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Simultaneous preparation of rabbit intestinal brush border and basolateral membrane vesicles
J Stein1, V Milovic, R Gerhard
12nd Medical Department, Johann Wolfgang Goethe University, Frankfurt/Main, Germany.
Research in Experimental Medicine. Zeitschrift Fur Die Gesamte Experimentelle Medizin Einschliesslich Experimenteller Chirurgie
|January 1, 1994
Summary
This study presents a reproducible method for isolating rabbit intestinal brush border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV). Both vesicle types demonstrated high purity and functional integrity for transport studies.
Area of Science:
- Cell Biology
- Membrane Biochemistry
- Gastrointestinal Physiology
Background:
- Intestinal epithelial cells possess distinct apical (brush border) and basolateral membrane domains.
- Understanding the transport functions of these membranes is crucial for physiology and pharmacology.
- Previous methods for isolating these vesicles often lacked reproducibility or purity.
Purpose of the Study:
- To develop a highly reproducible method for simultaneous isolation of rabbit intestinal BBMV and BLMV.
- To characterize the purity, protein composition, and functional properties of the isolated vesicles.
- To establish a model for studying transepithelial intestinal transport using membrane vesicles.
Main Methods:
- Simultaneous isolation of BBMV and BLMV from rabbit intestine.
- Purification of BLMV using continuous sorbitol gradient centrifugation.
- Separation of BBMV via repeated precipitation with magnesium and EGTA.
- Enzyme activity assays for Na+, K(+)-ATPase and K(+)-activated phosphatase.
- SDS gel electrophoresis and Western blotting with monoclonal antibodies for protein analysis.
- D-glucose transport assays to assess functional properties.
- Vesicle orientation determination using SDS/BSA permeabilization.
Main Results:
- Over ten-fold enrichment of Na+, K(+)-ATPase and K(+)-activated phosphatase in BLMV and BBMV, respectively.
- SDS-PAGE revealed distinct protein profiles for BBMV and BLMV, with villin specific to BBMV.
- Functional D-glucose transport was confirmed in both vesicle preparations.
- BLMV were predominantly oriented inside-out, while BBMV were right-side out.
Conclusions:
- The described method provides highly pure and functionally intact BBMV and BLMV.
- The isolated vesicles serve as a reliable model for studying intestinal transport mechanisms.
- This technique has potential applications in physiological and pharmacological transport studies.