| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Laboratory of Epithelial Cell Biology, Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, PA, USA
2 Department of Molecular Cell Physiology, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom
* To whom correspondence should be addressed. E-mail: whill{at}pitt.edu.
The intact Xenopus 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 to homogenate. Markers of late endosomes/lysosomes and mitochondria were not enriched and endoplasmic reticulum was enriched only modestly. Permeabilities of native plasma membrane to water and urea were 8.1 x 10-4 cm/s and 5.6 x 10-7 cm/s respectively, values which are sufficiently low to classify them as low permeability or barrier membranes. Phospholipid analysis by mass spectrometry showed the membrane, not including cholesterol, to be rich in phosphatidylcholine (35.8 mole%), sphingomyelin (25.8 mole%) and phosphatidylinositol (6.8 mole%). Cholesterol concentration was 20.7 mole%. Membrane vesicles isolated from oocytes expressing AQP1 exhibited four-fold higher water permeability in stopped-flow experiments. Oocytes expressing mouse urea transporter A3 exhibited 7.5-fold faster phloretin-inhibitable urea transport compared to 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 which will allow the study of water and solute transport kinetics as well as substrate selectivity in heterologously expressed proteins.
This article has been cited by other articles:
|
|
 |
|
  X.-D. Zhang, P.-Y. Tseng, and T.-Y. Chen ATP Inhibition of CLC-1 Is Controlled by Oxidation and Reduction J. Gen. Physiol., September 29, 2008; 132(4): 421 - 428. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Sha, W. Pearson, L. C. Burcea, D. A. Wigfall, P. H. Schlesinger, C. G. Nichols, and R. W. Mercer Human FXYD2 G41R mutation responsible for renal hypomagnesemia behaves as an inward-rectifying cation channel Am J Physiol Renal Physiol, July 1, 2008; 295(1): F91 - F99. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. MacIver, C. P. Smith, W. G. Hill, and M. L. Zeidel Functional characterization of mouse urea transporters UT-A2 and UT-A3 expressed in purified Xenopus laevis oocyte plasma membranes Am J Physiol Renal Physiol, April 1, 2008; 294(4): F956 - F964. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Milescu, J. Vobecky, S. H. Roh, S. H. Kim, H. J. Jung, J. I. Kim, and K. J. Swartz Tarantula Toxins Interact with Voltage Sensors within Lipid Membranes J. Gen. Physiol., October 29, 2007; 130(5): 497 - 511. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Leduc-Nadeau, K. Lahjouji, P. Bissonnette, J.-Y. Lapointe, and D. G. Bichet Elaboration of a novel technique for purification of plasma membranes from Xenopus laevis oocytes Am J Physiol Cell Physiol, March 1, 2007; 292(3): C1132 - C1136. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH |
| Visit Other APS Journals Online |
