Proteomic analysis of plasma membrane vesicles isolated from the rat renal cortex

Polarized epithelial cells are responsible for the vectorial transport of solutes and have a key role in maintaining body fluid and electrolyte homeostasis. Such cells contain structurally and functionally distinct plasma membrane domains. Brush border and basolateral membranes of renal and intestinal epithelial cells can be separated using a number of different separation techniques, which allow their different transport functions and receptor expressions to be studied. In this communication, we report a proteomic analysis of these two membrane segments, apical and basolateral, obtained from the rat renal cortex isolated by two different methods: differential centrifugation and free-flow electrophoresis. The study was aimed at assessing the nature of the major proteins isolated by these two separation techniques. Two analytical strategies were used: separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at the protein level or by cation-exchange high-performance liquid chromatography (HPLC) after proteolysis (i.e., at the peptide level). Proteolytic peptides derived from the proteins present in gel pieces or from HPLC fractions after proteolysis were sequenced by on-line liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several hundred proteins were identified in each membrane section. In addition to proteins known to be located at the apical and basolateral membranes, several novel proteins were also identified. In particular, a number of proteins with putative roles in signal transduction were identified in both membranes. To our knowledge, this is the first reported study to try and characterize the membrane proteome of polarized epithelial cells and to provide a data set of the most abundant proteins present in renal proximal tubule cell membranes.     

HCO3- transport in basolateral membrane vesicles isolated from rat renal cortex

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.     

Na+/HCO3-co-transport in basolateral membrane vesicles isolated from rabbit renal cortex

Recent studies suggest that the major pathway for exit of HCO3- across the basolateral membrane of the proximal tubule cell is electrogenic Na+/HCO3- co-transport. We therefore evaluated the possible presence of Na+/HCO3- co-transport in basolateral membrane vesicles isolated from the rabbit renal cortex. Imposing an inward HCO3- gradient induced the transient uphill accumulation of Na+, and imposing an outward Na+ gradient caused HCO3- -dependent generation of an inside-acid pH gradient as monitored by quenching of acridine orange fluorescence, findings consistent with the presence of Na+/HCO3- co-transport. In the absence of other driving forces, generating an inside-positive membrane potential by imposing an inward K+ gradient in the presence of valinomycin caused net Na+ uptake via a HCO3- -dependent pathway, indicating that Na+/HCO3- co-transport is electrogenic and associated with a flow of negative charge. Imposing transmembrane Cl- gradients did not appreciably affect HCO3- gradient-stimulated Na+ influx, suggesting that Na+/HCO3- co-transport is not Cl- -dependent. The rate of HCO3- gradient-stimulated Na+ influx was a simple, saturable function of the Na+ concentration (Km = 9.7 mM, Vmax = 160 nmol/min/mg of protein), was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (I50 = 100 microM), but was inhibited less than 10% by up to 1 mM amiloride. We could not demonstrate a HCO3- -dependent or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive component of Na+ influx in microvillus membrane vesicles. This study thus indicates the presence of a transport system mediating electrogenic Na+/HCO3- co-transport in basolateral, but not luminal, membrane vesicles isolated from the rabbit renal cortex. Analogous to the use of renal microvillus membrane vesicles to study Na+/H+ exchange, renal basolateral membrane vesicles may be a useful model system for examining the kinetics and possible regulation of Na+/HCO3- co-transport.     

Specific receptors for atrial natriuretic polypeptide on basolateral membranes isolated from rat renal cortex

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.     

Outer envelope membranes from chloroplasts are isolated as right-side-out vesicles

Outer envelope membranes were isolated from purified chloroplasts of pea leaves. The sidedness of the vesicles was analyzed by (i) aqueous polymer-two phase partitioning, (ii) the effect of limited proteolysis on the outer-envelope proteins (OEP) 86 and OEP 7 in intact organelles and isolated membranes, (iii) fluorescence-microscopy and finally (iv) binding of precursor polypeptides to isolated outer-membrane vesicles. The results demonstrate that purified outer envelope membranes occur largely (>90%) as right-side-out vesicles.Abbreviations FITC fluorescein isothiocyanate - IEP Pinner-envelope protein - OE outer-envelope protein - pSSU precursor form of the small subunit of ribulose bisphosphate carboxylaseoxygenase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We thank P. Å. Albertsson, Lund, Sweden, for introducing one of us (S. E.) to the technique of phase partitioning. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 246) and Fonds der Chemischen Industrie.     

Uptake of proline by brushborder vesicles isolated from human kidney cortex

Proline transport into renal brushborder membrane vesicles isolated from human kidney is mediated by two uptake systems. The high-affinity system is stimulated by a Na gradient and appears to be shared with glycine while the low-affinity system is not. Uptake curves of low concentrations of proline exhibit a Na-gradient-dependent overshoot indicative of electrogenic transport. The proline transport systems observed in isolated human renal brushborder membrane vesicles appear to have characteristics similar to those in rat kidney membranes.     

The characterization of chlorobium vesicles and membranes isolated from green bacteria

Summary The particulate fraction in cell-free extracts of three Chlorobium strains was examined. It contains two structural components: membrane fragments and chlorobium vesicles. These two components have been separated, and certain of their properties compared. Both consist largely of lipid and protein. The lipids of the membrane fraction are predominantly phospholipids, but include a glycolipid which contains galactose, rhamnose and an unidentified sugar. The vesicles are highly enriched in bacteriochlorophyll, and contain large amounts of a monogalactosyl diglyceride; their content of phospholipid is low. All the succinic and malic dehydrogenase activity of the particulate fraction is located in the membranes, whereas NADH- and NADPH-linked dye reductases occur in both membranes and vesicles.The vesicles in crude extracts are unstable upon storage; they rapidly lose their characteristic structure, accompanied by a destruction of bacteriochlorophyll. These changes can be prevented by brief treatment of extracts with glutaraldehyde.Many of the isolated vesicles contain from 10 to 25 regular intravesicular structures, which are about 10 nm wide and circular, with a central hole 3 nm in diameter.     

Transitional endoplasmic reticulum membranes and vesicles isolated from animals and plants

Summary The process of formation from endoplasmic reticulum and transfer to Golgi apparatus of small 50–70 nm transition vesicles has been reconstituted in a cell-free system. Fractions enriched in transition elements derived from part-rough, part-smooth transitional regions of the endoplasmic reticulum were prepared from elongation zones of hypocotyls of etiolated seedlings of soybean and coleoptiles of maize and were compared with those from rat liver. When activated with nucleoside triphosphate, cytosol and an ATP regenerating system, time- and temperature-dependent transfer of membranes to Golgi apparatus acceptor was demonstrated. The fractions enriched in transition elements were radioiodinated with¹²⁵I by the Bolton-Hunter procedure. Acceptor Golgi apparatus stacks were immobilized to nitrocellulose strips to facilitate analysis. In heterologous transfer experiments, the plant and animal acceptors and donors could be interchanged. The transfer was limited primarily by the donor (rat liver > soybean hypocotyl > maize coleoptiles) and determined secondarily by the source of the acceptor. The acceptor fractions were most efficacious when prepared from the same source as the donor. Thus, 50–70 nm vesicles bud from transitional endoplasmic reticulum elements of plants function in a manner similar to those of animal cells to transfer membrane materials to the Golgi apparatus. The recognition signals that determine vesicle fusion appear to be conserved both among species and between the plant and animal kingdoms to the extent that donor and acceptor sources may be interchanged with only small reductions in overall efficiency of transfer.Abbrevations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EDTA ethylenediaminetetraacetic acid     

l-Glutamine transport in native vesicles isolated from Ehrlich ascites tumor cell membranes

Native vesicles isolated from Ehrlich ascites tumor cells accumulate glutamine by means of Na⁺-dependent transport systems; thiocyanate seems to be the more effective anion. The apparent affinity constant for the process was 0.38 mM. The Arrhenius plot gave an apparent activation energy of 12.3 kJ/mol. The structural analogs of glutamine, acivicin (2.5 mM) and azaserine (2.5 mM), inhibited the net uptake by 67 and 70%, respectively. The sulfhydryl reagents mersalyl, PCMBS, NEM, and DTNB also inhibited net uptake, suggesting that sulfhydryl groups may be involved in the activity of the carrier protein. A strong inhibition was detected when the vesicles were incubated in the presence of alanine, cysteine, or serine; in addition, histidine, but not glutamate or leucine, had a negative effect on glutamine transport.     

Carboxypeptidase activity in synaptic vesicles isolated from striatum and cortex of calf brain

A carboxypeptidase activity has been found in synaptic vesicles (secretory granules) isolated from the cortex and striatum of calf brain which removes amino acids from the carboxy terminus of enkephalin-containing (EC) peptides. The formed enkephalin molecules are not further degraded by this enzyme activity. The preparations were found to be free of cytoplasmic and lysosomal constituents as determined by marker enzyme activities. The vesicle preparations of both cortex and striatum showed differences in the degradation velocities of the various EC peptides depending on size and charge of the amino acid present at the carboxy terminus. The pH optimum of the release of Met-enkephalin from Met-enkephalin-Arg6 has been shown to be between pH 5 and 6. The enzyme activity is inhibited by thiol-blocking agents such as p-hydroxymercuribenzoate and copper ions, but only slightly by metal-chelating agents.     

Sodium uptake mechanisms in brush-border membrane vesicles prepared from rabbit renal cortex

Amiloride-sensitive and amiloride-insensitive components of ²²Na⁺ uptake were examined in brush-border membrane vesicles prepared from rabbit renal cortex. Both components could be stimulated by interior-negative electrical potentials, demonstrating a sodium conductance pathway and an effect of electrical potential on the initial rate of Na⁺/H⁺ exchange.     

Transport of d-glucose by brush border membranes isolated from the renal cortex

The transport of d-glucose by brush border membranes isolated from the rabbit renal cortex was studied. At concentrations less than 2 mM, the rate of d-glucose uptake increased linearly with the concentration of the sugar. No evidence was found for a “high-affinity” (μM) saturable site. Saturation was indicated at concentrations of d-glucose greater than 5 mM. The uptake of d-glucose was stereospecific and selectively inhibited by d-galactose and other sugars. Phlorizin inhibited the uptake of d-glucose in the presence and absence of Na⁺. The glycoside was a potent inhibitor of the efflux of d-glucose. Preloading the brush border membrane vesicles with d-glucose, but not with l-glucose, accelerated exchange diffusion of d-glucose. These results demonstrate that the uptake of d-glucose by renal brush borders represents transport into an intravesicular space rather than solely binding. The rate of d-glucose uptake was increased when the Na⁺ in the extravesicular medium was high and the membranes were preloaded with a Na⁺-free medium. The rate of d-glucose uptake was inhibited by preloading the brush border membranes with Na⁺. These results are consistent with the Na⁺ gradient hypothesis for d-glucose transport in the kidney. Thus, the presence of a Na⁺-dependent facilitated transport of d-glucose in isolated renal brush border membranes is indicated. This finding is consistent with what is known of the transport of the sugar in more physiologically intact preparations and suggests that the membranes serve as an effective model system in examining the mechanism of d-glucose transport in the kidney.     

Sulphate-ion/sodium-ion co-transport by brush-border membrane vesicles isolated from rat kidney cortex

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.     

An ATP-driven proton pump in brush-border membranes from rat renal cortex

Summary The rate of ATP hydrolysis in ATP-preloaded plasma membrane vesicles derived from the luminal membrane of renal cortical tubules, and the rate of H⁺ secretion out of the same vesicles were investigated. Both were inhibited at low temperature, by the action of filipin, an antibiotic that complexes with cholesterol in plasma membranes, and by the action of blockers of mitochondrial F_o hydrogen channels, dicyclohexylcarbodiimide and Dio-9. Valinomycin in the presence of K⁺ showed a stimulatory effect, the protonophor carbonyl-cyanid-p-trifluormethoxy-phenylhydrazone stimulated the intravesicular ATP hydrolysis and apparently abolished acidification of the extravesccular medium. Lowering of the pH of the extravesicular medium retarded ATP hydrolysis, while readjustment of extra- and intravesicular pH accelerated ATP hydrolysis again. These findings strongly support the assumption that an ATP-driven proton pump is located in the luminal membrane of renal cortical tubules.     

Phenylalanine uptake in isolated renal brush border vesicles

The uptake of l-phenylalanine into brush border microvilli vesicles and basolateral plasma membrane vesicles isolated from rat kidney cortex by differential centrifugation and free flow electrophoresis was investigated using filtration techniques.Brush border microvilli but not basolateral plasma membrane vesicles take up l-phenylalanine by an Na⁺-dependent, saturable transport system. The apparent affinity of the transport system for l-phenylalanine is 6.1 mM at 100 mM Na⁺ and for Na⁺ 13 mM at 1 mM l-phenylalanine. Reduction of the Na⁺ concentration reduces the apparent affinity of the transport system for l-phenylalanine but does not alter the maximum velocity.In the presence of an electrochemical potential difference for Na⁺ across the membrane (η_Na0 >η_Na1) the brush border microvilli accumulate transiently l-phenylalanine over the concentration in the incubation medium (overshoot phenomenon). This overshoot and the initial rate of uptake are markedly increased when the intravesicular space is rendered electrically more negative by membrane diffusion potentials induced by the use of highly permeant anions, of valinomycin in the presence of an outwardly directed K⁺ gradient and of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence of an outward-directed proton gradient.These results indicate that the entry of l-phenylalanine across the brush border membrane into the proximal tubular epithelial cells involves cotransport with Na⁺ and is dependent on the concentration difference of the amino acid, on the concentration difference of Na⁺ and on the electrical potential difference. The exit of l-phenylalanine across the basolateral plasma membranes is Na⁺-independent and probably involves facilitated diffusion.     

Calcium ion transport across plasma membranes isolated from rat kidney cortex.

Basal-lateral-plasma-membrane vesicles and brush-border-membrane vesicles were isolated from rat kidney cortex by differential centrifugation followed by free-flow-electrophoresis. Ca2+ uptake into these vesicles was investigated by a rapid filtration method. Both membranes show a considerable binding of Ca2+ to the vesicle interior, making the analysis of passive fluxes in uptake experiments difficult. Only the basal-lateral-plasma-membrane vesicles exhibit an ATP-dependent pump activity which can be distinguished from the activity in mitochondrial and endoplasmic reticulum by virtue of the different distribution during free-flow electrophoresis and its lack of sensitivity to oligomycin. The basal-lateral plasma membranes contain in addition a Na+/Ca2+-exchange system which mediates a probably rheogenic counter-transport of Ca2+ and Na+ across the basal cell border. The latter system is probably involved in the secondary active Na+-dependent and ouabain-inhibitable Ca2+ reabsorption in the proximal tubule, the ATP-driven system is probably more important for the maintenance of a low concentration of intracellular Ca2+.