Sodium-bicarbonate cotransport occurs in rat kidney cortical membranes but not in rat small intestinal basolateral membranes.

Basolateral membrane vesicles were isolated from rat kidney cortex and small intestinal enterocytes. Both membrane preparations show ATP-dependent calcium uptake and cytochalasin B-sensitive D-glucose transport. In renal membranes, sodium influx is stimulated by bicarbonate; bicarbonate-dependent sodium flux is membrane-potential-dependent and inhibited by 4,4'-di-isothiocyanato-2, 2'-stilbenedisulphanic acid ('DIDS'). Small intestinal basolateral membranes do not show bicarbonate-dependent sodium fluxes.     

Identification of a fodrin-like protein in rat liver basolateral membranes

A 240 KDa calmodulin- and actin-binding protein has been identified in the plasma membrane of rat liver. This protein is mainly associated with subplasmamembrane fractions enriched in the basolateral domain and very little of it is found in the canalicular membrane fraction. An 80 KDa actin-binding protein is found only in the canalicular fraction.     

Preparative scale isolation of basal-lateral plasma membranes from rat intestinal epithelial cells.

A simple, efficient procedure is described for the preparative scale isolation of basal-lateral membranes from the rat intestinal epithelium. The intestinal mucosa was mildly homogenized and soluble protein and RNA were separated from the homogenate by differential centrifugation. The basal-lateral membranes were then separated from nuclei, mitochondria, and brush border membranes by differential centrifugation in a medium close to the equilibrium density of the basal-lateral membranes. Final purification of the basal-lateral membranes was achieved on a linear density gradient in a high-capacity zonal rotor. The final product (usually at least 40 mg protein) represented a 34% yield of basal-lateral membranes purified 18-fold with respect to protein, 26-fold with respect to brush border membranes, and 53-fold with respect to mitochondria.     

Mechanism of the sarcoplasmic reticulum calcium pump. Fluorometric study of the phosphorylated intermediates.

After removal of calcium ions bound to the high affinity sites the sarcoplasmic reticulum calcium pump can be phosphorylated by inorganic phosphate. The intrinsic fluorescence of the protein is used to follow conformational changes of the pump and an intensity change can be observed upon addition of phosphate. This effect is activated by internal calcium ($\text{K}\text{1}\text{2}\text{= 10 mM}$) and inhibited by external calcium ($\text{K}\text{1}\text{2}\text{= 0.4 μM}$) and the apparent affinity for phosphate is high (0.2 mM). We conclude that the change observed is linked to the formation of the gradient-dependent phosphorylated intermediate. It is compared with previous results concerning the enzymatic cycle of the pump.     

Proton pathways in rat renal brush-border and basolateral membranes

The quenching of acridine orange fluorescence was used to monitor the formation and dissipation of pH gradients in brush-border and basolateral membrane vesicles isolated from rat kidney cortex. The fluorescence changes of acridine orange were shown to be sensitive exclusively to transmembrane delta pH and not to membrane potential difference. In brush-border membrane vesicles, an Na+ (Li+)-H+ exchange was confirmed. At physiological Na+ concentrations, 40-70% of Na+-H+ exchange was mediated by the electroneutral Na+-H+ antiporter; the remainder consisted of Na+ and H+ movements through parallel conductive pathways. Both modes of Na+-H+ exchange were saturable, with half-maximal rates at about 13 and 24 mM Na+, respectively. Besides a Na+ gradient, a K+ gradient was also able to produce an intravesicular acidification, demonstrating conductance pathways for H+ and K+ in brush-border membranes. Experiments with Cl- or SO2-4 gradients failed to demonstrate measurable Cl--OH- or SO2-4-OH- exchange by an electroneutral antiporter in brush-border membrane vesicles; only Cl- conductance was found. In basolateral membrane vesicles, neither Na+(Li+)-H+ exchange nor Na+ or K+ conductances were found. However, in the presence of valinomycin-induced K+ diffusion potential, H+ conductance of basolateral membranes was demonstrated, which was unaffected by ethoxzolamide and 4,4'-diisothiocyanostilbene-2,2-disulfonic acid. A Cl- conductance of the membranes was also found, but antiporter-mediated electroneutral Cl--OH- or SO2-4-OH- exchange could not be detected by the dye method. The restriction of the electroneutral Na+-H+ exchanger to the luminal membrane can explain net secretion of protons in the mammalian proximal tubule which leads to the reabsorption of bicarbonate.     

Occlusion of divalent cations in the phosphorylated calcium pump of sarcoplasmic reticulum

The calcium pump of sarcoplasmic reticulum possesses high-affinity calcium-binding and ATP-binding sites. At 0 degrees C pH 6.8 and in the absence of calcium, about 3.5 nmol/mg of high-affinity ATP-binding sites are titrated with a dissociation constant, Kd, of 5 microM. In the presence of Ca2+, ATP phosphorylates the enzyme at a much lower concentration: K 1/2 = 100 nM. In the absence of ATP the calcium ions reversibly bind to the high-affinity calcium sites (6.5 nmol/mg); however the following is shown in this paper. 1. Phosphorylation of the enzyme in the presence of calcium leads to the immediate occlusion of the calcium ions bound to the high-affinity sites. 2. Two moles of calcium are occluded per mole of phosphoenzyme formed. 3. Occlusion can be reversed by ADP. 4. Transport is a slower process which occurs in the presence of Mg2+ at the same rate as the spontaneous decay of the phosphoenzyme. Experiments performed in the absence of magnesium reveal another divalent cation binding site which is probably directly involved in ATP and Pi binding. The nature of the cation bound to this site determines the stability and ADP-sensitivity of the phosphoenzyme.     

Activatory effect of calcium-binding protein regucalcin on ATP-dependent calcium transport in the basolateral membranes of rat kidney cortex

The effect of regucalcin, a calcium-binding protein, on ATP-dependent Ca2+ transport in the basolateral membranes isolated from rat kidney cortex was investigated. The prepared membranes were in inside-out oriented and membrane vesicles. Ca2+-ATPase activity in the basolateral membranes was progressively elevated by increasing concentrations of regucalcin (10-8 to 10-6 M) in the reaction mixture. This increase was dependent on Ca2+ addition. The activatory effect of regucalcin on the enzyme is inhibited by the presence of digitonin (5 × 10-6%) which can solubilize the membranous lipids. Moreover, the regucalcin effect was clearly abolished by the presence of vanadate (0.1 mM) or N-ethylmaleimide (5.0 mM). However, the effect of calmodulin (6 × 10-7 M) to increase Ca2+-ATPase activity was not significantly inhibited by vanadate or N-ethylmaleimide, indicating that the action mode of regucalcin differs from that of calmodulin. Also, the activatory effect of regucalcin on Ca2+-ATPase was appreciably inhibited by addition of dibutyryl cAMP (10-5 and 10-3 M), while inositol 1,4,5-trisphosphate (10-7 and 10-5 M) had no effect. Dibutyryl cAMP itself did not have an effect on the enzyme activity. Furthermore, the 45Ca2+ uptake by the basolateral membranes was clearly increased by the presence of regucalcin (10-7 and 10-6 M). This increase was completely blocked by the presence of vanadate (0.1 mM), N-ethylmaleimide (5.0 mM) or dibutyryl cAMP (10-4 and 10-3 M) in the reaction mixture. These results clearly demonstrate that regucalcin, which is expressed in rat kidney cortex, can increase Ca2+-ATPase activity and Ca2+ uptake in the basolateral membranes. Regucalcin may play a cell physiologic role as an activator in the ATP-dependent Ca2+ pumps in the basolateral membranes from rat kidney cortex.     

Quantitation of plasma membrane calcium pump phosphorylated intermediates by electrophoresis

P-ATPases are characterized by the formation of acid-stable phosphorylated intermediates (EP) during their reaction cycle. We have developed a microscale method to determine EP that involves the phosphorylation of the enzyme using [gamma-(32)P]ATP and precipitation with TCA; separation of the sample by SDS-PAGE, and measurement of the enzyme protein and (32)P-labeled EP by digital analysis of both the stained gel and its autoradiogram, respectively. The principal advantages of this method over typical procedures (filtration and centrifugation) are the low amount of enzyme required and the substantial decrease in the blank values and data scattering produced by unspecific phosphorylation and nonquantitative recovering of the enzyme. Application of this new method to a purified preparation of the plasma membrane calcium ATPase (PMCA) results in overcoming the difficulties of measuring EP at high ATP concentrations. A biphasic behavior of the substrate curve for EP was observed when the study was extended to ATP levels within the physiological range. Since, in principle, the method does not require the use of highly purified preparations, it could be helpful for the study of phosphorylated intermediates especially under conditions in which small amounts of protein are available, e.g., mutated variants of P-ATPases.     

Asymmetric distribution of gangliosides in rat renal brush-border and basolateral membranes

Highly enriched brush-border and basolateral membranes isolated from rat renal cortex were used to study the distribution of endogenous gangliosides in the two distinct plasma membrane domains of epithelial cells. These two membrane domains differed in their glycolipid composition. The basolateral membranes contained more of both neutral and acidic glycolipids, expressed on a protein basis. In both membranes, the neutral glycolipids corresponding to mono-, di-, tri- and tetraglycosylceramides were present. The basolateral membranes contained more diglycosylceramide than the brush-border membranes. The major gangliosides found were GM4, GM3, and GD3 with minor amounts of GM1 and GD1a. The latter were identified and quantified by sensitive iodinated cholera toxin binding assays. When the distribution of individual gangliosides was calculated as a percent of total gangliosides, the brush-border membranes were enriched with GM3, GM1 and GD1a compared to the basolateral membranes, which were enriched with GD3 and GM4. The observation of a distinct distribution of glycolipids between brush-border and basolateral membranes of the same epithelial cell suggests that there may be a specific sorting and insertion process for epithelial plasma membrane glycolipids. In turn, asymmetric glycolipid biogenesis may reflect differences in glycolipid function between the two domains of the epithelial plasma membrane.     

Basolateral plasma membranes of intestinal epithelial cells. Identification by lactoperoxidase-catalysed iodination and isolation after density perturbation with digitonin.

Lactoperoxidase-catalysed iodination was used to label intestinal epithelial cell sheets with 125I. The iodination was carried out under conditions that allowed little penetration of lactoperoxidase into the cells and membrane-bound 125I therefore provided an effective marker for following plasma-membrane fragments through subcellular-fractionation procedures. 2. After homogenization and isopycnic zonal centrifugation through sucrose gradients two peaks of membrane-bound 125I were detected. One coincided with brush border enzymes such as alkaline phosphatase, disaccharidases and L-leucine B-naphthylamidase, whereas the other was coincident with the major peak of (Na++K+)-stimulated ATPase (adenosine triphosphatase), which has been thought to be concentrated in the basolateral plasma membranes of these cells. Neither peak of 125I reflected the distribution of any marker for an intracellular organelle. 3. A larger proportion of the (Na++K+)-stimulated ATPase, and thus of the basolateral plasma-membrane material, was found in a crude 'mitochondrial' fraction. It was not readiily separated from mitochondria by conventional techniques of subcellular fractionation. 4. Treatment of the 'mitochondrial' fraction with digitonin increased the density of basolateral plasma membrane but had little effect on mitochondrial density. A purified preparation of digitonin-loaded basolateral plasma membranes was isolated at a density of 1.20-1.22 by isopycnic centrifugation. 5. The enzymic composition of this preparation of basolateral plasma membranes is compared with previous preparations isolated from intestinal mucosal 'scrape' materials and from isolated cells.     

Kinetic effects of calcium and ADP on the phosphorylated intermediate of sarcoplasmic reticulum ATPase

The decomposition of 32P phosphorylated enzyme intermediate formed by incubation of sarcoplasmic reticulum ATPase with [gamma-32P]ATP was studied following dilution of the reaction medium with a large excess of nonradioactive ATP. The phosphoenzyme decomposition includes two kinetic components. The fraction of intermediate undergoing slower decomposition is minimal in the presence of low (microM) Ca2+ and maximal in the presence of high (mM) Ca2+. A large fraction of phosphoenzyme undergoes slow decomposition when the Ca2+ concentration is high inside the vesicles, even if the Ca2+ concentration in the medium outside the vesicles is low. Parallel measurements of ATPase steady state velocity in the same experimental conditions indicate that the apparent rate constant for the slow component of phosphoenzyme decomposition is inadequate to account for the steady state ATPase velocity observed under the same conditions and cannot be the rate-limiting step in a single, obligatory pathway of the catalytic cycle. On the contrary, the steady state enzyme velocity at various Ca2+ concentrations is accounted for by the simultaneous contribution of both phosphoenzyme fractions undergoing fast and slow decomposition. Contrary to its slow rate of decomposition in the forward direction of the cycle, the phosphoenzyme pool formed in the presence of high Ca2+ reacts rapidly with ADP to form ATP in the reverse direction of the cycle. Detailed analysis of these experimental observations is consistent with a branched pathway following phosphoryl transfer from ATP to the enzyme, whereby the phosphoenzyme undergoes an isomeric transition followed by ADP dissociation, or ADP dissociation followed by the isomeric transition. The former path is much faster and is prevalent when the intravesicular Ca2+ concentration is low. When the intravesicular Ca2+ concentration rises, a pool of phosphoenzyme is formed by reverse equilibration through the alternate path. In the absence of ADP this intermediate decays slowly in the forward direction, and in the presence of ADP it decays rapidly in the reverse direction of the cycle.     

1 alpha, 25-Dihydroxy-vitamin D-3 regulates ATP-dependent calcium transport in basolateral plasma membranes of rat enterocytes

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 1 alpha,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.     

Somatostatin and intestinal calcium transport in the rat

In intact rats we studied the influence of low doses of intravenously (i.v.) administered somatostatin (SRIF) on the net absorption and the bidirectional fluxes (lumen-to-plasma, LP; plasma-to-lumen, PL) of calcium in the duodenum, jejunum, ileum and caecum. In the duodenum SRIF inhibited the LP-flux and the net absorption of Ca significantly at infusion rates of 0.75 and 1.0 microgram SRIF . kg-1 . h-1. The PL-flux was not altered by any of the SRIF doses administered. In the other gut segments studied (jejunum, ileum, caecum) neither the net absorption nor the bidirectional Ca fluxes were changed by i.v. SRIF. It is concluded that SRIF in the plasma levels achieved in this study has an influence on the duodenal calcium absorption (CaA) of the rat; questions regarding the mechanisms of this action as well as the physiological significance of our findings are as yet unresolved.     

Calcium transport and phosphorylated intermediate of (Ca2+ + Mg2+)-ATPase in plasma membranes of rat liver

We have identified and characterized calcium transport and the phosphorylated intermediate of the (Ca2+ + Mg2+)-ATPase in plasma membrane vesicles prepared from rat liver. The calcium transport did not absolutely require the presence of oxalate and was completely inhibited by 1 microM of ionophore A23187. Oxalate, which serves as a trapping agent in calcium uptake of skeletal muscle and liver microsomes, was not absolutely required to maintain the net accumulation of calcium. The Vmax and Km for calcium uptake were 35.2 +/- 10.1 pmol of calcium/mg of protein/min, and 17.6 +/- 2.5 nM of free calcium, respectively. Ten mM magnesium was required for the maximal accumulation of calcium. Substitution of 5 and 10 mM ADP, CTP, GTP, and UTP for ATP could not support calcium uptake. The calcium uptake was not affected by 0.5 mM ouabain, 20 mM azide, or 2 micrograms/ml of oligomycin but was inhibited in a dose-dependent fashion by vanadate, with a Ki of approximately 20 microM for vanadate. The substrate affinities and specificities of this calcium-transport activity suggest that it is closely associated with the (Ca2+ + Mg2+)-ATPase reported in the plasma membranes of liver (Lotersztajn, S., Hanoune, J., and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215). A calcium-stimulated and magnesium-dependent phosphoprotein was also demonstrated in the same membrane vesicles. The free calcium concentration at which its phosphorylation was half-maximal was 15.5 +/- 5.6 nM. Sodium fluoride, ouabain, sodium azide, oligomycin, adriamycin, and N,N'-dicyclohexylcarbodiimide did not affect its formation while vanadate at 100 microM inhibited the calcium-dependent phosphorylation by approximately 60%. The properties of this phosphoprotein suggest that it may be the phosphorylated intermediate of the (Ca2+ + Mg2+)-ATPase in the plasma membranes of rat liver.     

Binding proteins for linear renin-inhibiting peptides in basolateral plasma membranes of rat liver.

A linear hydrophobic peptide, (Code no. EMD 55068), a synthetic renin-antagonist, competitively inhibits the uptake of taurocholate and of another linear peptide (EMD 51921) but not of oleic acid, serine or thiamin hydrochloride into isolated rat liver cells. EMD 55068 was attached to a gel matrix at a position that is not involved in the protein ligand interaction. The gel matrix used did not interact nonspecifically with solubilized proteins from rat liver. The quantity of bound ligand was determined to be 3.6 mg/ml of gel matrix. In the fraction of EDTA extracted hydrophilic membrane-associated proteins, no binding proteins were detected. Affinity chromatography of integral plasma membrane proteins resulted in four protein bands with molecular masses of 46, 49, 53 and 56 kDa in SDS-PAGE. In contrast, solubilized plasma membrane proteins from AS-30D ascites hepatoma cells, which are unable to transport bile acids and linear peptides, did not bind specifically to the affinity matrix.     

Temperature adaptation of biological membranes: differential homoeoviscous responses in brush-border and basolateral membranes of carp intestinal mucosa

The effects of temperature acclimation of carp upon the hydrocarbon order of intestinal membranes has been determined. A fractionation technique has been developed for the simultaneous purification of brush-border and basolateral membrane fractions from the intestinal mucosa. The specific activity of alkaline phosphatase in the brush-border fraction was enhanced 6.4-fold over that of the initial homogenate, whilst the (Na(+)-K+)-stimulated ATPase was enhanced 5.8-fold in the basolateral fraction. The specific activities of NADPH-cytochrome-c reductase, succinate-cytochrome-c reductase and acid phosphatase were not increased in these two fractions. Membrane hydrocarbon order in membranes from 10 and 30 degrees C-acclimated carp has been compared by measuring the steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene over a range of temperatures. In the brush-border fraction, polarization was identical in both cold- and warm-acclimated groups, whilst large differences were observed in the basolateral fraction sufficient to offset approx. 75% of the temperature-induced ordering effects of cold. The fatty acid composition of the major phosphoglyceride fractions in the brush-border fraction was also largely unaffected by thermal acclimation, whilst the basolateral fraction showed significant increases in the proportion of unsaturated fatty acids in the cold. It is concluded that whilst the basolateral membrane of intestinal mucosa displays a large homoeoviscous response that correlates with a shift in lipid composition, the brush-border membrane does not. These findings are consistent with evidence of functional adaptations of the basolateral membrane during thermal acclimation (Gibson, J.S., Ellory, J.C. and Cossins, A.R. (1985) J. Exp. Biol. 114, 355-364).