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Isolation and characterization of the Xenopus oocyte plasma membrane: a new method for studying activity of water and solute transporters

Isolation and characterization of the Xenopus oocyte plasma membrane: a new method for studying... The intact Xenopus laevis oocyte is a useful model system for studying expressed water and solute transporters but suffers from a number of limitations, most notably large unstirred layers and other intracellular diffusion barriers. To overcome these, we have developed a method for isolating plasma membrane vesicles from oocytes. This approach facilitates more precise control of the intravesicular environment and virtually eliminates the problem of unstirred layers in kinetic experiments. The isolation procedure results in 50.6-fold enrichment of the plasma membrane marker alkaline phosphodiesterase compared with the homogenate. Markers of late endosomes/lysosomes and mitochondria were not enriched, and the endoplasmic reticulum was enriched only modestly. Permeabilities of native plasma membrane to water and urea were 8.1 x 10 –4 and 5.6 x 10 –7 cm/s, respectively, values that are sufficiently low to classify them as barrier membranes. Phospholipid analysis by mass spectrometry showed the membrane, not including cholesterol, to be rich in phosphatidylcholine (35.8 mole percent), sphingomyelin (25.8 mole percent), and phosphatidylinositol (6.8 mole percent). Cholesterol concentration was 20.7 mole percent. Membrane vesicles isolated from oocytes expressing aquaporin-1 exhibited fourfold higher water permeability in stopped-flow experiments. Oocytes expressing mouse urea transporter A3 (UT-A3) exhibited 7.5-fold faster phloretin-inhibitable urea transport compared with water-injected controls. There was no difference in water permeability between these membrane vesicles, suggesting that UT-A3 is not a water carrier. In conclusion, we describe an improved method for the isolation of the oocyte plasma membrane that will allow the study of water and solute transport kinetics as well as substrate selectivity in heterologously expressed proteins. aquaporin; urea; permeability; barrier; lipid composition Address for reprint requests and other correspondence: W. G. Hill, Laboratory of Epithelial Cell Biology, Renal-Electrolyte Div., Univ. of Pittsburgh School of Medicine, A1222 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261 (e-mail: whill@pitt.edu ) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png AJP - Renal Physiology The American Physiological Society

Isolation and characterization of the Xenopus oocyte plasma membrane: a new method for studying activity of water and solute transporters

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Publisher
The American Physiological Society
Copyright
Copyright © 2011 the American Physiological Society
ISSN
0363-6127
eISSN
1522-1466
DOI
10.1152/ajprenal.00022.2005
pmid
15741609
Publisher site
See Article on Publisher Site

Abstract

The intact Xenopus laevis oocyte is a useful model system for studying expressed water and solute transporters but suffers from a number of limitations, most notably large unstirred layers and other intracellular diffusion barriers. To overcome these, we have developed a method for isolating plasma membrane vesicles from oocytes. This approach facilitates more precise control of the intravesicular environment and virtually eliminates the problem of unstirred layers in kinetic experiments. The isolation procedure results in 50.6-fold enrichment of the plasma membrane marker alkaline phosphodiesterase compared with the homogenate. Markers of late endosomes/lysosomes and mitochondria were not enriched, and the endoplasmic reticulum was enriched only modestly. Permeabilities of native plasma membrane to water and urea were 8.1 x 10 –4 and 5.6 x 10 –7 cm/s, respectively, values that are sufficiently low to classify them as barrier membranes. Phospholipid analysis by mass spectrometry showed the membrane, not including cholesterol, to be rich in phosphatidylcholine (35.8 mole percent), sphingomyelin (25.8 mole percent), and phosphatidylinositol (6.8 mole percent). Cholesterol concentration was 20.7 mole percent. Membrane vesicles isolated from oocytes expressing aquaporin-1 exhibited fourfold higher water permeability in stopped-flow experiments. Oocytes expressing mouse urea transporter A3 (UT-A3) exhibited 7.5-fold faster phloretin-inhibitable urea transport compared with water-injected controls. There was no difference in water permeability between these membrane vesicles, suggesting that UT-A3 is not a water carrier. In conclusion, we describe an improved method for the isolation of the oocyte plasma membrane that will allow the study of water and solute transport kinetics as well as substrate selectivity in heterologously expressed proteins. aquaporin; urea; permeability; barrier; lipid composition Address for reprint requests and other correspondence: W. G. Hill, Laboratory of Epithelial Cell Biology, Renal-Electrolyte Div., Univ. of Pittsburgh School of Medicine, A1222 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261 (e-mail: whill@pitt.edu )

Journal

AJP - Renal PhysiologyThe American Physiological Society

Published: Jul 1, 2005

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