Preparation of basolateral membrane vesicles from rat enterocytes: influence of different gradient media.
An attempt was made to assess whether the choice of the gradient media could influence the yield of basolateral membrane vesicles isolated from the rat intestine as well as their functional characteristics. Crude membranes prepared in the same way were therefore centrifuged with 10% Percoll, on a discontinuous sucrose gradient or on a continuous sorbitol gradient. The protein yield was significantly higher with the Percoll gradient than with sucrose and sorbitol gradient centrifugation (2.7 +/- 1.0%; 0.4 +/- 0.1%; 0.6 +/- 0.2%, respectively). Enrichment in Na+,K+-ATPase was similar in all three preparations (8.50 +/- 2.34; 8.22 +/- 4.78; 8.20 +/- 2.08). However, contamination with brush border membranes was significantly higher after Percoll gradient centrifugation and negligible after the use of the other two gradient media. Transport of D-glucose in the BLM prepared by Percoll gradient centrifugation also indicated some contamination with functional brush-border membranes. An attempt to purify basolateral membrane vesicles after Percoll gradient centrifugation with Ca2+ precipitation, however, reduced the protein yield to less than 1%. We conclude that in the preparation of basolateral membrane vesicles from the rat enterocytes each of the gradient media may have certain advantages and disadvantages, which should be considered according to the purpose of the preparation.
Milovic
V
V
Second Medical Department, Johann Wolfgang Goethe University, Frankfurt/Main, Germany.
Stein
J
J
Ruppert
S
S
Zeuzem
S
S
Caspary
W F
WF
D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane; D002499:Centrifugation, Density Gradient / Q000379:methods*; D005260:Female; D005947:Glucose; D007422:Intestines / Q000166:cytology* / Q000648:ultrastructure; D008297:Male; D011205:Povidone; D051381:Rats; D011921:Rats, Inbred WKY; D012822:Silicon Dioxide; D013012:Sorbitol; D013395:Sucrose
D013012:Sorbitol; D013395:Sucrose; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone; D005947:Glucose