A rapid method for the reconstitution of Na+-dependent neutral amino acid transport from bovine renal brush-border membranes

Lynch, Angela M.; McGivan, J D

doi:10.1042/bj2440503

Cited by 22 publications

(26 citation statements)

References 33 publications

(25 reference statements)

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“…This extract usually contained 50-60% of the original protein. Crude phospholipids were prepared from fresh egg yolk as described and were stored under N2 at -20°C (12,13). To prepare liposomes, 300 mg of phospholipids were dried under N2 and resuspended in 3 ml of 20 mM NaHepes, pH 7.4/100 mM KCl/1 mM EDTA.…”

Section: Methodsmentioning

confidence: 99%

Functional reconstitution of the lysosomal sialic acid carrier into proteoliposomes.

Mancini¹,

Beerens²,

Galjaard³

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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The lysosomal carrier for the acidic monosaccharides sialic acid and glucuronic acid was solubilized from rat liver lysosomal membranes and reconstituted into phospholipid vesicles. Membrane proteins were extracted from lysosomal membranes with Triton X-100. Upon removal of the detergent by absorption on Amberlite XAD-2 beads, the solubilized proteins were incorporated in egg yolk phospholipids. The reconstituted proteoliposomes show proton-driven carriermediated uptake of acidic monosaccharides. The reconstituted carrier was compared in several characteristics with the transporter as present in the native lysosomal membrane. Transporter substrate affinity (Kt for glucuronic acid = 0.4 mM) and specificity for acidic monosaccharides are completely retained.The proteoliposomes also demonstrate trans-stimulation properties with both substrates sialic acid and glucuronic acid. The transporter is inhibited, both in its native and in the reconstituted state, by the sulfhydryl-modifying agents p-chloromercuribenzoic acid, N-ethylmaleimide, and phenyl isothiocyanate. In native membrane vesicles, arginine and histidine modifiers phenylglyoxal and diethyl pyrocarbonate inactivated transport in a substrate-protectable manner. In reconstituted proteoliposomes, similar inhibition was observed. However, protection by substrates was achieved only after treatment with phenylglyoxal. These data suggest that arginine or histidine residues or both are present at or near the substrate binding site of the carrier. Possibly, other essential histidines become exposed in the reconstituted state. The successful functional reconstitution of the lysosomal sialic acid carrier represents an important step towards its purification and its detailed molecular characterization.

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Section: Methodsmentioning

confidence: 99%

Functional reconstitution of the lysosomal sialic acid carrier into proteoliposomes.

Mancini¹,

Beerens²,

Galjaard³

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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“…Egg yolk phospholipids were isolated as described previously [18,191. MEGA-10 was synthesized according to Hildreth [20].…”

Section: Methodsmentioning

confidence: 99%

“…Artificial liposomes were prepared essentially by the method of Lynch and McGivan [18]. Briefly, 5% (mol/mol) cholesterol was added to egg yolk phospholipid dissolved in chloroform and the chloroform evaporated under N2.…”

Section: Reconstitutionmentioning

confidence: 99%

“…Undispersed lipid material was removed by centrifuging at 100000 g and 4°C for 30 min. Phospholipid concentration in the suspension was assayed as described previously [18].…”

Section: Reconstitutionmentioning

confidence: 99%

“…Fractionation and reconstitution of Na+-dependent phosphate transport from bovine BBMV Na+-dependent phosphate transport was reconstituted from solubilised bovine BBMV in artificial phospholipid membranes by a modification of a method previously used for reconstitution of amino acid transport [18]. It was found that the incorporation of 5% cholesterol in the lipid was essential to obtain transport activity.…”

Section: Phosphate Transport In Isolated Bovine Renal Brush Border Mementioning

confidence: 99%

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Adaptive regulation of Na⁺‐dependent phosphate transport in the bovine renal epithelial cell line NBL‐1

Helps

McGivan

1991

European Journal of Biochemistry

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1. Na+-dependent phosphate transport activity from bovine brush border membrane vesicles (BBMV) was reconstituted into artificial phospholipid membranes. It was shown, using lectin affinity chromatography, that transport activity was mediated by a glycoprotein. 2. The bovine kidney epithelial cell line NBL-1 expresses a Na+-dependent phosphate transporter which shows adaptive regulation in response to various media phosphate concentrations. Cells incubated in low-phosphate medium and medium containing 10 mM phosphate have V,,, values of 6.34 0.18 and 2.95 k 0.1 nmol . mg-' . 3 min-' and K , values of 17.1 3.0 and 36.3 k 7.14 pM for phosphate transport, respectively. Thus there is a significant (P < 0.001) decrease in the K,,, and increase in the V,,, of transport when cells are transferred from 10 mM phosphate medium to low-phosphate medium. 3. Adaptation of phosphate transport in NBL-1 cells to low-phosphate medium was abolished by tunicamycin, an inhibitor of protein glycosylation, indicating that the transporter in these cells, like that in bovine BBMV, is a glycoprotein. 4. Cells were adapted to low-phosphate medium in the presence of [3H]mannose. Under these conditions a single membrane protein of molecular mass 55 kDa was labelled in the absence but not the presence of tunicamycin. Little labelling occurred if the cells were maintained in high-phosphate medium. 5. On the basis of these observations, it is proposed that the phosphate transporter of NBL-1 cells is a 55-kDa glycoprotein.Phosphate is accumulated across the brush border membrane of renal proximal tubule cells via a Na'/ phosphate cotransport system. Na+-dependent phosphate transport has been extensively studied in isolated brush border membrane vesicles from rabbit and rat kidney [l -31. The symport of phosphate and Na+ is electroneutral with a stoichiometry of 2Na+/phosphate at physiological pH values.More recently, established renal epithelial cell lines have been employed as model systems to study the adaptation and regulation of phosphate transport. In particular in OK cells (derived from opossum kidney) phosphate transport is inhibited by exposure of cells to parathyroid hormone and stimulated by insulin-like growth factor I and insulin; mechanisms underlying these hormonal effects have been investigated [4-71. In both OK cells and in the LLC-PK1 cell line derived from pig kidney, exposure of cells to a medium containing low phosphate concentrations cause an adaptive increase in phosphate transport characterised by an increased V,,, [4,8, 91.The molecular identity of the phosphate transporter has been proposed by several groups. Wuarin et al. [lo] have identified several possible transport proteins in OK cells on the basis of substrate protection of phosphate transport from inhibition by the relatively specific inhibitor N-acetyl-

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pH-Dependent Intestinal Transport of Monocarboxylic Acids: Carrier-Mediated and H+-Cotransport Mechanism Versus pH-Partition Hypothesis

Tsuji

Simanjuntak²,

Tamai³

1990

Journal of Pharmaceutical Sciences

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A rapid method for the reconstitution of Na+-dependent neutral amino acid transport from bovine renal brush-border membranes

Cited by 22 publications

References 33 publications

Functional reconstitution of the lysosomal sialic acid carrier into proteoliposomes.

Functional reconstitution of the lysosomal sialic acid carrier into proteoliposomes.

Adaptive regulation of Na⁺‐dependent phosphate transport in the bovine renal epithelial cell line NBL‐1

pH-Dependent Intestinal Transport of Monocarboxylic Acids: Carrier-Mediated and H+-Cotransport Mechanism Versus pH-Partition Hypothesis

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A rapid method for the reconstitution of Na+-dependent neutral amino acid transport from bovine renal brush-border membranes

Cited by 22 publications

References 33 publications

Functional reconstitution of the lysosomal sialic acid carrier into proteoliposomes.

Functional reconstitution of the lysosomal sialic acid carrier into proteoliposomes.

Adaptive regulation of Na+‐dependent phosphate transport in the bovine renal epithelial cell line NBL‐1

pH-Dependent Intestinal Transport of Monocarboxylic Acids: Carrier-Mediated and H+-Cotransport Mechanism Versus pH-Partition Hypothesis

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Adaptive regulation of Na⁺‐dependent phosphate transport in the bovine renal epithelial cell line NBL‐1