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J Biber

Publications and source records attributed to J Biber.

At least 199 records · Page 11Linked to original sources
PubMed1984

Intravesicular NAD has no effect on sodium-dependent phosphate transport in isolated renal brush border membrane vesicles.

The effects of intravesicular NAD on Na+-dependent 32Pi uptake were investigated in isolated rat kidney brush border membrane vesicles (BBMV). NAD was introduced into the vesicles by osmotic shock, and extravesicular NAD was removed by passing the vesicles through a anion exchange column. The effectiveness of the osmotic shock procedure and the hydrolysis of extra- and intravesicular NAD were controlled by enzymatic analysis and thin layer chromatography. ADP-ribosylation of the membrane proteins was analyzed in vesicles osmotically shocked in the presence of either [adenylate-32P]-NAD or [adenine-2,8-3H]-NAD by SDS-polyacrylamide gel electrophoresis. It was found that the Na+-dependent Pi uptake was inhibited when the BBMV were incubated with NAD at alkaline pH, which resulted in rapid NAD hydrolysis. When NAD was present in the intravesicular space only, the Na+-dependent Pi uptake was not inhibited. 32P from NAD was rapidly incorporated into a number of brush border membrane proteins, but no incorporation of 3H-adenine could be detected. The results provide evidence that NAD does not inhibit Pi transport by a direct interaction with the cytoplasmic side of the brush border membrane. No evidence of ADP-ribosylation of the brush border membrane protein(s) was found.

Animals↗
PubMed1983

Sodium-dependent transport of phosphate in LLC-PK1 cells.

Transport of phosphate has been studied in subconfluent monolayers of LLC-PK1 cells. It was found that this transport system shows similar characteristics to those observed in the kidney. Uptake of phosphate is mediated by a Na+-dependent, substrate-saturable process with an apparent Km value for phosphate of 96 +/- 15 mumol/l. Kinetic analysis of the effect of Na+ indicated that at (pH 7.4) two sodium ions are cotransported with one HOP4(2-) ion (Hill coefficient 1.5) with an apparent Km value for sodium of 56 mmol/l. Pi uptake is inhibited by metabolic inhibitors (ouabain and FCCP). In the pH range of 6.6 of 7.4 Pi uptake rate does not change significantly, indicating that both the monovalent and the divalent form of phosphate are accepted by the transport system. It is suggested that phosphate is transported by LLC-PK1 cells together with sodium (2 Na+:1 HPO4(2-) in an electroneutral manner down a favourable sodium gradient.

Animals↗
PubMed1983

Isolation of basolateral membranes from columnar cells of the proximal colon of the guinea pig.

A method for an analytical isolation of plasma membranes from columnar cells (colonocytes) of the proximal colon of the guinea pig is described. Isolation of the colonocytes was performed by a mild EDTA-chelation method. After homogenization, two subsequent sucrose gradient centrifugations (isokinetic and isopycnic) yielded a plasma-membrane fraction which was enriched 12-fold in (Na+ + K+)-ATPase activity and 8-fold in adenylate cyclase activity. It is suggested that the purified membrane fraction consists mainly of basolateral membranes of the colonocytes. Due to the lack of suitable marker enzymes, no evidence for enrichment of the brush-border membranes was obtained. Histochemical studies demonstrated that alkaline phosphatase is absent from the luminal membrane of the surface epithelial cells of the proximal colon of the guinea pig.

Animals↗
PubMed1983

The effect of cyclic nucleotides and cholera toxin on in vivo and in vitro phosphorylation of small intestinal brush border membranes.

The effect of cyclic nucleotides and cholera toxin on the phosphorylation of the brush border membrane proteins of the rat jejunum was studied. Phosphorylation was analyzed by autoradiography of brush border membrane proteins separated by SDS-polyacrylamide gel electrophoresis. Phosphorylation was performed either in vivo by perfusion of the jejunum with [32P]orthophosphate followed by an analysis of the isolated membranes or in vitro by phosphorylation of isolated brush border membranes by [gamma-32P]ATP in the presence of saponin. The addition of cholera toxin (10 micrograms/ml) or dibutyryl-cAMP (5 mmol/l) to the perfusate was unable to produce significant changes in the phosphoprotein pattern. On the other hand, cAMP (at 5 mumol/l) induced an increase of the phosphorylation of a 86 kDa protein when freshly isolated brush border membranes were phosphorylated by [gamma-32P]ATP. However, the same effect could also be induced by low concentrations of cGMP (0.1 mumol/l). It is concluded that brush border membranes from rat jejunum do not contain cAMP-dependent protein kinase activity and that cAMP-dependent protein phosphorylation of this membrane does probably not represent the final event of cholera toxin-induced secretion.

Animals↗
PubMed1983

Small-intestinal Na+/D-glucose cotransport. Inactivation of sugar transport and phlorizin binding by thiol-group and amino-group reagents.

It has previously been shown that mercurials acting from the cytoplasmic side or from within the hydrophobic part of the membrane inactivate the small intestinal Na+/D-glucose cotransporter by blocking essential SH-groups (Klip, A., Grinstein, S. and Semenza, G. (1979) Biochim. Biophys. Acta 558, 233-245). Another (set of) sulfhydryl(s) which are critical for phlorizin binding and sugar transport function and which may lie on the luminal side of the brush border membrane, can be blocked by DTNB and 4,4'-dithiopyridine but not by N-ethylmaleimide. In addition, modification of amino groups by fluorescamine, reductive methylation and (under certain conditions) DIDS also lead to inactivation of the carrier's binding and transport functions. No evidence was obtained that any of the above groups is directly involved in the binding of either Na+/D-glucose or phlorizin, since none of these compounds prevented inactivation of the cotransporter.

Animals↗
PubMed1983

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↗
PubMed1983

Phosphorylation of rat kidney proximal tubular brush border membranes. Role of c-AMP dependent protein phosphorylation in the regulation of phosphate transport.

A possible correlation between cyclic-AMP dependent protein phosphorylation and altered sodium dependent transport of inorganic phosphate was analyzed in isolated rat renal proximal tubular brush border membrane vesicles. In transiently opened vesicles (opened by an osmotic shock), the addition of gamma-32P-ATP leads to 32P-incorporation into several membrane proteins. The simultaneous addition of cyclic-AMP leads to increased phosphorylation of several proteins (e.g. apparent molecular weights: 40 kD, 46 kD, 55 kD). The addition of ATP, GTP and ITP to the osmotic shock medium leads to an (non-specific) inhibition of the sodium gradient dependent phosphate uptake. No further inhibition of the sodium dependent phosphate transport was observed when membrane vesicles were phosphorylated by ATP in the presence of cyclic-AMP. These data show a lack of correlation between cyclic-AMP dependent protein phosphorylation and altered sodium gradient dependent phosphate transport. Thus, there is no experimental support for the involvement of cyclic-AMP dependent protein phosphorylation as one of the final events in the regulation of phosphate transport across the rat renal proximal tubular brush border membrane.

Animals↗
PubMed1983

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↗
PubMed1983

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↗
PubMed1983

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↗
PubMed1982

Calcium uptake in isolated hepatic plasma-membrane vesicles.

A liver plasma-membrane fraction capable of Ca2+ uptake was isolated. The fraction exhibited high Na+, K+-ATPase, low glucose-6 phosphatase activity, and transported alanine in a Na+-dependent fashion. The uptake of Ca2+ was ATP-dependent; UTP, GTP, or CTP did not substitute for ATP. The presence of oxalate did not significantly alter the rate of uptake. The pH optimum of the reaction was basic (no uptake was visible at pH 6.8). These properties are at variance with those of the endoplasmic reticulum Ca2+ uptake system, which is oxalate-dependent, and has an acid pH optimum. The ATP-dependent Ca2+ uptake has a Km(Ca2+) of 1.4 X 10(-8) M and a Vmax of transport of 30 nmol X mg protein-1 X min-1. No conclusive results were obtained on the calmodulin-sensitivity of the process: addition of calmodulin to the vesicles did not stimulate uptake, and the anti-calmodulin drug trifluoperazine had no inhibitory effect. However, another anti-calmodulin drug (R24571) had a limited, but statistically significant, inhibitory action. A partial release of the accumulated Ca2+ from the vesicles could be induced by the addition of Na+, and incubation of the vesicles in a high Na+ medium (as compared to high K+ medium) resulted in lower (about 25%) calcium uptake. Partial release of the accumulated Ca2+ could be induced also by the addition of H+. The releasing effect of H+, taken together with the absence of Ca2+ uptake at acid pH, suggests the possibility of a H+/Ca2+ exchange.

Animals↗
PubMed1981

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↗
PubMed1980

Interaction of the sugar carrier of intestinal brush-border membranes with HgCl2.

HgCl2 was used as an inhibitor and potential label for the glucose carrier of intestinal brush-border membranes. Half-maximal inhibition of Na+-dependent D-glucose uptake was reached with micromolar concentrations of HgCl2 when the protein concentration was 1.2 mg/ml. Similar concentrations were found to inhibit the binding of [3H]phlorizin, a reversible competitive inhibitor of sugar transport. Inhibition was reversed by dithioerythritol but only marginally by EDTA. The data support the involvement of a sulfhydryl group in the inhibitory process. Deoxycholate-extracted membranes, which are enriched in specific phlorizin binding activity, were used for labeling studies using 203HgCl2. The polypeptides were separated by gel electrophoresis and analyzed by protein staining and autoradiography. Non-specific 203HgCl2 labeling was minimized by pre-treatment with sulfhydryl reagents which do not inhibit phlorizin binding. Several bands, which are lost from the autoradiographic pattern during a negative purification of the phlorizin binding sites, could be ruled out as essential components of the sugar carrier. The polypeptide profile was also analyzed following proteolysis, which abolished phlorizin binding. Those radioactive bands of which apparent Mr values were alterd by the treatment were considered as possible candidates. Finally, samples in which inhibition was reversed by thiols were also studied. The possible identity of the polypeptide(s) involved in glucose translocation is disussed in the light of these observations.

Animals↗