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1.
Summary A new procedure for the rapid isolation of renal cortical brush-border and basolateral membranes from the same homogenate is described. Brush-border membranes isolated using Mg2+-EGTA precipitation were enriched 18-fold for leucine aminopeptidase and had a recovery of 32.5%. Basolateral membrane fractions were isolated using a discontinuous sucrose gradient and showed an enrichment of 10.7-fold and recovery of 12.8% using (Na+, K+)-ATPase as a marker enzyme. Lipid analysis using two-dimensional TLC separation of phospholipids and gas liquid chromatography for cholesterol showed marked differences in the lipid composition of the brush-border and basolateral membranes. The brush-border membrane had increased sphingomyelin, phosphatidylserine, ethanolamine plasmalogens, and an increased cholesterol-to-phospholipid and sphingomyelin-to-phosphatidylcholine ratio compared to the basolateral membrane. The relative turnover of total membrane and individual phospholipid species using a double isotope ratio method was carried out. Phospholipids were labeled with either phosphorus 32 and 33 or acetate (3H, 1-14C). The relative turnover of phospholipid species and cholesterol differed strikingly. Phosphatidylcholine showed a high turnover, phosphatidylethanolamine and phosphatidylinositol had intermediate values and sphingomyelin, phosphatidylserine and cholesterol had low relative turnover rates. The order of phospholipid class relative turnover was independent of the labeled precursor used. The brush-border membrane had a significantly reduced relative turnover rate for total membrane phospholipids, sphingomyelin and cholesterol compared to the basolateral membrane. These data show marked differences in the lipid composition and relative turnover rates of the phospholipid species of the brush-border and basolateral membranes. They provide a biochemical basis for the recently reported differences in brush-border and basolateral membrane fluidity and suggest independent cellular regulation of brush-border and basolateral membrane lipids.  相似文献   

2.
Summary Cysteine-sensitive alkaline phosphatase and/or ouabain-sensitive Na+, K+-ATPase were studied by ultrastructure cytochemistry in epithelial cells of proximal and distal kidney tubules. Alkaline phosphatase reactivity was confined to the surface of the microvillous luminal cell membrane of proximal tubule cells, whereas distal tubules and collecting ducts were unreactive. The Na+, K+-ATPase reactivity was localized evenly along the cytoplasmic side of the basolateral cell membrane of cells of proximal and distal tubules and in collecting ducts. In the proximal tubules, where the activity was strongest, the Na+, K+-ATPase deposits were also found in the 10–50 nm gap between the cell membrane and the cisternae of tubulo-cisternal endoplasmic reticulum (TER) underlying a major part of the basolateral cell membrane. The restriction of Na+, K+-ATPase sites, which are involved in extrusion of Na+ from the cell, to a narrow cytoplasmic compartment located between the cell membrane and the cisternae of TER, is consistent with a transport role for the TER.  相似文献   

3.
《Insect Biochemistry》1991,21(7):749-758
The present study confirms previous reports of the presence of (Na+ + K+)-ATPase and anion-stimulated ATPase activity in Malpighian tubules of Locusta. In addition, the presence of a K+-stimulated, ouabain-insensitive ATPase activity has been identified in microsomal fractions. Differential and sucrose density-gradient centrifugation of homogenates has been used to separate membrane fractions which are rich in mitochondria, apical membranes and basolateral membranes; as indicated by the presence of succinate dehydrogenase and the presence or absence of non-specific alkaline phosphatase activity, respectively. Relatively high specific (Na+ + K+)-ATPase activity was associated with the basolateral membrane-rich fractions with only low levels of this activity being associated with the apical membrane-rich preparation. K+-stimulated ATPase activity was also associated, predominantly, with the basolateral membrane-rich fractions. However, comparison of the distribution of this activity with that of the (Na+ + K+)-ATPase suggests that the two enzymes did not co-separate. The possibility that the K+-stimulated ATPase was not associated with the basolateral plasma membrane is discussed.Anion-stimulated ATPase activity was found in the apical and basolateral membrane-rich fractions and in the fraction contaning mainly mitochondria. Nevertheless, the fact that this bicarbonate-stimulated activity did not co-separate with succinate dehydrogenase activity suggests that it was not exclusively mitochondrial in origin. These results are consistent with physiological studies indicating a basolateral (Na+ + K+)-ATPase but do not support the K+-stimulated ATPase as a candidate for the apical electrogenic pump. The possible role of the bicarbonate-stimulated ATPase activity in ion transport across both the basolateral and apical cell membranes is discussed.  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.
Basolateral plasma membranes of rat small intestinal epithelium were purified by density gradient centrifugation followed by zonal electrophoresis on density gradients. Crude basolateral membranes were obtained by centrifugation in which the marker enzyme, (Na+ + K+)-ATPase, was enriched 10-fold with respect to the initial homogenate. The major contaminant was a membrane fraction derived from smooth endoplasmic reticulum, rich in NADPH-cytochrome c reductase activity. The crude basolateral membrane preparation could be resolved into the two major components by subjecting it to zonal electrophoresis on density gradients. The result was that (Na+ + K+)-ATPase was purified 22-fold with respect to the initial homogenate. Purification with respect to mitochondria and brush border membranes was 35- and 42-fold, respectively. Resolution of (Na+ + K+)-ATPase from NADPH-cytochrome c reductase by electrophoresis was best with membrane material from adult rats between 180 and 250 g. No resolution between the two marker enzymes occurred with material from young rats of 125 to 140 g. These results demonstrate that zonal electrophoresis on density gradients, a simple and inexpensive technique, has a similar potential to free-flow electrophoresis.  相似文献   

6.
A technique currently used for isolation of brush border membranes from renal and intestinal epithelium that involves vigorous tissue homogenization and sedimentation of non-luminal membranes in the presence of Mg2+ has been adapted to rat liver. Liver plasma membranes so prepared consisted almost exclusively of vesicles by electron microscopy, showed some contamination with endoplasmic reticulum and minimal contamination with mitochondria or Golgi by marker enzymes, were highly enriched in alkaline phosphatase, Mg2+-ATPase, and 5′-nucleotidase activity compared with homogenate, and showed little enrichment in (Na+,K+)-ATPase. Comparison of this enzymatic profile with cytochemical studies localizing (Na+,K+)-ATPase and alkaline phosphatase to the sinusoidal/lateral and canalicular membranes, respectively, suggested that these membranes were predominantly of canalicular origin. They had a lower (Na+ + K+)-ATPase specific activity, lower lipid content, and higher cholesterol to phospholipid molar ratio than a conventional plasma membrane preparation believed to be enriched in canaliculi. Moreover, it was possible to measure movement of d-[3H]glucose into an osmotically sensitive space bounded by these membrane vesicles.  相似文献   

7.
Purified lamb kidney Na+, K+-ATPase, consisting solely of the Mτ = 95,000 catalytic subunit and the Mτ~- 44,000 glycoprotein, was solubilized with Triton X-100 and incorporated into unilamellar phospholipid vesicles. Freeze-fracture electron microscopy of the vesicles showed intramembranous particles of approximately 90–100 Å in diameter, which are similar to those seen in the native Na+,K+-ATPase fraction. Digestion of the reconstituted proteins with neuraminidase indicated that the glycoprotein moiety of the Na+,K+-ATPase was asymmetrically oriented in the reconstituted vesicles, with greater than 85% of the total sialic acid directed toward the outside of the vesicles. In contrast, in the native Na+,K+-ATPase fraction, the glycoprotein was symmetrically distributed. Purified glycoprotein was also asymmetrically incorporated into phospholipid vesicles using Triton X-100 and without detergents as described by R. I. MacDonald and R. L. MacDonald (1975, J. Biol. Chem.250, 9206–9214). The glycoprotein-containing vesicles were 500–1000 Å in diameter, unilamellar, and, in contrast to the vesicles containing the Na+,K+-ATPase, did not contain the 90- to 100-Å intramembranous particles. These results indicate that the intramembranous particles observed in the native Na+,K+-ATPase and in the reconstituted Na+,K+-ATPase are not due to the glycoprotein alone, but represent either the catalytic subunit, or the catalytic plus the glycoprotein subunit.  相似文献   

8.
Prostagladin A2, which prevents intestinal ulcers produced by administration of nonsteroidal antiinflammatory compounds such as indomethacin, inhibited the Na+,K+-ATPase activity in basolateral plasma membrane of rat intestine significantly. Prostaglandin A2 inhibited mainly the Na+-dependent phosphorylation step in the overall reaction of Na+,K+-ATPase. This decrease of the Na+,K+-ATPase activity by prostaglandin A2 was due to the decrease of Vmax of the enzyme and of the affinity of the enzyme for Na+. It was also suggested that the presence of both Δ5,6 and Δ10,11 structure of prostaglandin A2 may be necessary for the inhibition of the Na+,K+-ATPase activity.  相似文献   

9.
Basolateral plasma membrane vesicles of rat small intestinal epithelium accumulate calcium through an ATP-dependent pumping system. The activity of this system is highest in duodenum and decreases towards the ileum. This distribution along the intestinal tract is similar as the active calcium absorption capacity of intact intestinal epithelial segments. ATP-dependent calcium uptake in basolateral membrane vesicles from duodenum and ileum increased significantly after repletion of young vitamin D-3-deficient rats with 1α,25-dihydroxy-vitamin D-3. Ca2+-ATPase activity in duodenal basolateral membranes increased to the same extend as ATP-dependent calcium transport, but (Na+ + K+)-ATPase activity remained unaltered.  相似文献   

10.
Immunocytochemical localization of Na+, K+-ATPase in the rat kidney   总被引:1,自引:0,他引:1  
Summary To determine if rat kidney Na+, K+-ATPase can be localized by immunoperoxidase staining after fixation and embedding, we prepared rabbit antiserum to purified lamb kidney medulla Na+, K+-ATPase. When sodium dodecylsulfate polyacrylamide electrophoretic gels of purified lamb kidney Na+, K+-ATPase and rat kidney microsomes were treated with antiserum (1200), followed by [125I]-Protein A and autoradiography, the rat kidney microsomes showed a prominent radioactive band coincident with the -subunit of the purified lamb kidney enzyme and a fainter radioactive band which corresponded to the -subunit. When the Na+, K+-ATPase antiserum was used for immunoperoxidase staining of paraffin and plastic sections of rat kidney fixed with Bouin's, glutaraldehyde, or paraformaldehyde, intense immunoreactive staining was present in the distal convoluted tubules, subcapsular collecting tubules, thick ascending limb of the loops of Henle, and papillary collecting ducts. Proximal convoluted tubules stained faintly, and the thin portions of the loops of Henle, straight descending portions of proximal tubules, and outer medullary collecting ducts did not stain. Staining was confined to basolateral surfaces of tubular epithelial cells. No staining was obtained with preimmune serum or primary antiserum absorbed with purified lamb kidney Na+, K+-ATPase, or with osmium tetroxide postfixation. We conclude that the basolateral membranes of the distal convoluted tubules and ascending thick limb of the loops of Henle are the major sites of immunoreactive Na+, K+-ATPase concentration in the rat kidney.Supported by Grant AM 17047 from NIH and by the Veterans Administration  相似文献   

11.
The endolymphatic sac (ES) is a part of the membranous labyrinth. ES is believed to perform endolymph absorption, which is dependent on several ion transporters, including Na+/K+/2Cl cotransporter type 2 (NKCC-2) and Na+/K+-ATPase. NKCC-2 is typically recognized as a kidney-specific ion transporter expressed in the apical membrane of the absorptive epithelium. NKCC-2 expression has been confirmed only in the rat and human ES other than the kidney, but the detailed localization features of NKCC-2 have not been investigated in the ES. Thus, we evaluated the specific site expressing NKCC-2 by immunohistochemical assessment. NKCC-2 expression was most frequently seen in the intermediate portion of the ES, where NKCC-2 is believed to play an important role in endolymph absorption. In addition, NKCC-2 expression was also observed on the apical membranes of ES epithelial cells, and Na+/K+-ATPase coexpression was observed on the basolateral membranes of ES epithelial cells. These results suggest that NKCC-2 performs an important role in endolymph absorption and that NKCC-2 in apical membranes and Na+/K+-ATPase in basolateral membranes work coordinately in the ES in a manner similar to that in renal tubules. (J Histochem Cytochem 58:759–763, 2010)  相似文献   

12.
Summary The effects of prolonged cold exposure of Syrian hamsters on liver membrane (Na+/K+-ATPase activity and on liver intracellular K+ levels was examined. Membrane preparations from cold-acclimated hamsters (6°C for 3 weeks) exhibited significantly higherV max values for (Na+/K+)-ATPase and significantly greater ouabain binding. These data support the view that in the liver of these cold-exposed hamsters, there is an increase in the number of operational pumps. The fact that the intact liver cells (isolated via liver perfusion) from the cold-acclimated hamsters: (a) did not have higher concentrations of intracellular K+ (despite the presence of more operational pumps); and (b) exhibited greater rates of K+ loss when the pumps were inhibited by maximal ouabain suggests that the K+ leak across the liver cell plasma membrane is increased in the cold-acclimated hamsters. Although the physiological significance of these results needs further evaluation, these membrane changes may be of adaptive value for hibernation.Abbreviations CA cold-acclimated - P i inorganic phosphate - KRB Krebs-Ringer-bicarbonate buffer - BSA bovine serum albumin - ECF extracellular fluid - ICF intracellular fluid - dcs dry cell solid - N nitrogen  相似文献   

13.
Summary A method has been developed for the simultaneous isolation of basolateral plasma membrane vesicles from surface and crypt cells of rabbit distal colon epithelium by sequential use of differential sedimentation, isopycnic centrifugation and Ficoll 400 barrier centrifugation. The protein yield was high (total 0.81 mg/g mucosa) and surface and crypt cell-derived basolateral membrane fractions have been purified 34- and 9-fold with respect to the homogenate. The pattern of marker enzyme enrichments revealed only minor contamination by subcellular organelles. Latency of ouabain-sensitive (Na+, K+)-ATPase activity prior and after trypsin treatment of membranes indicated a vesicle configuration of sealed right side-out: sealed inside-out: leaky of approximately 211. The presence of sealed vesicles was also evident from the osmotic sensitivity of thed-[1-14C] mannitol equilibrium space determined with either fraction. Although considerably different in protein profile, surface and crypt basolateral membranes were similar in cholesterol to phospholipid molar ratio and membrane fluidity as determined by steady-state fluorescence polarization.Stopped-flow light scattering experiments revealed a rather low water permeability of the membranes with a permeability coefficient of 6 m/sec at 35°C, which is one order of magnitude lower than reported for small intestinal plasma membranes. Both membrane fractions have been shown to effectively generate outward uphill potassium ion gradients, a process that is energized by ATP and inhibited by the membrane-permeant cardiacglycoside digitoxin. These characteristics are consistent with the activity of a (Na+, K+) pump operating in inside-out vesicles.  相似文献   

14.
Kidney proximal tubules are a key segment in the reabsorption of solutes and water from the glomerular ultrafiltrate, an essential process for maintaining homeostasis in body fluid compartments. The abundant content of Na+ in the extracellular fluid determines its importance in the regulation of extracellular fluid volume, which is particularly important for different physiological processes including blood pressure control. Basolateral membranes of proximal tubule cells have the classic Na+ + K+-ATPase and the ouabain-insensitive, K+-insensitive, and furosemide-sensitive Na+-ATPase, which participate in the active Na+ reabsorption. Here, we show that nanomolar concentrations of ceramide-1 phosphate (C1P), a bioactive sphingolipid derived in biological membranes from different metabolic pathways, promotes a strong inhibitory effect on the Na+-ATPase activity (C1P50 ≈ 10 nM), leading to a 72% inhibition of the second sodium pump in the basolateral membranes. Ceramide-1-phosphate directly modulates protein kinase A and protein kinase C, which are known to be involved in the modulation of ion transporters including the renal Na+-ATPase. Conversely, we did not observe any effect on the Na+ + K+-ATPase even at a broad C1P concentration range. The significant effect of ceramide-1-phosphate revealed a new potent physiological and pathophysiological modulator for the Na+-ATPase, participating in the regulatory network involving glycero- and sphingolipids present in the basolateral membranes of kidney tubule cells.  相似文献   

15.
It is concluded that Ca2+ transport across the basolateral membranes of the ionocytes in killifish skin is mediated for the major part by a Na+/Ca2+-exchange mechanism that is driven by the (transmembrane) Na+ gradient established by Na+/K+-ATPase. The conclusion is based, firstly, on the biochemical evidence for the presence of a Na+/Ca2+-exchanger next to the Ca2+-ATPase in the basolateral membranes of killifish gill cells. Secondly, the transcellular Ca2+ uptake measured in an Ussing chamber setup was 85% and 80% reduced in freshwater (FW) and SW (SW) opercular membranes, respectively, as the Na+ gradient across the basolateral membrane was directly or indirectly (by ouabain) reduced. Thapsigargin or dibutyryl-cAMP/IBMX in SW opercular membranes reduced Ca2+ influx to 46%, comparable to the effects seen in FW membranes [reduction to 56%; Marshall et al. 1995a]. Basal Ca2+ influx across the opercular membrane was 48% lower in membranes from fish adapted to SW than in membranes from fish adaptated to FW. Branchial Na+/K+-ATPase activity was two times higher in SW adapted fish. Accepted: 29 October 1996  相似文献   

16.
Summary An antibody to the 96 kD -subunit of the Na+, K+ -ATPase from Bufo marinus has been used in immunostaining rat kidney and salivary glands. Intense staining was observed on basolateral membranes of distal tubules of the kidney and striated ducts of the three major salivary glands. Less intense staining was seen on the basolateral membranes of parotid acinar cells, but no staining was seen on the acinar cells of submandibular or sublingual glands. These sites of staining have been shown, by other methods, to posses substantial Na+, K+ -ATPase, indicating that the antibody recognizes antigenic determinants of the sodium pump highly conserved in the course of evolution. In addition, staining with this antibody was observed at the apical region of cells of the proximal straight tubule and of the papillary collecting duct in the kidney. Absorption studies suggest that the apical antigenic determinants are the same or closely related to each other but are distinct from basolateral antigenic determinants.  相似文献   

17.
Luminal brush border and contraluminal basal-lateral segments of the plasma membrane from the same kidney cortex were prepared. The brush border membrane preparation was enriched in trehalase and γ-glutamyltranspeptidase, whereas the basal-lateral membrane preparation was enriched in (Na+ + K+)-ATPase. However, the specific activity of (Na+ + K+)-ATPase in brush border membranes also increased relative to that in the crude plasma membrane fraction, suggesting that (Na+ + K+)-ATPase may be an intrinsic constituent of the renal brush border membrane in addition to being prevalent in the basal-lateral membrane. Adenylate cyclase had the same distribution pattern as (Na+ + K+)-ATPase, i.e. higher specific activity in basal-lateral membranes and present in brush border membranes. Adenylate cyclase in both membrane preparations was stimulated by parathyroid hormone, calcitonin, epinephrine, prostaglandins and 5′-guanylylimidodiphosphate. When the agonists were used in combination enhancements were additive. In contrast to the distribution of adenylate cyclase, guanylate cyclase was found in the cytosol and in basal-lateral membranes with a maximal specific activity (NaN3 plus Triton X-100) 10-fold that in brush border membranes. ATP enhanced guanylate cyclase activity only in basal-lateral membranes. It is proposed that guanylate cyclase, in addition to (Na+ + K+)-ATPase, be used as an enzyme “marker” for the renal basal-lateral membrane.  相似文献   

18.
Summary The effects of temperature and pressure on Na+/K+-adenosine triphosphatases (Na+/K+-ATPases) from gills of marine teleost fishes were examined over a range of temperatures (10–25°C) and pressures (1–680 atm). The relationship between gill membrane fluidity and Na+/K+-ATPase activity was studied using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The increase in temperature required to offset the membrane ordering effects of high pressure was 0.015–0.025°C·atm-1, the same coefficient that applied to Na+/K+-ATPase activities. Thus, temperature-pressure combinations yielding the same Na+/K+-ATPase activity also gave similar estimates of membrane fluidity. Substituion of endogenous lipids with lipids of different composition altered the pressure responses of Na+/K+-ATPase. Na+/K+-adenosine triphosphatase became more sensitive to pressure in the presence of chicken egg phosphatidylcholine, but phospholipids isolated from fish gills reduced the inhibition by pressure of Na+/K+-ATPase. Cholesterol increased enzyme pressure sensitivity. Membrane fluidity and pressure sensitivity of Na+/K+-ATPase were correlated, but the effects of pressure also dependent on the source of the enzyme. Our results suggest that pressure adaptation of Na+/K+-ATPase is the result of both changes in the primary structure of the protein and homeoviscous adaptation of the lipid environment.Abbreviations EDTA; DPH 1,6-diphenyl-1,3,5-hexatriene - PC phosphatidylcholine - PL phospholipid - SDH succinate dehydrogenase  相似文献   

19.
We have previously shown that peptide neurotensin inhibits cerebral cortex synaptosomal membrane Na+, K+-ATPase, an effect fully prevented by blockade of neurotensin NT1 receptor by antagonist SR 48692. The work was extended to analyze neurotensin effect on Na+, K+-ATPase activity present in other synaptosomal membranes and in CNS myelin and mitochondrial fractions. Results indicated that, besides inhibiting cerebral cortex synaptosomal membrane Na+, K+-ATPase, neurotensin likewise decreased enzyme activity in homologous striatal membranes as well as in a commercial preparation obtained from porcine cerebral cortex. However, the peptide failed to alter either Na+, K+-ATPase activity in cerebellar synaptosomal and myelin membranes or ATPase activity in mitochondrial preparations. Whenever an effect was recorded with the peptide, it was blocked by antagonist SR 48692, indicating the involvement of the high affinity neurotensin receptor (NT1), as well as supporting the contention that, through inhibition of ion transport at synaptic membrane level, neurotensin plays a regulatory role in neurotransmission.  相似文献   

20.
The basolateral membranes of kidney proximal tubule cells have (Na++K+)-ATPase and Na+-ATPase activities, involved in Na+ reabsorption. We showed that ceramide (Cer) modulates protein kinase A (PKA) and protein kinase C (PKC), which are involved in regulating ion transporters. Here we show that ceramide, promotes 60% inhibition of Na+-ATPase activity (I50 ≈ 100 nM). This effect was completely reversed by inhibiting PKA but did not involve the classic PKC signaling pathway. In these membranes we found the Cer-activated atypical PKC zeta (PKCζ) isoform. When PKCζ is inhibited, Cer ceases to inhibit the Na+-ATPase, allowing the cAMP/PKA signaling pathway to recover its stimulatory effect on the pump. There were no effects on the (Na++K+)-ATPase. These results reveal Cer as a potent physiological modulator of the Na+-ATPase, participating in a regulatory network in kidney cells and counteracting the stimulatory effect of PKA via PKCζ.  相似文献   

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