To C H, Hodson S A
Department of Optometry and Radiography, Hong Kong Polytechnic University, Hung Hom Kowloon, Hong Kong.
Yan Ke Xue Bao. 1999 Mar;15(1):22-8.
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.
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.
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.
A rapid method of isolating the bovine RPE PMEF has been devised which involved a single centrifugation procedure in a Percoll gradient.
设计一种利用Percoll离心介质快速分离牛视网膜色素上皮（RPE）细胞富含质膜组分（PMEF）的方法。
用紧密配合的Dounce匀浆器将牛RPE匀浆，并在16.7%的Percoll梯度中离心20分钟。根据RPE颗粒组分的蛋白质浓度、钠/钾-ATP酶和碳酸氢盐刺激的ATP酶活性对其进行表征。
总蛋白回收率为RPE匀浆的88.7%。在第一条带处鉴定出核层。根据其碳酸氢盐刺激的ATP酶活性，线粒体组分位于第二层。第三和第四条带富含质膜，其钠/钾-ATP酶活性分别为31.5和34.6μmol/mg/h。钠/钾-ATP酶活性约为RPE匀浆的六倍。
已设计出一种快速分离牛RPE PMEF的方法，该方法涉及在Percoll梯度中进行单次离心操作。
