Organic and Inorganic Solute Transport in Renal and Intestinal Membrane Vesicles Preserved in Liquid Nitrogen

Stevens, Bruce R.; Wright, Stephen H.; Hirayama, Bruce; Gunther, R D; Ross, Helen J.; Harms, Verna; Nord, Edward P.; Kippen, Ian; Wright, Ernest M.

doi:10.3109/09687688209065436

Cited by 63 publications

(25 citation statements)

References 10 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Glucose transport activity was measured during a 10-sec influx period with and without NaCi, using a rapid-mix/rapid-filtration technique (7). The characteristic transport properties of brush border membrane vesicles (7,8,12) were preserved by using the cryoprotectant buffer at temperatures ranging from -80 to -196TC, as also verified by Beliveau et al (9,10). The Na'-dependent cotransporter activity (J) at each radiation dose was the difference in total transport measured in NaCl (JtotaI) minus transport measured in choline chloride buffer (JNa-indePendent).…”

Section: Methodsmentioning

confidence: 99%

Intestinal brush border membrane Na+/glucose cotransporter functions in situ as a homotetramer.

Stevens

Fernandez

Hirayama

et al. 1990

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

View full text Add to dashboard Cite

The functional unit molecular size of the intestinal brush border membrane-bound Na+/glucose cotransporter was determined by radiation inactivation. Purified brush border membrane vesicles preserved in cryoprotectant buffer were irradiated (-1350C) with high-energy electrons from a 13-MeV (1 eV = 1.602 x 10-'9 J) linear accelerator at doses from 0 to 70 Mrad (1 rad = 0.01 Gy). After each dose, the cotransporter was investigated with respect to (i) Na'-dependent transport activity and (ii) immunologic blot analysis with antibodies against the cloned rabbit intestinal cotransporter. Increasing radiation decreased the maximal Na'-dependent cotransporter activity JfaX without affecting apparent Km. The size of the transporting functional unit was 290 ± 5 kDa. Immunologic blot analysis of brush border membranes gave a single band of Mr 70,000, which decreased in intensity with increased radiation dose and gave a target size of 66 ± 11 kDa. We conclude that activity of the intestinal Na+/glucose cotransporter in situ in the brush border membrane requires the simultaneous presence of four intact, independent, identical subunits arranged as a homotetramer.The intestinal brush border membrane Na+/glucose cotransporter is among the most thoroughly studied eukaryotic systems demonstrating secondary active (ion-coupled) transport. Biochemical and immunological experiments have identified the cotransporter as a polypeptide of Mr 70,000-75,000 on SDS/PAGE reducing gels (1-3), and the cloned cotransporter has a predicted molecular mass of 73 kDa (5, 6). However, information regarding structure-function relationships of the cotransporter in situ in the native membrane is lacking. In this study we used high-energy electron radiation inactivation to investigate the membrane-bound in situ size of the cotransporter. Our results indicate that the cotransporter functions in the membrane as a 290-kDa homotetramer comprised of four independent 73-kDa subunits. METHODS AND MATERIALSBrush border membrane vesicles were prepared from male New Zealand White rabbit small intestines by a 10 mM MgCl2 precipitation procedure (7,8). The membrane vesicles were enriched =15-fold in the brush border membrane marker enzymes alkaline phosphatase, leucine aminopeptidase, sucrase, and 'y-glutamyltranspeptidase. Vesicles (20 ,ug of protein per ,l) were equilibrated in a cryoprotectant buffer (9, 10) that contained 14% (vol/vol) glycerol, 1.4% (wt/vol) D-sorbitol, 150 mM KCI, and 5 mM Hepes-Tris (pH 7.5). Beliveau et al. (9, 10) have established that this buffer preserves glucose and phosphate transport, and Na+/H+ antiport in kidney brush border membrane vesicles. Aliquots of the vesicle suspension were frozen in 2-ml glass ampules (type 12012; Kimble, Toledo, OH). The sealed ampules were then kept at -80'C and transported on dry ice. The membranes were irradiated at -1350C with a beam of 13 MeV (1 eV = 1.602 x 10-1' J) electrons produced by a linear accelerator, as described elsewhere (11). After irradiation the ampules were held at -800C until...

show abstract

Section: Methodsmentioning

confidence: 99%

Intestinal brush border membrane Na+/glucose cotransporter functions in situ as a homotetramer.

Stevens

Fernandez

Hirayama

et al. 1990

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

View full text Add to dashboard Cite

show abstract

“…Membranes prepared by these methods showed an increase in alkaline phosphatase specific activity of 10 (renal)-and 20 (intestinal)-fold over the initial homogenate. Vesicles were suspended in 300 mM-mannitol buffered to pH 7-5 with 10 mM-Tris-HEPES at a protein concentration of 10-15 mg/ml., and were preserved in liquid N2 until use (Stevens et al 1982b). The electrical potential difference (p.d.)…”

Section: Methodsmentioning

confidence: 99%

Kinetics of sodium‐dependent solute transport by rabbit renal and jejunal brush‐border vesicles using a fluorescent dye.

Schell¹,

Stevens²,

Wright³

1983

The Journal of Physiology

View full text Add to dashboard Cite

SUMMARY1. The kinetics of Na-coupled solute transport by renal and jejunal brush-border vesicles in the rabbit were examined using the potential-sensitive fluorescent dye diS-C3-(5).2. All organic solutes known to be transported across these membranes by Na-coupled mechanisms increase the fluorescence of the dye in the presence of Na, but not K. An increase in fluorescence (AF) corresponds to a depolarization of the electrical potential difference (5-60 mV) across the brush-border membrane in the intact cell. AF was independent of the valency of the transported solute.3. The fluorescence response was saturable, and for twelve solutes the K,, i.e. the concentration of the substrate generating 50 % of the maximal response, agreed quite closely with the Kt values reported from tracer studies.4. For six solutes increasing the Na concentration decreased K,, and this agrees with the effect of Na on the kinetics of succinate transport in renal vesicles.5. We conclude that D-glucose, neutral amino acids and imino acids are co-transported with Na across both renal and jejunal brush-border membranes, and that carboxylic acids, fl-amino acids, and dibasic amino acids are co-transported with Na across the renal, but not jejunal, membranes.

show abstract

“…Also, vesicle shrinkage is nonideal when vesicles of low intravesicular osmolality are exposed to excessive osmotic gradients as in the case of Fig. 1A, and this can lead to a nonzero extrapolation artifact; this phenomenon has been observed in fresh membrane vesicle preparations (7). The vesicles retained their initial influx rates during at least 180 min following reconstitution (Fig.…”

Section: Fig 4 the Effects Of Initial Gradients And Namentioning

confidence: 92%

“…Storage of membranes has been previously attempted by freezing in solutions of dithiothreitol (17), ethylene glycol (18), or dimethyl sulfoxide (19) or in liquid N 2 (7). However, lyophilization of membranes containing amino acid transport polypeptides appears to be the method of choice on several grounds.…”

Section: Fig 4 the Effects Of Initial Gradients And Namentioning

confidence: 99%

See 1 more Smart Citation

Sodium-Dependent Amino Acid Transport Is Preserved in Lyophilized Reconstituted Apical Membranes from Intestinal Epithelium

Stevens

Preston

1998

Analytical Biochemistry

View full text Add to dashboard Cite

Organic and Inorganic Solute Transport in Renal and Intestinal Membrane Vesicles Preserved in Liquid Nitrogen

Cited by 63 publications

References 10 publications

Intestinal brush border membrane Na+/glucose cotransporter functions in situ as a homotetramer.

Intestinal brush border membrane Na+/glucose cotransporter functions in situ as a homotetramer.

Kinetics of sodium‐dependent solute transport by rabbit renal and jejunal brush‐border vesicles using a fluorescent dye.

Sodium-Dependent Amino Acid Transport Is Preserved in Lyophilized Reconstituted Apical Membranes from Intestinal Epithelium

Contact Info

Product

Resources

About