Triton X-100 lost the ability to bind folic acid or the 40,000-Da folate binding protein. These differences in molecular weight and rebinding capacity may be explained by the existence of a receptor for folate binding protein which was extracted by Triton X-100, but not by NaCl. The greater concentration of folate binding protein in the renal tubule cell brush border membrane preparations as compared to those from basolateral membranes ascribes, for the first time, a functional role for folate binding protein in the renal reabsorption of folates which is required to prevent loss of folate in the urine and perhaps in the membrane transport of folates in general. 相似文献
4.
A rapid and reproducible method has been developed for the simultaneous isolation of basolateral and brush-border membranes from the rabbit renal cortex. The basolateral membrane preparation was enriched 25-fold in (Na+ + K+)-ATPase and the brush-border membrane fraction was enriched 12-fold in alkaline phosphatase, whereas the amount of cross-contamination was low. Contamination of these preparations by mitochondria and lysosomes was minimal as indicated by the low specific activities of enzyme markers, i.e., succinate dehydrogenase and acid phosphatase. The basolateral fraction consisted of 35–50% sealed vesicles, as demonstrated by detergent (sodium dodecyl sulfate) activation of (Na+ + K+)-ATPase activity and [3H]ouabain binding. The sidedness of the basolateral membranes was estimated from the latency of ouabain-sensitive (Na+ + K+)-ATPase activity assayed in the presence of gramicidin, which renders the vesicles permeable to Na+ and K+. These studies suggest that nearly 90% of the vesicles are in a right-side-out orientation. 相似文献
5.
A rapid and reproducible method has been developed for the simultaneous isolation of basolateral and brush-border membranes from the rabbit renal cortex. The basolateral membrane preparation was enriched 25-fold in (Na+ + K+)-ATPase and the brush-border membrane fraction was enriched 12-fold in alkaline phosphatase, whereas the amount of cross-contamination was low. Contamination of these preparations by mitochondria and lysosomes was minimal as indicated by the low specific activities of enzyme markers, i.e., succinate dehydrogenase and acid phosphatase. The basolateral fraction consisted of 35-50% sealed vesicles, as demonstrated by detergent (sodium dodecyl sulfate) activation of (Na+ + K+)-ATPase activity and [3H]ouabain binding. The sidedness of the basolateral membranes was estimated from the latency of ouabain-sensitive (Na+ + K+)-ATPase activity assayed in the presence of gramicidin, which renders the vesicles permeable to Na+ and K+. These studies suggest that nearly 90% of the vesicles are in a right-side-out orientation. 相似文献
6.
Biotin uptake mechanisms in brush-border and basolateral membrane vesicles isolated from rabbit kidney cortex 总被引:1,自引:0,他引:1
Biotin transport was studied using brush-border and basolateral membrane vesicles isolated from rabbit kidney cortex. An inwardly directed Na+ gradient stimulated biotin uptake into brush-border membrane vesicles and a transient accumulation of the anion against its concentration gradient was observed. In contrast, uptake of biotin by basolateral membrane vesicles was found to be Na+-gradient insensitive. Generation of a negative intravesicular potential by valinomycin-induced K+ diffusion potentials or by the presence of Na+ salts of anions of different permeabilities enhanced biotin uptake by brush-border membrane vesicles, suggesting an electrogenic mechanism. The Na+ gradient-dependent uptake of biotin into brush-border membrane vesicles was saturable with an apparent Km of 28 microM. The Na+-dependent uptake of tracer biotin was significantly inhibited by 50 microM biotin, and thioctic acid but not by 50 microM L-lactate, D-glucose, or succinate. Finally, the existence in both types of membrane vesicles of a H+/biotin- cotransport system could not be demonstrated. These results are consistent with a model for biotin reabsorption in which the Na+/biotin- cotransporter in luminal membranes provides the driving force for uphill transport of this vitamin. 相似文献
7.
The mechanism of HCO3- translocation across the proximal tubule basolateral membrane was investigated by testing for Na+-HCO3- cotransport using isolated membrane vesicles purified from rat renal cortex. As indicated by 22Na+ uptake, imposing an inwardly directed HCO3- concentration gradient induced the transient concentrative accumulation of intravesicular Na+. The stimulation of basolateral membrane vesicle Na+ uptake was specifically HCO3(-)-dependent as only basolateral membrane-independent Na+ uptake was stimulated by an imposed hydroxyl gradient in the absence of HCO3-. No evidence for Na+-HCO3- cotransport was detected in brush border membrane vesicles. Charging the vesicle interior positive stimulated net intravesicular Na+ accumulation in the absence of other driving forces via a HCO3(-)-dependent pathway indicating the flow of negative charge accompanies the Na+-HCO3- cotransport event. Among the anion transport inhibitors tested, 4-4'-diisothiocyanostilbene-2,2'-disulfonic acid demonstrated the strongest inhibitor potency at 1 mM. The Na+-coupled transport inhibitor harmaline also markedly inhibited HCO3- gradient-driven Na+ influx. A role for carbonic anhydrase in the mechanism of Na+-HCO3- cotransport is suggested by the modest inhibition of HCO3- gradient driven Na+ influx caused by acetazolamide. The imposition of Cl- concentration gradients had a marked effect on HCO3- gradient-driven Na+ influx which was furosemide-sensitive and consistent with the operation of a Na+-HCO3- for Cl- exchange mechanism. The results of this study provide evidence for an electrogenic Na+-HCO3- cotransporter in basolateral but not microvillar membrane vesicles isolated from rat kidney cortex. The possible existence of an additional basolateral membrane HCO3(-)-translocating pathway mediating Na+-HCO3- for Cl- exchange is suggested. 相似文献
8.
Specific receptors for atrial natriuretic polypeptide on basolateral membranes isolated from rat renal cortex 总被引:1,自引:0,他引:1
R Hori K Inui H Saito Y Matsukawa K Okumura K Nakao N Morii H Imura 《Biochemical and biophysical research communications》1985,129(3):773-779
The binding of alpha-human atrial natriuretic polypeptide (alpha-hANP) to brush border and basolateral membranes isolated from the rat renal cortex was studied at 0 degree C by a rapid filtration technique. Specific binding of 125I-alpha-hANP to basolateral membranes reached a steady state at 4 hr. The binding to brush border membranes was maximal at 5-15 min and then rapidly decreased. The analysis of incubation mixtures with basolateral membranes revealed little degradation of 125I-alpha-hANP during the 4-hr incubation, while there was extensive degradation of the ligand with brush border membranes during the 30-min incubation. High affinity binding of 125I-alpha-hANP was demonstrated on basolateral membranes but not on brush border membranes. These data suggest that specific receptors for alpha-hANP are localized on basolateral membranes of the renal cortex. 相似文献
9.
Shoji T Suzuki H Kusuhara H Watanabe Y Sakamoto S Sugiyama Y 《American journal of physiology. Gastrointestinal and liver physiology》2004,287(4):G749-G756
The mechanism for the cellular extrusion of organic anions across the intestinal basolateral membrane was examined using isolated membrane vesicles from rat jejunum, ileum, and colon. It was found that 17beta-estradiol 17beta-D-glucuronide (E217betaG) is taken up in an ATP-dependent manner into the basolateral membrane vesicles (BLMVs) but not into the brush-border or microsomal counterparts. The ATP-dependent uptake of E217betaG into BLMVs from jejunum and ileum was described by a single component with a Km value of 23.5 and 8.31 microM, respectively, whereas that into the BLMVs from colon was described by assuming the presence of high (Km=0.82 microM)- and low-affinity (Km=35.4 microM) components. Taurocholate, 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole glucuronide and taurolithocholate sulfate, but not leukotriene C4, were significantly taken up by the BLMVs. In addition to such substrate specificity, the inhibitor sensitivity of the ATP-dependent transport in BLMVs was similar to that of rat multidrug resistance-associated protein 3 (Mrp3), which is located on the basolateral membrane of enterocytes. Together with the fact that the rank order of the extent of the expression of Mrp3 (jejunum < ileum < colon) is in parallel with that of the extent of the transport of ligands, these results suggest that the ATP-dependent uptake of organic anions into isolated intestinal BLMVs is at least partly mediated by Mrp3. 相似文献
10.
Sulphate-ion/sodium-ion co-transport by brush-border membrane vesicles isolated from rat kidney cortex
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Uptake of SO(4) (2-) into brush-border membrane vesicles isolated from rat kindey cortex by a Ca(2+)-precipitation method was investigated by using a rapid-filtration technique. Uptake of SO(4) (2-) by the vesicles was osmotically sensitive and represented transport into an intra-vesicular space. Transport of SO(4) (2-) by brush-border membranes was stimulated in the presence of Na(+), compared with the presence of K(+) or other univalent cations. A typical ;overshoot' phenomenon was observed in the presence of an NaCl gradient (100mm-Na(+) outside/zero mm-Na(+) inside). Radioactive-SO(4) (2-) exchange was faster in the presence of Na(+) than in the presence of K(+). Addition of gramicidin-D, an ionophore for univalent cations, decreased the Na(+)-gradient-driven SO(4) (2-) uptake. SO(4) (2-) uptake was only saturable in the presence of Na(+). Counter-transport of Na(+)-dependent SO(4) (2-) transport was shown with MoO(4) (2-) and S(2)O(3) (2-), but not with PO(4) (2-). Changing the electrical potential difference across the vesicle membrane by establishing different diffusion potentials (anion replacement; K(+) gradient+/-valinomycin) was not able to alter Na(+)-dependent SO(4) (2-) uptake. The experiments indicate the presence of an electroneutral Na(+)/SO(4) (2-)-co-transport system in brush-border membrane vesicles isolated from rat kidney cortex. 相似文献
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S V Jakobsson 《Experimental cell research》1974,84(1):319-334
13.
Many roles have been suggested for N-acetyl-aspartate in brain function because of it being located almost exclusively in that organ. However, its true role remains to be demonstrated. We show here that N-acetyl-aspartate: 1) binds to a hydrophobic protein fraction from the cerebral cortex of the rat, which specifically bindsl-aspartate,l-glutamate, and -amino-butyric acid; and 2) has a marked inhibitory effect on the aspartate binding sites of this proteolipid fraction. Structural analogs of N-acetyl-asparate, i.e. N-carbamyl-aspartate and N-methyl-aspartate also inhibit thel-aspartate binding by the brain protein fraction used. 相似文献
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The transport of dipeptides and beta-lactam antibiotics across the rat renal basolateral membrane was examined. The initial uptake of glycylsarcosine and cefadroxil by rat renal basolateral membrane vesicles was inhibited by the presence of all the di- and tripeptides and beta-lactam antibiotics that were tested in this study. However, the uptake of both substrates was not inhibited by glycine, an amino acid. The initial uptake of zwitterionic beta-lactam antibiotics, cefadroxil, cephradine, and cephalexin, was stimulated by preloaded glycylsarcosine (countertransport effect). On the other hand, the uptake of dianionic beta-lactam antibiotics, ceftibuten and cefixime, was not affected. A concentration-dependent initial uptake of glycylsarcosine and cefadroxil suggested the existence of a carrier-mediated mechanism, whereas the transport of ceftibuten did not show any saturated uptake. The transporter that participates in the permeation of dipeptides and beta-lactam antibiotics across basolateral membranes showed lower affinity than did PEPT1 and PEPT2. This is the first study that showed an evidence for a peptide transporter, expressed in the rat renal basolateral membrane, that recognizes zwitterionic beta-lactam antibiotics using basolateral membrane vesicles isolated from normal rat kidney. 相似文献
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HCO3- exit across the basolateral membrane of the kidney proximal tubule cell is mediated via an electrogenic Na+:HCO3- cotransporter. We have studied the effect of pH on the activity of this cotransport system in basolateral membrane vesicles isolated from rabbit renal cortex. At constant internal pH 6.0, increasing the external pH and [HCO3-] increased the rate of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive 22Na+ influx into the vesicles. To determine the role of internal pH on the activity of the Na+:HCO3- cotransport system, the influx of 22Na+ via HCO3-dependent Na(+)-Na+ exchange was measured in the absence of an initial pH and [HCO3-] gradient (pH(i) = pH(o), 5% CO2). Increasing the pH from 6.8 to 7.2 increased whereas, increasing the pH from 7.4 to 8.0 decreased the rate of 22Na+ influx via this exchange. Increasing pH at constant [HCO3-] (pH(i) = pH(o) = 8.0, 1.5% CO2 versus pH(i) = pH(o) = 7.2, 10% CO2) reduced the influx of 22Na+ via HCO3-dependent Na(+)-Na+ exchange. Increasing pH at constant [CO3(2-)](pH(i) = pH(o) = 8.0, 1.5% CO2 versus pH(i) = pH(o) = 7.2, 60% CO2) was associated with reduced 22Na+ uptake. Decreasing the pH (pH(i) = pH(o) = 6.3, 60% CO2 versus pH(i) = pH(o) = 7.2, 5% CO2) was associated with a reduced rate of HCO3(-)-dependent Na(+)-Na+ exchange. We conclude that the Na+:HCO3- cotransporter displays a significant pH sensitivity profile with the cotransporter being more functional at pH 7.0-7.4 and less active at more acid or alkaline pH. In addition, the results suggest that the pH sensitivity arises at the inner surface of the basolateral membrane. 相似文献
18.
Specific binding sites for corticosterone in isolated cells and plasma membrane from rat liver 总被引:3,自引:0,他引:3
Miguel Trueba Iñaki Ibarrola Kepa Ogiza Aida Marino José María Macarulla 《The Journal of membrane biology》1991,120(2):115-124
Summary The specific binding of [3H]corticosterone to hepatocytes is a nonsaturable, reversible and temperature-dependent process. The binding to liver purified plasma membrane fraction is also specific, reversible and temperature dependent but it is saturable. Two types of independent and equivalent binding sites have been determined from hepatocytes. One of them has high affinity and low binding capacity (K
D=8.8nm andB
max=1477 fmol/mg protein) and the other one has low affinity and high binding capacity (K
D=91nm andB
max=9015 fmol/mg). In plasma membrane only one type of binding site has been characterized (K
D=11.2nm andB
max=1982 fmol/mg). As it can be deduced from displacement data obtained in hepatocytes and plasma membrane the high affinity binding sites are different from the glucocorticoid, progesterone nuclear receptors and the Na+,K+-ATPase digitalis receptor. Probably it is of the same nature that the one determinate for [3H]cortisol and [3H]corticosterone in mouse liver plasma membrane. Beta-and alpha-adrenergic antagonists as propranolol and phentolamine did not affect [3H]corticosterone binding to hepatocytes and plasma membranes; therefore, these binding sites are independent of adrenergic receptors. The binding sites in hepatocytes and plasma membranes are not exclusive for corticosterone but other steroids are also bound with very different affinities. 相似文献
19.
Mitsuru Sugawara 《生物化学与生物物理学报:生物膜》2003,1609(1):39-44
The transport of dipeptides and β-lactam antibiotics across the rat renal basolateral membrane was examined. The initial uptake of glycylsarcosine and cefadroxil by rat renal basolateral membrane vesicles was inhibited by the presence of all the di- and tripeptides and β-lactam antibiotics that were tested in this study. However, the uptake of both substrates was not inhibited by glycine, an amino acid. The initial uptake of zwitterionic β-lactam antibiotics, cefadroxil, cephradine, and cephalexin, was stimulated by preloaded glycylsarcosine (countertransport effect). On the other hand, the uptake of dianionic β-lactam antibiotics, ceftibuten and cefixime, was not affected. A concentration-dependent initial uptake of glycylsarcosine and cefadroxil suggested the existence of a carrier-mediated mechanism, whereas the transport of ceftibuten did not show any saturated uptake. The transporter that participates in the permeation of dipeptides and β-lactam antibiotics across basolateral membranes showed lower affinity than did PEPT1 and PEPT2. This is the first study that showed an evidence for a peptide transporter, expressed in the rat renal basolateral membrane, that recognizes zwitterionic β-lactam antibiotics using basolateral membrane vesicles isolated from normal rat kidney. 相似文献
20.
[3H]Pteroylglutamic acid (PteGlu) uptake was studied using brush-border membrane vesicles isolated from rat kidney. Results on the uptake of [3H]PteGlu by brush-border membrane vesicles incubated in media of increasing osmolarities demonstrated that uptake was contributed by two components, intravesicular transport and membrane binding. Both the components of the uptake exhibited similar pH dependence, with maxima at pH 5.6, and were found to be saturable mechanisms with Km values of 6.7.10(-7) and 11.2.10(-7) M, respectively. These studies show that PteGlu is transported by isolated rat kidney brush-border membrane vesicles in a manner consistent with a saturable system and that a binding component may be functionally associated with this. 相似文献