Isolation and characterization of renal cortical membranes using an aqueous two-phase partition technique.
The aqueous two-phase partition technique is a simple, rapid and inexpensive method for the fractionation of membrane preparations. Aqueous two-phase partitioning separates according to surface properties such as charge and hydrophobicity, making it complementary to established centrifugation techniques, which separate on the basis of density. Although aqueous two-phase partitioning has been successfully applied to animal tissues, there are limited data on the functional properties of the isolated membranes. We have applied the aqueous two-phase partition technique to rat renal brush-border membrane vesicles and sheets. Our aim was to remove organelle contamination while maintaining the functional properties of the membranes. Evidence from marker enzyme analysis and electron microscopy supports the conclusion that renal brush-border membranes are fractionated separate from the mitochondria and endoplasmic reticulum. This separation procedure did not alter the Na(+)-dependent transport of brush-border membrane vesicles. Na(+)-D-glucose symporter and Na(+)-H+ antiporter activity in the fractionated preparation increased to the same extent as did the enrichment of enzyme markers for brush-border membranes.
Hammond
T G
TG
Department of Medicine, University of Wisconsin Medical School, Madison 53792.
Majewski
R R
RR
Onorato
J J
JJ
Brazy
P C
PC
Morré
D J
DJ
D000704:Analysis of Variance; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000648:ultrastructure*; D003911:Dextrans; D004798:Enzymes / Q000032:analysis; D007672:Kidney Cortex / Q000378:metabolism / Q000648:ultrastructure*; D008297:Male; D008854:Microscopy, Electron; D008871:Microvilli / Q000378:metabolism / Q000648:ultrastructure*; D011092:Polyethylene Glycols; D051381:Rats; D017207:Rats, Sprague-Dawley; D017923:Sodium-Hydrogen Exchangers; D014867:Water
D002352:Carrier Proteins; D003911:Dextrans; D004798:Enzymes; D017923:Sodium-Hydrogen Exchangers; D014867:Water; D011092:Polyethylene Glycols