Biogenesis of endogenous plasma membrane proteins in epithelial cells.
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Animals↗
Biomedical subjects
B Stieger
Publications and source records attributed to B Stieger.
Dissection of the asynchronous transport of intestinal microvillar hydrolases to the cell surface.
Novel subcellular fractionation procedures and pulse-chase techniques were used to study the intracellular transport of the microvillar membrane hydrolases sucrase-isomaltase and dipeptidylpeptidase IV in the differentiated colon adenocarcinoma cell line Caco-2. The overall rate of transport to the cell surface was two fold faster for dipeptidylpeptidase IV than for sucrase-isomaltase, while no significant differences were observed in transport rates from the site of complex glycosylation to the brush border. The delayed arrival of sucrase-isomaltase in the compartment where complex glycosylation occurs was only in part due to exit from the endoplasmic reticulum. A major slow-down could be ascribed to maturation in and transit of this enzyme through the Golgi apparatus. These results suggest that the observed asynchronism is due to more than one rate-limiting step along the rough endoplasmic reticulum to trans-Golgi pathway.
Biological Transport↗
A novel marker glycoprotein for the microvillus membrane of surface colonocytes of rat large intestine and its presence in small-intestinal crypt cells.
Murine mAbs were produced against purified microvillus membranes of rat colonocytes in order to establish a marker protein for this membrane. The majority of antibodies binding to the colonic microvillus membrane recognized a single protein with a mean apparent Mr of 120 kD in both proximal and distal colon samples. The antigen is membrane bound as probed by phase-partitioning studies using Triton X-114 and by the sodium carbonate extraction procedure and is extensively glycosylated as assessed by endoglycosidase F digestion. Localization studies in adult rats by light and electron microscopy revealed the microvillus membrane of surface colonocytes as the principal site of the immunoreaction. The antigen was not detectable in kidney or liver by immunoprecipitation but was present in the small intestine, where it was predominantly confined to the apical membrane of crypt cells and much less to the microvillus membrane of differentiated enterocytes. During fetal development, the antigen appears first in the colon at day 15 and 1-2 d later in the small intestine. In both segments, it initially covers the whole luminal surface but an adult-like localization pattern develops soon after birth. The antibodies were also used to develop a radiometric assay for the quantification of the antigen in subcellular fractions of colonocytes in order to assess the validity of a previously developed method for the purification of colonic brush-border membranes (Stieger, B., A. Marxer, and H.P. Hauri. 1986. J. Membr. Biol. 91:19-31.). The results suggest that we have identified a valuable marker glycoprotein for the colonic microvillus membrane, which in adult rats may also serve as a marker for early differentiation of enterocyte progenitor cells in small-intestinal crypt cells.
Age Factors↗
Biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation.
We have used pulse-chase metabolic radiolabeling with L-[35S]methionine in conjunction with subcellular fractionation and specific protein immunoprecipitation techniques to compare the posttranslational transport pathways taken by endogenous domain-specific integral proteins of the rat hepatocyte plasma membrane in vivo. Our results suggest that both apical (HA 4, dipeptidylpeptidase IV, and aminopeptidase N) and basolateral (CE 9 and the asialoglycoprotein receptor [ASGP-R]) proteins reach the hepatocyte plasma membrane with similar kinetics. The mature molecular mass form of each of these proteins reaches its maximum specific radioactivity in a purified hepatocyte plasma membrane fraction after only 45 min of chase. However, at this time, the mature radiolabeled apical proteins are not associated with vesicles derived from the apical domain of the hepatocyte plasma membrane, but instead are associated with vesicles which, by several criteria, appear to be basolateral plasma membrane. These vesicles: (a) fractionate like basolateral plasma membrane in sucrose density gradients and in free-flow electrophoresis; (b) can be separated from the bulk of the likely organellar contaminants, including membranes derived from the late Golgi cisternae, transtubular network, and endosomes; (c) contain the proven basolateral constituents CE 9 and the ASGP-R, as judged by vesicle immunoadsorption using fixed Staphylococcus aureus cells and anti-ASGP-R antibodies; and (d) are oriented with their ectoplasmic surfaces facing outward, based on the results of vesicle immunoadsorption experiments using antibodies specific for the ectoplasmic domain of the ASGP-R. Only at times of chase greater than 45 min do significant amounts of the mature radiolabeled apical proteins arrive at the apical domain, and they do so at different rates. Approximate half-times for arrival are in the range of 90-120 min for aminopeptidase N and dipeptidylpeptidase IV whereas only 15-20% of the mature radiolabeled HA 4 associated with the hepatocyte plasma membrane fraction has become apical even after 150 min of chase. Our results suggest a mechanism for hepatocyte plasma membrane biogenesis in vivo in which all integral plasma membrane proteins are shipped first to the basolateral domain, followed by the specific retrieval and transport of apical proteins to the apical domain at distinct rates.
Animals↗
Isolation of brush-border membranes from rat and rabbit colonocytes: is alkaline phosphatase a marker enzyme?
A method for the isolation of brush-border membranes of large intestinal epithelial cells was developed, which is based on the purification of intact brush-border caps by Percoll density-gradient centrifugation followed by separation of the vesiculated brush-border membranes on sucrose gradients. The procedure has two major advantages in comparison to known methods: its first step does not depend on the determination of marker enzymes and the method is applicable to rats as well as rabbits without major modifications. Due to the lack of an accepted marker for the colonic brush-border membrane the validity of the isolation procedure was tested by its application to the small intestine. Rat small intestinal brush-border membranes were enriched 21-fold when compared to the homogenate. The method was used to evaluate alkaline phosphatase as a marker enzyme for the colonic brush-border membrane. The results suggest that alkaline phosphatase is not exclusively localized in the brush-border membrane since this enzyme was also associated with membranes having different physical properties.
Alkaline Phosphatase↗
Sodium-proton exchange in colon brush-border membranes.
Apical membrane vesicles were prepared from proximal and distal segments of the large intestine of the rat by a method based on morphological criteria and were used to determine 22Na uptake. In both preparations an outwardly directed proton gradient stimulated 22Na uptake. In proximal colon a decrease in vesicular volume induced by an increased media osmolarity led to diminished 22Na uptake at 90 min; a significant (50-60%) portion of uptake represented binding. Initial uptake was linear for 10 s and extrapolated through zero, indicating minimal extravesicular binding. Initial uptake was a saturable function of medium Na concentration. In both preparations initial influx of 0.1 mM NaCl was inhibited by amiloride (0.1-1.0 mM), 15 mM NaCl, 15 mM LiCl, and 15 mM NH4Cl. From the characteristics of the initial 22Na influx we conclude that the apical membrane from colonocytes of proximal and distal segments contains a Na-H exchange with properties similar to those described in other epithelia.
Amiloride↗
Biosynthesis of rat liver cytochrome P-450 in mitochondria-associated rough endoplasmic reticulum and in rough microsomes in vivo.
The hypothesis of a preferential biosynthesis of a major phenobarbital inducible form of hepatic cytochrome P-450 (P-450b) in mitochondria-associated rough endoplasmic reticulum (RERmito) was tested by measuring incorporation rates of [35S]methionine and delta-amino[3H]levulinate into the hemoprotein in adult rats. RERmito, rough microsomes (RM representing RER not associated with mitochondria) and smooth microsomes (SM) were quantitatively isolated from the same homogenate by rate zonal centrifugation and their content of P-450b determined by rocket immunoelectrophoresis. P-450b was isolated by immunoprecipitation from detergent-solubilized membrane fractions. The time course and rate of incorporation of [35S] methionine into immunoprecipitable P-450b of RERmito and of RM were similar at all time points studied (2-15 min) both under conditions of maximal induction (4 injections of phenobarbital in 4 days) and after a single injection of phenobarbital. The incorporation of [35S]methionine into P-450b of SM was slower at early time points (2-8 min) but similar to RERmito and RM after 15 min. In contrast, at short labeling periods (less than 8 min) more delta-amino[3H]levulinate was incorporated into P-450b of RERmito than into P-450b of RM and SM. No significant accumulation of free apocytochrome P-450b was found in either membrane fraction. These data indicate a close coordination of the biosynthesis and assembly of apocytochrome P-450b and its prosthetic heme but do not support the hypothesis of a major functional role of MITO X RER complexes in the synthesis of microsomal cytochrome P-450b.
Animals↗
Na/H- and Cl/OH-exchange in rat jejunal and rat proximal tubular brush border membrane vesicles. Studies with acridine orange.
The quenching of the acridine orange fluorescence was used to monitor the formation and/or dissipation of a delta pH in brush border vesicles isolated from rat kidney cortex or rat jejunum. Similar findings were obtained with both brush border membrane vesicle preparations. Acridine orange fluorescence was quenched by a preset delta pH (intravesicular acid) or by the ionophore (valinomycin/CCCP) dependent development of a delta pH (intravesicular acid) under conditions of potassium efflux. Under sodium efflux conditions, an acidification of the intravesicular space occurred: a) due to indirect (electrical) coupling of sodium and proton fluxes; b) due to directly coupled sodium/proton exchange. The initial rate of the dissipation of a preset delta pH was accelerated by pulse injections of sodium in a saturable manner; lithium partially replaced sodium. The sodium dependent acceleration in the rate of dissipation of a preset delta pH was not altered by replacing gluconate with chloride. Amiloride was an inhibitor of directly coupled sodium/proton exchange. An inwardly directed chloride gradient did not induce intravesicular acidification. The initial rate of the dissipative proton fluxes (preset delta pH) was slightly accelerated by an outwardly directed chloride gradient. Sodium/proton exchange dependent acidification of the intravesicular space was not altered by replacing gluconate with chloride. These results clearly document the existence of sodium/proton exchange in both renal and intestinal brush border membrane vesicles. In contrast, Cl/OH exchange--under our experimental conditions--must have a much smaller rate than Na/H exchange.
Acridine Orange↗
Demonstration of sodium-dependent, electrogenic substrate transport in rat small intestinal brush border membrane vesicles by a cyanine dye.
The cyanine dye DiS-C2(5) was tested as an indicator for changes in membrane potential of subfractionated rat jejunal brush border membrane vesicles. The fluorescence of this dye increased with inside positive and decreased with inside negative potentials. The sensitivity to inside negative potentials was greater than to inside positive potentials. The addition of L-alanine, L-phenylalanine, L-methionine, D-galactose and D-glucose in the presence of sodium provoked a transient fluorescence increase indicating an inside positive membrane potential due to electrogenic, sodium-coupled transport. Besides the sodium-dependence, the dye reflected stereo-specificity and saturability of D-glucose transport. When D-glucose loaded vesicles were incubated in D-glucose-free medium, a decrease in fluorescence was observed indicating that D-glucose efflux is also electrogenic.
Amino Acids↗
Heterogeneity of brush-border-membrane vesicles from rat small intestine prepared by a precipitation method using Mg/EGTA.
Brush border membrane vesicles from rat small intestine were isolated by a Mg/EGTA precipitation method. Further fractionation either by free flow electrophoresis or by sucrose density gradient centrifugation leads to subfractions which differ with respect to enzyme enrichment factors, transport properties for D-glucose and protein pattern analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A relative enrichment of (Na+ + K+)-ATPase is found in one fraction, whereas in another fraction maltase, aminopeptidase M and alkaline phosphatase are relatively enriched. The fractions show different properties of D-glucose transport under tracer exchange conditions and a different inhibition of D-glucose transport by phlorizin and phloretin. These results indicate that the vesicles obtained from rat small intestine by this cation precipitation method are not homogeneous. The inhomogeneity cannot be due to a crosscontamination by membranes other than from the cell envelopment, as none of the fractions show a significant enrichment of succinate--cytochrome c oxidoreductase, KCN-resistant NADH oxidoreductase or glucosaminidase. The inhomogeneity might be due either to a crosscontamination by basal-lateral membranes or to membranes derived from epithelial cells not yet fully differentiated.
Animals↗
The application of a potential-sensitive cyanine dye to rat small intestinal brush border membrane vesicles.
The sensitivity of the fluorescent dye, 3,3'-diethylthiadicarbocyanine (DiS-C2(5], was too low for the detection of membrane potential changes in rat small intestinal membrane vesicles. Only after adding LaCl3 or after fractionation of the intestinal membranes by free-flow electrophoresis could the dye be used to monitor electrogenic Na+-dependent transport systems. It is concluded that the response of this potential-sensitive dye is influenced by the negative surface charge density of the vesicles.
Animals↗
Transport of L-lysine by rat renal brush border membrane vesicles.
L-3H-lysine uptake into brush border membrane vesicles was measured by a rapid filtration technique. A significant binding of L-lysine at the vesicle interior was observed. Extrapolating initial linear uptake to zero incubation time did not indicate binding of the amino acid to the external membrane surface. Sodium stimulated the L-lysine uptake specifically. Experiments in the presence of potassium/valinomycin induced diffusion potentials, and experiments with a potential sensitive fluorescent dye documented an electrogenic uptake mechanism for L-lysine only in the presence of sodium. Sodium independent uptake proceeds via an electroneutral pathway. Transstimulation experiments show carrier mediated uptake in the presence and absence of sodium. An outwardly directed proton-gradient stimulated L-lysine uptake in the presence and absence of sodium. Saturation of L-lysine uptake was observed in the presence and absence of sodium. In the absence of sodium, L-lysine uptake was inhibited by L-arginine, L-cystine, L-phenylalanine and L-methionine. The sodium dependent uptake was inhibited by L-arginine and L-cysteine; small inhibition by L-phenylalanine was observed. In the presence or absence of sodium, L-lysine uptake was inhibited neither by D-lysine nor by L-glutamic acid. These results document carrier mediated transport of L-lysine via (a) transport mechanism(s) not obligatory requiring sodium.
Amino Acids↗
Transport of L-cystine by rat renal brush border membrane vesicles.
Brush border membranes were isolated from rat renal cortex by a divalent cation precipitation method. L-35S-cystine uptake into the vesicles was measured by a rapid filtration method. Covalent incorporation of tracer into membrane proteins was observed after prolonged incubations. At short incubation periods (1 min) binding was small and allowed an analysis of transmembrane transport. To guarantee transport of L-cystine, the experiments were performed in the presence of the oxidant diamide. Sodium stimulated L-cystine uptake specifically. A potassium/valinomycin induced inside negative diffusion potential stimulated sodium dependent L-cystine transport. Thus, transport is potential sensitive in the presence of sodium. At low substrate and inhibitor concentrations, L-cystine transport was inhibited by L-lysine, L-ornithine and L-arginine but not by D-lysine in the presence and absence of sodium. At higher inhibitor concentration, the neutral amino acids L-phenylalanine and L-leucine also inhibited L-cystine uptake, but only the sodium dependent uptake. These inhibition experiments suggest that L-cystine is transported by the brush border membrane by a transport system for basic amino acids not necessarily requiring sodium. In addition, transport of L-cystine can also proceed via sodium dependent transport pathways for neutral amino acids. In the concentration range tested (up to 0.225 mmoles/l), no saturation of L-cystine transport was observed in the presence and absence of sodium.
Amino Acids↗
Transport of L-cysteine by rat renal brush border membrane vesicles.
Brush border membranes were isolated from rat renal cortex by a divalent cation precipitation method. L-35S-cysteine uptake into the vesicles was measured by a rapid filtration method. Only minimal binding of the amino acid to the vesicles was observed. Sodium stimulates L-cysteine uptake specifically. Anion replacement experiments, experiments in the presence of potassium/valinomycin-induced diffusion potential as well as experiments with a potential-sensitive fluorescent dye document an electrogenic sodium-dependent uptake mechanism for L-cysteine. Tracer replacement experiments as well as the fluorescence experiments indicate a preferential transport of L-cysteine. Transport of L-cysteine is inhibited by L-alanine and L-phenylalanine but not by L-glutamic acid and the L-basic amino acids. Initial, linear influx kinetics provide evidence for the existence of two transport sites. The results suggest (a) sodium-dependent mechanism(s) for L-cysteine shared by other neutral amino acids.
Alanine↗
Co- and counter-transport mechanisms in brush border membranes and basal-lateral membranes of intestine and kidney.
One way to obtain a deeper understanding of the complex function of the small intestinal and renal proximal tubular epithelium is to dissect it into single components and then, having defined the components under well-controlled conditions, try to describe the behaviour of the whole system on the basis of the properties of the single components. Brush border and basal-lateral membranes can be isolated by different methods, including free flow electrophoresis, differential and gradient centrifugation. Transport can be analysed in vesiculated membrane fractions by tracer techniques and spectrophotometric techniques. Different sodium-solute co-transport mechanisms were identified in the brush border membrane. Until now, studies with vesicles failed to document a sodium-chloride co-transport mechanism satisfactorily. On the other hand, a sodium/proton and a chloride/hydroxyl exchange mechanism were documented. These two exchange mechanisms could represent partial reactions of the postulated electroneutral sodium-chloride co-transport. In addition to different sodium-independent transport systems, the basal-lateral membrane contains an ATP-driven transport system for calcium as well as a sodium/calcium exchange mechanism. Studies with membrane vesicles isolated from animals which have been exposed to different dietary conditions or in which the parathyroid hormone or 1.25(OH)2VitD3 level has been altered show altered transport of calcium and inorganic phosphate. Thereby, it might be possible to identify the biochemical mechanisms involved in transport regulation.
Acid-Base Equilibrium↗
A high yield preparation for rat kidney brush border membranes. Different behaviour of lysosomal markers.
Rat kidney cortex slices were homogenized with a polytron in a isoosmotic medium containing 5 mmol/l EGTA. By two precipitations with MgCl2 (12 mmol/l) and differential centrifugation, brush border membranes were purified. The brush border marker enzymes alkaline phosphatase and aminopeptidase M were found to be enriched 17.0 +/- 5.3-fold and 16.7 +/- 3.7-fold, respectively. By this method, a high yield of brush border membranes was obtained (48.3 +/- 7.9% for alkaline phosphatase; 47.0 +/- 9.5% for aminopeptidase M). The acid phosphatase was enriched 5-fold, whereas other lysosomal enzymes (glucosaminidase, glucuronidase, cathepsin D) were enriched only 0.2-fold. Acid phosphatase activity could not be washed out, but could be separated from alkaline phosphatase and leucine aminopeptidase by means of free flow electrophoresis and sucrose density gradient centrifugation. Vesicles prepared by the presently described Mg/EGTA-method show better transport properties, compared to vesicles prepared by the calcium method of Evers et al. (Evers, C., Haase, W., Murer, H. and Kinne, R. (1978) Membrane Biochem. 1, 203-219), whereas by SDS-polyacrylamide gel electrophoresis, no differences in the protein patterns were observed.
Acid Phosphatase↗
Structure-effect relationships of amiodarone analogues on the inhibition of thyroxine deiodination.
OBJECTIVES: Amiodarone (AMI) has proven to be a potent anti-arrhythmic compound. Due to the structural similarity between AMI and thyroid hormone, it is possible that the drug could inhibit the activity of the 5'-thyroxine-deiodinase. METHODS: AMI analogues resulting from (1) dealkylation, (2) deiodination and (3) deamination were synthesised and used as inhibitors in an in vitro biotransformation reaction of thyroxine (T4) to 3,3',5'-triiodothyronine (T3). Using high-performance liquid chromatography and ultraviolet detection for quantifying T3, it was found that the 5'-T4 deiodinase type I was involved in the reaction. On separate occasions, AMI or an AMI analogue was added to the reaction as an inhibitor. RESULTS: All studied AMI analogues inhibited 5'-T4 deiodination competitively (Ki value range 25-360 microM). In the concentration range of 1-1000 microM, AMI and its N-desethylated, deiodinated analogues inhibited 5'-T4 deiodination very weakly. AMI analogues with a hydroxyl group at the 4-position were strong inhibitors. Moreover, diiodo-AMI analogues inhibited 5'-T4 deiodination more strongly than their corresponding monoiodo- or deiodinated derivatives. CONCLUSION: It is likely that the degraded products of AMI could be responsible for thyroid dysfunction toxicosis in AMI therapy.
Amiodarone↗