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G Burckhardt

Publications and source records attributed to G Burckhardt.

At least 91 records · Page 5Linked to original sources
PubMed1985

Peptide transport in rabbit intestinal brush-border membrane vesicles studied with a potential-sensitive dye.

Peptide transport in purified rabbit intestinal brush-border membrane vesicles has been studied using a potential-sensitive fluorescent dye, di-S-C3(5). Transport of dipeptides is accompanied by an increase in the fluorescence of the dye in the presence and absence of Na+, indicating electrogenic, Na+-independent peptide transport. Dipeptides containing D-amino acids also increase the fluorescence, showing that these peptides too possess significant affinity for the peptide transport system. beta-Alanylglycylglycine and prolylglycylglycine, very much like the dipeptides, increase the fluorescence even in the absence of Na+ which demonstrates the Na+-independent, electrogenic transport of tripeptides. However, concentrations needed for half-maximal fluorescence changes are higher for tripeptides than for dipeptides suggesting different affinities for the carriers. The studies, in addition, provide evidence for the existence of more than one carrier system for translocation of small peptides in rabbit intestinal brush-border membrane.

Animals↗
PubMed1985

Two binding modes of netropsin are involved in the complex formation with poly(dA-dT).poly(dA-dT) and other alternating DNA duplex polymers.

Using CD measurements we show that the interaction of netropsin to poly(dA-dT).poly(dA-dT) involves two binding modes at low ionic strength. The first and second binding modes are distinguished by a defined shift of the CD maximum and the presence of characteristic isodichroic points in the long wavelength range from 313 nm to 325 nm. The first binding mode is independent of ionic strength and is primarily determined by specific interaction to dA.dT base pairs. Employing a netropsin derivative and different salt conditions it is demonstrated that ionic contacts are essential for the second binding mode. Other alternating duplexes and natural DNA also exhibit more or less a second step in the interaction with netropsin observable at high ratio of ligand per nucleotide. The second binding mode is absent for poly(dA).poly(dT). The presence of a two-step binding mechanism is also demonstrated in the complex formation of poly(dA-dT).poly(dA-dT) with the distamycin analog consisting of pentamethylpyrrolecarboxamide. While the binding mode I of netropsin is identical with its localization in the minor groove, for binding mode II we consider two alternative interpretations.

Binding Sites↗
PubMed1985

ATP-dependent H+ pump in membrane vesicles from rat kidney cortex.

The presence of membrane vesicles containing an ATP-driven H+ pump was demonstrated in rat kidney cortex homogenate using the delta pH-sensitive dye acridine orange (AO). These vesicles were purified by differential and Percoll density gradient centrifugation. ATP-driven H+ uptake was about 20-fold enriched compared with the homogenate. Determination of marker enzyme activities indicated that these vesicles do not originate from brush border and basolateral membranes, lysosomes, endoplasmic reticulum, mitochondria, Golgi membranes, or red blood cells. The identity with brush border membranes was further excluded by the absence of Na+-H+ exchange. Renal cortical endocytotic vesicles that had taken up horseradish peroxidase or fluorescein isothiocyanate-labeled dextran (FITC-dextran) after injection of these substances into rats in vivo comigrated with the H+ pump activity on the Percoll gradient. Similar characteristics of the H+ pump demonstrated by the AO method and by fluorescence changes of in vivo trapped FITC-dextran proved the identity of H+ pump-containing vesicles with endocytotic vesicles. ATP-driven H+ uptake into endocytotic vesicles was stimulated by Cl- and weakly inhibited by oligomycin. N-ethylmaleimide, dicyclohexylcarbodiimide, and Dio-9 were stronger inhibitors. Histochemical studies revealed that horseradish peroxidase-filled endocytotic vesicles are localized in the apical region of proximal tubule cells. An H+ pump with similar characteristics, but much lower activity, was found in brush border membranes, basolateral membranes, and mitochondria isolated by standard techniques, suggesting a possible contamination of these preparations with endocytotic vesicles.

Acridine Orange↗
PubMed1984

Characteristics of glycylsarcosine transport in rabbit intestinal brush-border membrane vesicles.

Glycylsarcosine was found to be very resistant to hydrolysis by brush-border membrane vesicles from rabbit intestine. The dipeptide was transported intact into an osmotically responsive intravesicular space. The initial uptake rate of glycylsarcosine into these vesicles was greater in mannitol medium compared to that in the presence of an inward gradient of either Na+ or other monovalent cations. When vesicles preloaded with glycylsarcosine were incubated in a peptide-free medium, there was a rapid efflux of the dipeptide and the t1/2 for the process was less than 2 min. An inside-negative K+ diffusion potential generated by valinomycin stimulated glycylsarcosine uptake even in the absence of Na+. Experiments with the potential-sensitive dye DiS-C3 (5) showed that glycylsarcosine depolarized the brush-border membrane in the presence and absence of Na+. Imposition of an inward proton gradient stimulated the initial uptake rates of glycylsarcosine while the equilibrium uptake was not affected. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone decreased this proton gradient-induced stimulation. An inward proton gradient increased the Vmax of the transport system (10.8 +/- 0.8 nmol/min/mg of protein when [pH]o = [pH]i = 5.5; and 20.8 +/- 2.2 nmol/min/mg of protein when [pH]o = 5.5 and [pH]i = 7.8), without significantly affecting the apparent Kt (17.3 +/- 1.4 mM versus 19.5 +/- 2.0 mM). Glycyl-L-proline uptake was inhibited by glycylsarcosine and KI for the process was 20.8 +/- 3.0 mM. A relatively lower KI (2.8 +/- 1.2 mM) was obtained for the inhibition of glycylsarcosine uptake by glycyl-L-proline. The uptake of glycyl-L-proline and glycylsarcosine was strongly inhibited by L-carnosine, glycyl-L-leucine, and L-prolylglycine. With each inhibitory peptide, the KI values for the inhibition of glycyl-L-proline uptake and of glycylsarcosine uptake were comparable. Preloading the vesicles with unlabeled glycylsarcosine stimulated the uptake of labeled glycyl-L-proline. These data suggest that in rabbit intestinal brush-border membrane vesicles (i) glycylsarcosine and proton(s) are co-transported, (ii) this process results in a net transport of positive charge across the membrane and, (iii) a single transport system is involved in the translocation of glycyl-L-proline and glycylsarcosine.

Animals↗
PubMed1984

Effect of the preparation method on Na+-H+ exchange and ion permeabilities in rat renal brush-border membranes.

The delta pH-dependent quenching of Acridine orange was used to characterize Na+-H+ exchange and K+ and H+ conductances in brush-border membrane vesicles isolated by precipitation with either CaCl2 or MgCl2 from rat kidney cortex. A transmembrane pH difference of 2.5 units (inside acidic) was imposed and the initial rate of its dissipation was followed after injecting a puls of tetramethylammonium gluconate (control) or sodium or potassium gluconate. In membranes isolated by CaCl2, the Na+-H+ exchange was partially electroneutral (45% to 77% of the total exchange) and the rest was due to electrically coupled Na+ and H+ movements through conductive pathways in the membranes. In membranes prepared by MgCl2, the rate of total Na+-H+ exchange was about twice as high as that in membranes obtained by CaCl2 precipitation. However, total and electroneutral exchanges were equal indicating negligible electrically coupled Na+ and H+ movements in these membranes. K0.5 for Na+ in all preparations was in the same range, being in average 30 mM. Amiloride was a competitive inhibitor of Na+-H+ exchange in membranes obtained with both preparations; Ki values ranged between 0.1 and 0.58 mM. The rates of delta pH-dissipation with K+ gradients (+/- valinomycin) were by 50% to 150% higher in membranes prepared with CaCl2 than in membranes isolated with MgCl2 indicating much higher H+ and K+ conductances in membranes obtained with CaCl2. Therefore, the rate of Na+-H+ exchange as well as the conductances for various ions in the isolated brush-border membranes depend on membrane preparation.

Amiloride↗
PubMed1984

Sodium-dependent dicarboxylate transport in rat renal basolateral membrane vesicles.

Dicarboxylate transport in basolateral membrane vesicles prepared from rat kidney cortex was studied using 3H-methylsuccinate as a substrate. A sodium gradient (out greater than in) simulated methylsuccinate uptake and led to a transient overshoot. Lithium inhibited methylsuccinate uptake in the presence of sodium. The dependence of methylsuccinate uptake on sodium concentration indicated the interaction of more than one sodium ion with the transporter. Half-maximal stimulation was observed at 24 mmol/l sodium. Sodium-driven methylsuccinate uptake was electrogenic carrying a net positive charge. The basolateral dicarboxylate transport system exhibited an optimum at pH 7.0-7.5. In contrast, the sodium-dependent dicarboxylate transport system of brush border membranes depended much less on pH and had no optimum in the tested range. Cis-inhibition studies showed a preference of the system for dicarboxylates in the trans-configuration (fumarate) over cis-dicarboxylates (maleate). Citrate was accepted but oxalate and L-glutamate were not. DIDS exhibited a small inhibition. Among the monocarboxylates, gluconate and pyruvate inhibited methylsuccinate uptake whereas probenecid and p-aminohippurate (1 mmol/l) were without effect. The data indicate the presence of a sodium-dependent transport system in the basolateral membrane which accepts tricarboxylic acid cycle intermediates. This system is most likely not identical to the transport system responsible for organic anion secretion.

Animals↗
PubMed1984

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↗
PubMed1984

Properties of an anion exchanger in rat renal basolateral membrane vesicles.

Sulfate uptake in basolateral membrane vesicles from rat kidney cortexes was studied to test for the presence of an anion exchanger in these membranes. A pH difference (pHout = 6.4, pHin = 8.4) and a sodium gradient (out greater than in, pHout = pHin = 7.4) provided the driving force for intravesicular sulfate accumulation above the equilibrium content ("overshoot"). Sulfate uptake in vesicles preloaded with 25 mM of sulfate, thiosulfate, phosphate, chloride, or bicarbonate, or 10 mM of formate, acetate, L-lactate, pyruvate, p-aminohippurate, or oxalate was stimulated with respect to uptake in unpreloaded vesicles. Probenecid inhibited these trans stimulations. When the uptake of 0.1 mM labeled sulfate was determined in the presence of 5 mM of various anions in the incubation medium, cis inhibitions were observed when sulfate was driven by a proton gradient (out greater than in), a sodium gradient (out greater than in), or a p-aminohippurate gradient (in greater than out). All anions that trans stimulated sulfate uptake also showed a cis inhibition. Only small cis inhibitions were seen with maleate and succinate. No inhibition by any of the anions was found when sulfate uptake was studied in the absence of driving cation or anion gradients. Our data indicate a common exchanger for inorganic and organic anions that can additionally be driven by a sodium gradient and a pH difference. This exchanger may be involved in reabsorption and secretion of anions in the proximal tubule of the rat kidney.

Animals↗
PubMed1983

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.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1983

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↗
PubMed1983

Apparent inhibition of Na+/H+ exchange by amiloride and harmaline in acridine orange studies.

Amiloride and harmaline were tested as inhibitors of proton movements in brush-border membrane vesicles from rat kidney cortex. Transmembrane pH differences were visualized using acridine orange. Fluorescence quenching due to Na+ gradient-driven intravesicular acidification was inhibited by amiloride and harmaline. However, a similar inhibition was observed for the Na+ gradient-driven electrogenic proton movements in the presence of gramicidin. Moreover, amiloride and harmaline decreased the fluorescence signal of electrogenic proton movements driven by a K+ gradient in the presence of valinomycin. The degree of inhibition of intravesicular acidification by both drugs was concentration dependent. Half-maximal inhibition (I50) of Na+/H+ exchange and K+ gradient-driven proton movements occurred at 0.21 and 0.6 amiloride, respectively. The I50 for harmaline was 0.21 mM in both cases. Amiloride also decreased the initial quenching of acridine orange fluorescence due to a preset pH gradient without affecting the rate of dissipation of the pH gradient. This effect was independent of the buffer capacity. In contrast, harmaline seemed to dissipate pH gradient in the same way as a permeant buffer. Amiloride and harmaline led to a concentration-dependent fluorescence decrease even in aqueous solution. The results suggest an interaction of amiloride and harmaline with acridine orange which overlaps a possible specific inhibition of Na+/H+ exchange by these drugs.

Acridine Orange↗
PubMed1983

Inhibition of bile salt transport in brush-border membrane vesicles from rat small intestine by photoaffinity labeling.

The uptake of photolabile bile acid derivative, (7,7-azo-3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24 oyl)-2-aminoethanesulfonate (7,7-azo-TC), was investigated in rat ileal brush-border membrane vesicles. The uptake of 7,7-azo-TC showed a transient vesicle to medium ratio greater than one in the presence of a Na+ gradient. The Na+-dependent uptake of 7,7-azo-TC was inhibited by taurocholate and vice versa. 130 microM 7,7-azo-TC inhibited Na+-dependent taurocholate uptake by 50%. The degree of inhibitory power on taurocholate uptake was taurodeoxycholate greater than cholate greater than 7,7-azo-TC. Photoaffinity labeling of membrane vesicles with 7,7-azo-TC irreversibly inhibited Na+-dependent taurocholate and D-glucose transport but not Na+-dependent L-alanine transport. Kinetic and photoaffinity labeling experiments indicate that this representative photoaffinity probe interacts with the ileal Na+, bile salt cotransporter and may be used to identify polypeptide components of this transport system.

Affinity Labels↗
PubMed1983

Bile salt-binding polypeptides in brush-border membrane vesicles from rat small intestine revealed by photoaffinity labeling.

Photoaffinity labeling of small intestinal brush-border membrane vesicles with photolabile bile salt derivatives was performed to identify bile salt-binding polypeptides in these membranes. The derivatives used in this study were the sodium salts of 7,7-azo-3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oic acid, 3 beta-azido-7 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oic acid, their respective taurine conjugates, and (11 xi-azido-12-oxo-3 alpha, 7 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-aminoethanesulfonic acid. With ileal brush-border membrane vesicles, photoaffinity labeling resulted in the identification of 5 polypeptides with apparent molecular weights of 125,000, 99,000, 83,000, 67,000, and 43,000. The extent of labeling depended on the photolabile derivative employed. In jejunal brush-border membrane vesicles, polypeptides with apparent molecular weights of 125,000, 94,000, 83,000, 67,000, and 43,000 were labeled. The results indicate that the binding polypeptides involved in bile salt transport in ileal brush-border membrane vesicles are 1) similar with one exception to those concerned with bile salt transport in jejunal brush-border membranes, and 2) markedly different from those previously shown to be concerned with bile salt transport in plasma membranes of hepatocytes.

Affinity Labels↗
PubMed1983

Studies on histone H1 condensing properties in complexes with DNA and polydeoxyribonucleotides using netropsin as a probe.

The binding of histone H1 with DNA and synthetic DNA duplex polymers with respect to its property to induce higher ordered structures has been studied using the DNA binding antibiotic netropsin as a probe. It was shown that the formation of distinct steps of different condensed structures (double-fibers, cable- and stem-like forms) is influenced by the ionic strength. CD titration data of DNA-H1 complexes with netropsin at 20 mM NaCl indicated no change in the binding to strong affinity sites (dA X dT clusters) as compared to free DNA's, while weak netropsin binding regions are strongly affected by competition interaction with H1. At low histone concentration the presence of netropsin favours the formation of double fibers. CD and electron microscopic findings indicated that at 20 mM NaCl the occurrence of condensed structures of DNA histone H1 complexes is not dependent on the base content. The major groove interaction of H1 most probably plays the major role in the formation of higher ordered structures. However, the minor groove binding might be involved as a secondary event. A hierarchy of relevant morphological structures observed for DNA-H1 complexes is presented.

Chemical Phenomena↗
PubMed1982

Lack of intestinal transport of [3H]-demethylphalloin: comparative studies with phallotoxins and bile acids on isolated small intestinal cells and ileal brush border membrane vesicles.

Several earlier studies suggested that the uptake of phallotoxins by liver cells is a carrier mediated process using a transport system normally handling bile acids (see Frimmer 1982). In this study we have shown whether ileal cells, well known to transport bile acids too, are able to take up phallotoxins. Isolated epithelial cells prepared from guinea pig ileum accumulated [14C]-cholate, whereas [3H]-demethylphalloin ([3H]-DMP) was not taken up. The same observation was made with isolated jejunal cells but the uptake of [14C]-cholate was much slower. [3H]-DMP, however, was partly bound to intestinal cells. This process was not inhibited by cholate, iodipamide, oligomycin and carbonylcyano-chlorophenylhydrazone (CCCP), compounds known to decrease the uptake of phallotoxins into liver cells. Substituting Na+ for choline+ and also Cl- for SCN- did not influence the binding of [3H]-DMP. Frozen intestinal cells from the guinea pig bound two time more [3H]-DMP after thawing compared with intact cells. Supplementary uptake experiments on isolated brush border membrane vesicles from rat ileum revealed that phalloidin does not inhibit taurocholate uptake and that taurocholate does not interfere with [3H]-DMP binding. The results suggest that [3H]-demethylphalloin is not recognized by the bile acid carrier of the guinea pig and the rat ileum. It is concluded that the transport system for bile acids present in ileal cell is different from that of liver cells.

Alkaloids↗
PubMed1981

Binding of netropsin to DNA in complexes with polypeptides containing repetitive lysine sequences.

The interaction of the antibiotic netropsin with calf thymus DNA, T4 DNA and poly(dA-dT) . poly(dA-dT) in complexes with sequential polypeptides containing repetitive lysine sequences and histone H1 was investigated using circular dichroism spectroscopy and equilibrium dialysis. Both soluble DNA-polypeptide complexes and insoluble complexes showed binding of netropsin. The possibility of displacement of polypeptides from DNA binding sites by competition with netropsin molecules was eliminated by experiments using 14C-labelled polypeptides. From the analysis of CD titration behavior as well as from the results of equilibrium dialysis studies it follows that netropsin does not compete with polypeptides for DNA binding sites, which suggests that these two ligands occupy different sites. Various explanations for minor differences in the CD behavior of the bound netropsin in the saturation region are also discussed.

Amino Acid Sequence↗
PubMed1981

The influence of pH on phosphate transport into rat renal brush border membrane vesicles.

Sodium-dependent transport of inorganic phosphate into brush border membrane vesicles is strongly influenced by altering pH of the incubation medium (pHo). At constant total phosphate concentration an increase in pHo leads to an increase in the uptake of inorganic phosphate. Uptake of inorganic phosphate, however, is not affected by the intravesicular pH (pHi) or by transmembrane pH differences (pHo--pHi). If initial phosphate uptake is studied as a function of total phosphate concentration in the medium the half saturation concentration increases when pHo is raised from 6.3--6.9 but remains unaltered between pHo 6.9 and 7.8 Vmax increases about 3-fold between pHo 6.3 and 6.9 and by a factor of about 1.6 between pHo 6.9 and 7.4. The pHo- dependence of phosphate uptake is diminished by increasing sodium concentrations. Altering transmembrane electrical potential difference by potassium + valinomycin-induced diffusion potentials or by anion replacement fails to demonstrate electrogenicity of sodium-phosphate cotransport. Experiments using a potential-sensitive fluorescent dye, however, indicate a vesicle inside positive electrical potential difference when inorganic phosphate is added. The phosphate-induced alterations in the electrical potential difference are sodium-dependent and more pronounced at low pHo values. Together with earlier observations these results suggest that translocation of inorganic phosphate across the proximal tubular brush border membrane is mediated by cotransport of 2 sodium ions with one either monovalent or divalent phosphate molecule according to its availability in the tubular fluid. The pH sensitivity of this transport system is rather due to alterations in the transport system itself than to pH- dependent alterations in the ratio of monovalent to divalent phosphate.

Animals↗