Separation of basolateral plasma membranes from smooth endoplasmic reticulum of the rat enterocyte by zonal electrophoresis on density gradients

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.     

Redox reactions of tonoplast and plasma membranes isolated from soybean hypocotyls by free-flow electrophoresis

Redox reactions were studied in more than 90% pure tonoplast and plasma membranes isolated by free-flow electrophoresis from soybean (Glycine max) hypocotyls. Both types of membrane contained a b-type cytochrome (_max = 561 nm) and a noncovalently bound flavin, two possible components of a transmembrane electron-transport chain. Isolated tonoplast and plasma membranes reduced ferricyanide, indophenol and various iron complexes with NADH or NADPH as electron donors. The redox activity was inhibited in tonoplast membranes by about 60% by 10 μM p-chloromercuribenzene sulfonate, 8% by 500 μM lanthanum nitrate and 10% by 100 μM nitrophenyl acetate. In contrast, the redox activity of isolated plasma membranes was inhibited by about 60% by 500 μM lanthanum nitrate or 100 μM nitrophenyl acetate, but only 25% by 10 μM p-chloromercuribenzene sulfonate. The results show that both tonoplast and plasma membranes of soybean contain active electron-transport systems, but that the two systems respond differently to inhibitors.     

Fluorescence anisotropy from diphenylhexatriene in rat liver plasma membranes

Rat livers were fractionated to obtain intracellular membrane preparations and a highly purified preparation of bile canaliculi. The fraction containing bile canaliculi was homogenized and subfractionated to give fractions representing fragments of contiguous membrane and of canalicular microvilli. The relative purity and extent of contamination of each preparation was determined. When the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene was incorporated into aliquots of each fraction at the same probe: lipid ratio and the steady-state anisotropy of its fluorescence measured, it was found that the plasma membrane preparations were much more ordered than the intracellular membrane preparations. Of the plasma membrane preparations, that containing the canalicular microvilli was the most ordered, even allowing for any contribution of contaminants. Thus the microvillus membrane of the bile canaliculus appears to be the most ordered domain of the plasma membrane of the hepatocyte. The high order in this domain may be a factor in reducing the susceptibility to bile salt damage during bile secretion, since it is this region which is exposed to high concentrations of bile salts in vivo.     

Role of rat organic anion transporter 3 (Oat3) in the renal basolateral transport of glutathione

The tripeptide GSH is important in maintenance of renal redox status and defense against reactive electrophiles and oxidants. Previous studies showed that GSH is transported across the basolateral plasma membrane (BLM) into the renal proximal tubule by both sodium-coupled and sodium-independent pathways. Substrate specificity and inhibitor studies suggested the function of several carriers, including organic anion transporter 3 (Oat3). To test the hypothesis that rat Oat3 can function in renal GSH transport, the cDNA for rat Oat3 was expressed as a His6-tagged protein in E. coli, purified from inclusion bodies and by Ni2+-affinity chromatography, and reconstituted into proteoliposomes. cDNA-expressed and reconstituted Oat3 transported both GSH and p-aminohippurate (PAH) in exchange for 2-oxoglutarate (2-OG) and 2-OG and PAH in exchange for GSH, and PAH uptake was inhibited by both probenecid and furosemide, consistent with function of Oat3. mRNA expression of Oat3 and several other potential carriers was detected by RT-PCR in rat kidney cortex but was absent from NRK-52E cells, a rat proximal tubular cell line. Basolateral uptake of GSH in NRK-52E cells showed little PAH- or 2-OG-stimulated uptake. We conclude that Oat3 can function in GSH uptake and that NRK-52E cells possess a low background rate of GSH uptake, making these cells a good model for overexpression of specific, putative GSH carriers.     

A simple method for the isolation of basolateral plasma membrane vesicles from rat kidney cortex Enzyme activities and some properties of glucose transport

A procedure for preparing basolateral membrane vesicles from rat renal cortex was developed by differential centrifugation and Percoll density gradient centrifugation, and the uptake of d-[³H]glucose into these vesicles was studied by a rapid filtration technique. (Na⁺ + K⁺)-ATPase, the marker enzyme for basolateral membranes, was enriched 22-fold compared with that found in the homogenate. The rate of d-glucose uptake was almost unaffected by Na⁺ gradient (no overshoot).     

Intrinsic protein phosphorylation in synaptic plasma membrane fragments from the rat General characteristics and migration behaviour on polyacrylamide gels of the main phosphate acceptors

Preparations enriched in synaptic membrane fragments from rat cerebral cortex contain protein kinases which phosphorylate membrane proteins in reactions dependent on cAMP, Ca²⁺ (in the absence or presence of calmodulin) or independent of these factors. In these present work characteristics of the main phosphorylated acceptors were studied and compared with the results of other investigations. Apparent molecular weights were estimated by determining electrophoretic mobility on gels of different acrylamide concentration. Irregular migration behaviour was detected by measuring free mobilities from Ferguson plots. Certain phosphate acceptors were found to exhibit anomalously low free mobilities and it was concluded that estimates of molecular weight for these acceptors were unreliable.     

Isolation of plasma membranes from rat lungs. Effect of age on the subcellular distribution of adenylate cyclase activity

A simple and rapid method of isolating plasma membranes from rat lungs is described. The method involves homogenization of tissue in isotonic sucrose-buffered medium followed by differential and sucrose density gradient centrifugation. Plasma membranes obtained by this procedure were essentially free from other subcellular contamination. Plasma membranes isolated from 2-day-old rat lungs showed 6 to 7-fold purification of adenylate cyclase and 5′-nucleotidase activities compared to the original homogenate In contrast, plasma membranes from 35-day-old rat lungs showed no purification of adenylate cyclase activity although 5′-nucleotidase activity showed similar enrichment. These results suggest that adenylate cyclase activity is not a reliable marker for plasma membranes from adult rat lungs.     

Partial purification and properties of the cytoplasmic factors modulating adenylate cyclase activity in rat lung plasma membranes

The adult rat lung supernatant contains some factors which markedly enhance adenylate cyclase activity in membranes (Nijjar, M.S. (1979) Biochim. Biophys. Acta 584, 43–50). These factors were separated into two less active components (peaks 1 and 2) by DEAE-cellulose chromatography. However, their recombination restored the full activation of adenylate cyclase. Further purification and characterization of these factors revealed that the activation in peak 1 contained two proteins of low (14 500) and high (65 000) molecular weight whereas the activator in peak 2 contained only one protein of 65 000. The kinetics of adenylate cyclase activation revealed that both the K_m and V values were affected. The data also demonstrate that calmodulin was not involved in the cytoplasmic activation of adenylate cyclase in rat lungs.     

Simultaneous isolation of brush border and basolateral membrane from rabbit enterocytes. Presence of brush border hydrolases in the basolateral membrane of rabbit enterocytes

By a slight modification of the procedure described by Gratecos et al. (Gratecos, D., Knibiehler, M., Benoit, V. and Sémériva, M. (1978) Biochim. Biophys. Acta 512, 508–524), the basolateral and brush border membranes of rabbit enterocytes have been purified concomitantly from the same aliquot of mucosa. The two types of membrane have been obtained with the same yield (15%) and enrichment of specific markers (18-fold).The presence in the basolateral membrane of hydrolases known to be specific of the brush border membrane has been confirmed by using immunological techniques.     

Analysis and purification of the blood-sinusoidal domain of rat liver plasma membrane

Methods are described for the analysis and purification of the blood-sinusoidal domains of rat liver plasma membranes using a combination of sucrose and Ficoll density gradient centrifugation. Use has been made of ¹²⁵I-labelled wheat-germ agglutinin and hormone-stimulated adenylate cyclase to identify the blood sinusoidal fraction, which may be resolved from Golgi and endoplasmic reticulum markers on Ficoll gradients.     

The mapping of three subfractions of endoplasmic reticulum membranes isolated from L-929 cells by the use of spin probes

Summary This paper concerns the estimation of microviscosity parameters in smooth, light rough and heavy rough endoplasmic reticulum subfractions isolated from L-929 cells. Electron spin resonance using three probes was utilized in order to make estimations of rotational correlation times. The highest microviscosity was found in the smooth fraction. The lipid bilayer is less viscous and the annular one more rigid in heavy rough compared to light rough membranes. The individual membrane subfractions differ with regard to their portrait of thermoinduced structural transitions. The highest number of such transitions was detected in smooth membranes. There were no low-temperature transitions (relative to physiological temperature) or common thermoinduced structural rearrangements of the lipids in the heavy rough subfraction, a membrane fraction characteristic of transformed cells. The results show that each membrane subfraction is characterized by an intrinsic series of thermoinduced structural transitions, which, in combination with an estimation of microviscosity, yields a portrait of the structural state of the membrane lipids.     

Binding and degredation of insulin by plasma membranes from bovine liver isolated by a large scale preparation

With the large-scale preparation described, as much as 1 kg of bovine liver can be processed, giving a yield of more than 1 g plasma membrane protein. From analytical and morphological criteria the plasma membrane fraction isolated mainly derives from bile-canalicular and contiguous areas of the hepatocytes.The insulin binding activity is quite similar to insulin receptors in otherr cell systems and membrane preparations. Insulin-degrading activity is very low in the isolated plasma fraction. Most of degrading activity is located in a microsomal membrane fraction. Neverthless the K_m and the pH dependence of the insulin-degrading activity in both fractions are nearly identical.From these studies we conclude that binding and degradation of insulin are two independent processes located on different cell organelles.     

Asymmetric synthesis followed by transmembrane movement of phosphatidylethanolamine in rat liver endoplasmic reticulum

Studies with phospholipase C have indicated that two-thirds of the phosphatidylethanolamine of rat liver endoplasmic reticulum is located in the inner leaflet of the membrane bilayer. Phosphatidyl[¹⁴C]ethanolamine is synthesised in microsomes incubated with CDP[¹⁴C]ethanolamine. Using phospholipase C as a probe we have observed that the labelled phospholipid is initially (1–2 min) concentrated in the ‘outer leaflet’ of the membrane bilayer. The specific activity of this pool of phosphatidylethanolamine was 3.5 times that of the inner leaflet. If, however, the microsomes were opened with 0.4% taurocholate or the French pressure cell to make both sides of the bilayer available to phospholipase C, the phosphatidylethanolamine behaves as a single pool for hydrolysis. On longer incubation, up to 30 min, with CDP[¹⁴C]ethanolamine the specific activity of the outer leaflet phosphatidylethanolamine becomes close to that of the inner leaflet. In chase experiments, in which microsomal phosphatidylethanolamine was labelled by incubation with CDP[¹⁴C]ethanolamine for 1 min, the reaction stopped by addition of calcium, and the microsomes isolated by centrifugation and reincubated, labelled phosphatidylethanolamine was transferred from the ‘outer leaflet’ to the ‘inner leaflet’, so that both were equally labelled. These observations suggest that phosphatidylethanolamine is synthesised at the cytoplasmic leaflet of the endoplasmic reticulum and subsequently transferred across the membrane to the cisternal leaflet of the bilayer. Transmembrane movement is apparently temperature-dependent and independent of continued synthesis of phosphatidylethanolamine.     

Location of rat liver iodothyronine deiodinating enzymes in the endoplasmic reticulum

The iodothyronine-deiodinating enzymes (iodothyronine-5- and 5′-deiodinase) of rat liver were found to be located in the parenchymal cells. Differential centrifugation of rat liver homogenate revealed that the deiodinases resided mainly in the microsomal fraction. The subcellular distribution pattern of these enzymes correlated best with glucose-6-phosphatase, a marker enzyme of the endoplasmic reticulum. Plasma membranes, prepared by discontinuous sucrose gradient centrifugation, were found to contain very little deiodinating activity. Analysis of fractions obtained during the course of plasma membrane isolation showed that the deiodinases correlated positively with glucose-6-phosphates (r >/ 0.98) and negatively with the plasma membrane marker 5′-nucleotidase (r ranging between ?0.88 and ?0.97). It is concluded that the iodothyronine-deiodinating enzymes of rat liver are associated with the endoplasmic reticulum.     

Differentiation of Ca pumps linked to plasma membrane and endoplasmic reticulum in the microsomal fraction from intestinal smooth muscle

ATP promotes ⁴⁵Ca uptake by the microsomal fraction from the longitudinal smooth muscle of guinea-pig ileum and this uptake is stimulated by oxalate. As the microsomal fraction is made up of various subcellular entities, we examined the localization of the Ca²⁺-transport activity by density gradient centrifugation, taking advantage of the selective effect of digitonin (at low concentration) on the density of plasmalemmal elements. When the ⁴⁵Ca-uptake activity was measured in the absence of oxalate, its behavior in subfractionation experiments closely paralleled that of the plasmalemmal marker 5′-nucleotidase. In contrast, the additional Ca²⁺-transport activity elicited by oxalate behaved like NADH-cytochrome c reductase, a putative endoplasmic reticulum marker. The endoplasmic reticulum vesicles constituted only a small part of the membranes in the microsomal fraction, which explains that their Ca²⁺-storage capacity was not detectable in the absence of Ca²⁺-trapping agent. Low digitonin concentrations selectively increased the Ca²⁺ permeability of the plasmalemmal vesicles. The two Ca²⁺-transport activities were further differentiated by their distinct sensitivities to K⁺, vanadate and calmodulin. In this respect, the oxalte-insensitive and oxalate-stimulated Ca²⁺-transport systems resembled, respectively, the sarcolemmal and sarcoplasmic reticulum Ca²⁺ pumps in cardiac and skeletal muscle, in accordance with the subcellular locations established by density gradient centrifugation.     

Reconstitution of a passive Ca2+-transport pathway from the basolateral plasma membrane of rat parotid gland acinar cells

We have previously reported that rat parotid gland basolateral plasma membrane vesicles (BLMV) have a relatively high affinity Ca²⁺ transport pathway and an unsaturable Ca²⁺ flux component (Lockwich et al., 1994. J. Membrane Biol. 141:289–296). In this study, we have solubilized BLMV with octylglucoside (1.5%) and have reconstituted the solubilized proteins into proteoliposomes (PrL) composed of E. coli bulk phospholipids, by using a detergent dilution method. PrL exhibited 3–5-fold higher ⁴⁵Ca²⁺ influx than control liposomes (without protein). Ca²⁺ uptake into PrL was dependent on the [protein] in PrL and steady state [Ca²⁺] in PrL was in equilibrium with external [Ca²⁺]. These data demonstrate that a passive, protein-mediated Ca²⁺ transport has been reconstituted from BLMV into PrL. ⁴⁵Ca²⁺ influx into liposomes did not saturate with increasing [Ca²⁺] in the assay medium. In contrast, PrL displayed saturable ⁴⁵Ca²⁺ influx and exhibited a single Ca²⁺ flux component with an apparent K _ca=242 ± 50.9 m and V _max=13.5 ± 1.14 nmoles Ca²⁺/mg protein/ minute. The K _ca of Ca²⁺-transport in PrL was similar to that of the high affinity Ca²⁺ influx component in BLMV while the V _max was about 4-fold higher. The unsaturable Ca²⁺ flux component was not detected in PrL. ⁴⁵Ca²⁺ influx in PrL was inhibited by divalent cations in the order of efficacy, Zn²⁺>Mn²⁺>Co²⁺=Ni²⁺, and appeared to be more sensitive to lower concentrations of Zn²⁺ than in BLMV. Consistent with our observations with BLMV, the carboxyl group reagent N,N-dicyclohexylcarbodiimide (DCCD) inhibited the reconstituted Ca²⁺ transport in PrL. Importantly, in both BLMV and PrL, DCCD induced a 40–50% decrease in V _max of Ca²⁺ transport without an alteration in K _ca. These data strongly suggest that the high affinity, passive Ca²⁺ transport pathway present in BLMV has been functionally reconstituted into PrL. We suggest that this approach provides a useful experimental system towards isolation of the protein(s) involved in mediating Ca²⁺ influx in the rat parotid gland basolateral plasma membrane.We thank Dr. Bruce Baum for his constant support and encouragement. We also thank Ms. Grace Park and all our colleagues for their assistance during the course of this work.     

Purification of plasma membranes from leaves of conifer and deciduous tree species by phase partitioning and free-flow electrophoresis

Purified plasma membrane fractions were obtained from leaves of Picea abies L., Pinus sylvestris L., Fagus sylvatica L. and Quercus robur L., whereas plasma membranes from Pinus halepensis Mill, proved to be more difficult to obtain, perhaps due to the higher content of volatile substances in this plant species. Plasma membranes were purified by both phase partitioning and free-flow electrophoresis from microsomal fractions and identified on the basis of biochemical and in some cases morphological and cytochemical markers. Electron micrographs revealed that membrane vesicles from Pinus sylvestris exhibited a very clear dark-light-dark pattern and measurements of membrane thickness showed that it ranged from 6 to 10 nm. Most membranes were 8 nm thick and stained with phosphotungstic acid at low pH, both typical characteristics of the plasma membrane. Enzymatic identification of plasma membranes consisted in the determination of the vanadate-sensitive ATPase (EC 3.6.1.3) activity. The specific activity in the upper phase (U₂) fraction was 10–25 times higher than those in the lower phase and microsomal fractions, depending on plant species. 1,3-β-glucan synthase II (EC 2.4.1.3), another putative plasma membrane marker, was not detected in the plasma membrane-enriched fractions of conifer needles and showed a very low specific activity in membranes of deciduous trees. Contamination by membranes of other origin was determined by analysis of membrane markers: cytochrome c oxidase (EC 1.9.3.1) for mitochondria, inosine diphosphatase (EC 3.6.1.6) for Golgi apparatus, cytochrome c reductase (EC 1.6.2.4) for endoplasmic reticulum, and pyrophosphatase (EC 3.6.1.1) for tonoplasts. The main, but relatively low contamination, was due to tonoplasts, as determined by the activity of pyrophosphatase. Plasma membrane characteristics were quite different depending on the season during which needles were taken. Membrane preparations of better quality were more easily obtained from samples taken during winter.     

The migration of labeled phosphatidylcholine from the nuclear-associated endoplasmic reticulum to plasma membranes in L-929 cells

Summary The nuclear-associated endoplasmic reticulum of L-929 cells was found to contain the highest amount of labeled phosphatidylcholine after a 60 min incubation with¹⁴C-choline. Radioactivity was otherwise distributed relatively evenly among other membrane-containing organelles (nuclei, mitochondria, plasma membranes and endoplasmic reticulum membranes). During a 120 min chase following removal of isotope and addition of cold choline chloride, there was a considerable reduction in labeled phosphatidylcholine in the NER and nuclei. The decrease in radioactivity in these fractions was matched by an almost identical increase in the fraction containing mitochondria and plasma membranes. Separation of mitochondria and plasma membranes by centrifugation on discontinuous gradients showed that¹⁴C-choline labeled phosphatidylcholine appeared most rapidly in the plasma membranes. The results indicate that phospholipid molecules migrate within a short period of time from their site of synthesis in the NER to plasma membranes.     

Kinetic properties of Na+/Ca2+ exchange in basolateral plasma membranes of rat small intestine

The presence of an Na⁺/Ca²⁺ exchange system in basolateral plasma membranes from rat small intestinal epithelium has been demonstrated by studying Na⁺ gradient-dependent Ca²⁺ uptake and the inhibition of ATP-dependent Ca²⁺ accumulation by Na⁺. The presence of 75 mM Na⁺ in the uptake solution reduces ATP-dependent Ca²⁺ transport by 45%, despite the fact that Na⁺ does not affect Ca²⁺-ATPase activity. Preincubation of the membrane vesicles with ouabain or monensin reduces the Na⁺ inhibition of ATP-dependent Ca²⁺ uptake to 20%, apparently by preventing accumulation of Na⁺ in the vesicles realized by the Na⁺-pump. It was concluded that high intravesicular Na⁺ competes with Ca²⁺ for intravesicular Ca²⁺ binding sites. In the presence of ouabain, the inhibition of ATP-dependent Ca²⁺ transport shows a sigmoidal dependence on the Na⁺ concentration, suggesting cooperative interaction between counter transport of at least two sodium ions for one calcium ion. The apparent affinity for Na⁺ is between 15 and 20 mM. Uptake of Ca²⁺ in the absence of ATP can be enhanced by an Na⁺ gradient (Na⁺ inside > Na⁺ outside). This Na⁺ gradient-dependent Ca²⁺ uptake is further stimulated by an inside positive membrane potential but abolished by monensin. The apparent affinity for Ca²⁺ of this system is below 1 μM. In contrast to the ATP-dependent Ca²⁺ transport, there is no significant difference in Na⁺ gradient-dependent Ca²⁺ uptake between basolateral vesicles from duodenum, midjejunum and terminal ileum. In duodenum the activity of ATP-driven Ca²⁺ uptake is 5-times greater than the Na⁺/Ca²⁺ exchange capacity but in the ileum both systems are of equal potency. Furthermore, the Na⁺/Ca²⁺ exchange mechanism is not subject to regulation by 1α,25-dihydroxy vitamin D-3, since repletion of vitamin D-deficient rats with this seco-steroid hormone does not influence the Na⁺/Ca²⁺ exchange system while it doubles the ATP-driven Ca²⁺ pump activity.     

Differential time-course of induction of rat liver gamma-glutamyltransferase and drug-metabolizing enzymes in the endoplasmic reticulum, Golgi and plasma membranes after a single phenobarbital injection. Evaluation of protein variations by two-dimensional electrophoresis

This study was conducted to follow as a function of time the activity of gamma-glutamyltransferase in the various membranes of rat liver cells after a single dose of phenobarbital (PB) (75 mg kg-1 body weight). Gamma-glutamyltransferase induction was maximal 24 h after PB treatment in both the rough endoplasmic reticulum and the plasma membranes. This pattern of induction differed from that of some drug metabolizing enzymes. While total cytochrome P-450 content was enhanced mainly in endoplasmic reticulum until 48 h after PB treatment, UDP-glucuronosyltransferase activity was not greatly altered by PB under the same conditions. The comparison of two-dimensional electrophoretic polypeptide profiles of each subcellular membrane isolated from control and phenobarbital-treated rats revealed important variations induced by PB. In plasma membranes, the heaviest subunit (apparent Mr = 60 x 10(3)) of hepatic gamma-glutamyltransferase was provisionally identified as a collection of polypeptide which differ only by their pI. The concentration of these polypeptides was smaller in the endoplasmic reticulum where they were of lower apparent molecular mass. This suggests that the gamma-glutamyltransferase precursor is already processed at the level of the endoplasmic reticulum but it is still not completely mature or glycosylated. Five days of continuous PB treatment induced by appearance of new gamma-glutamyltransferase isoforms in plasma membranes. We demonstrate that after a single injection of PB, gamma-glutamyltransferase activity increases simultaneously with some drug-metabolizing enzymes, such as total cytochrome P-450 but not with others, such as UDP-glucuronosyltransferases.