A simple method of rat renal brush border membrane preparation using polyethylene glycol precipitation.
A simple method for preparation of brush border membranes (BBM) from rat kidney using polyethylene glycol (PEG) precipitation has been described. This method avoids the use of cations for the preparation, which might alter membrane lipid composition. These preparations were assessed for enrichment of marker enzymes, contamination by subcellular structures, lipid composition and transport function. An enrichment of 11.8910-fold of alkaline phosphatase, 13.9500-fold of amino peptidase and 13.6500-fold of gamma-glutamyl transpeptidase and an approximate yield of 60% were seen in the final membrane preparation as compared to the homogenate. There was very little contamination of basolateral membranes, peroxisomes, microsomes or lysosomes in the final membrane preparation. Analysis of sugars indicated high content of fucose and sailic acid as compared to hexoses. Isolated membranes appeared as vesicles as seen by electron microscopy. Lipid analysis indicated the presence of various neutral and phospholipids with a high content of sphingomyelin along with a cholesterol/phospholipid ratio of 0.4850. The isolated membrane vesicles were able to transport glucose. This study has shown a simple method of renal brush border membrane preparation, which is comparatively pure and functionally active.
Basivireddy
Jayasree
J
Department of Gastrointestinal Sciences, The Wellcome Trust Research Laboratory, Christian Medical College and Hospital, IDA Scudder road, Vellore 632004, India.
Balasubramanian
K A
KA
D000818:Animals; D002374:Catalase / Q000378:metabolism; D002458:Cell Fractionation; D011232:Chemical Precipitation; D002784:Cholesterol / Q000378:metabolism; D005989:Glycerides / Q000378:metabolism; D006601:Hexoses / Q000378:metabolism; D006867:Hydrolases / Q000378:metabolism; D007668:Kidney / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D008871:Microvilli / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D011092:Polyethylene Glycols; D051381:Rats; D005723:gamma-Glutamyltransferase / Q000378:metabolism
D005989:Glycerides; D006601:Hexoses; D011092:Polyethylene Glycols; D002784:Cholesterol; D002374:Catalase; D005723:gamma-Glutamyltransferase; D006867:Hydrolases; D019158:N-Acetylneuraminic Acid