A rapid method of preparing the plasma membrane enriched fraction of the bovine retinal pigment epithelial cells.
OBJECTIVE
To devise a rapid method of isolating the plasma membrane enriched fraction (PMEF) of the bovine retinal pigment epithelial (RPE) cells with Percoll centrifugation medium.


METHODS
The bovine RPE was homogenised with a tight fit Dounce homogeniser and centrifuged in a 16.7% Percoll gradient for 20 minutes. The RPE particulate fractions were characterised in terms of their protein concentrations, Na/K-ATPase and bicarbonate stimulated ATPase activities.


RESULTS
The total protein recovery was 88.7% of the RPE homogenate. The nucleus layer was identified at the first band. The mitochondrial fraction was at the second layer according to its bicarbonate stimulated ATPase activity. The 3rd and 4th bands were enriched with plasma membranes and their Na/K-ATPase activities were 31.5 and 34.6 mumol/mg/h respectively. The Na/K-ATPase activities were about six times that of the RPE homogenate.


CONCLUSIONS
A rapid method of isolating the bovine RPE PMEF has been devised which involved a single centrifugation procedure in a Percoll gradient.
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Department of Optometry and Radiography, Hong Kong Polytechnic University, Hung Hom Kowloon, Hong Kong. ORCHTO@POLYU.EDU.HK
Hodson
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D000818:Animals; D002417:Cattle; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D008565:Membrane Proteins / Q000032:analysis; D010857:Pigment Epithelium of Eye / Q000201:enzymology / Q000648:ultrastructure*; D011205:Povidone; D012822:Silicon Dioxide; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D008565:Membrane Proteins; C016039:Percoll; D012822:Silicon Dioxide; D000254:Sodium-Potassium-Exchanging ATPase; D011205:Povidone