IOVS Circulation
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mircheff, A. K.
Right arrow Articles by Bok, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mircheff, A. K.
Right arrow Articles by Bok, D.

Investigative Ophthalmology & Visual Science, Vol 31, 863-878, Copyright © 1990 by Association for Research in Vision and Ophthalmology

ARTICLES AND REPORTS

Isolation and provisional identification of plasma membrane populations from cultured human retinal pigment epithelium

AK Mircheff, SS Miller, DB Farber, ME Bradley, WT O'Day and D Bok
Department of Physiology & Biophysics, University of Southern California School of Medicine, Los Angeles 90033.

We have attempted to isolate samples of apical and basal-lateral plasma membranes from cultured fetal human RPE. Cells from confluent, dome- forming cultures were disrupted with a Dounce apparatus. Nuclei and melanin granules were sedimented by centrifugation at 2600 g for 10 min. The supernates were layered over gradients of 17.5-65% sorbitol and centrifuged at 122,000 g for 5 hr. Fractions were grouped into "density windows" on the basis of their biochemical marker contents. Na,K-ATPase and alkaline phosphatase overlapped but did not precisely parallel one another, suggesting associations with two partially separated membrane populations; in density window I, alkaline phosphatase was enriched 4.3-fold, and Na,K-ATPase was enriched 1.7- fold, whereas in window II the corresponding enrichment factors were 7.7 and 6.7. These markers were well resolved from a mitochondrial marker, but they were overlapped by endoplasmic reticulum and Golgi markers. Additional density gradient centrifugations, performed after samples had been suspended in 55% sorbitol, further separated alkaline phosphatase- and Na,K-ATPase-containing membranes from endoplasmic reticulum and Golgi membranes, yielding alkaline phosphatase and Na,K- ATPase cumulative enrichment factors of 6.8 and 2.5 for the sample from window I and 9.3 and 10.9 for the sample from window II. Subsequent phase partitioning analysis of the sample from window I further enriched an alkaline-phosphatase-rich membrane population, which is believed to represent the RPE basal-lateral membranes. The sample from density window II contained two membrane populations, both enriched in Na,K-ATPase, alkaline phosphatase, and galactosyltransferase, and both of which appear to be derived from the apical plasma membrane. SDS-PAGE and Western blotting confirmed a correlation between Na,K-ATPase catalytic activity and Na,K-ATPase alpha subunit immunoreactivity.


This article has been cited by other articles:


Home page
 IOVSHome page
A. Maminishkis, S. Chen, S. Jalickee, T. Banzon, G. Shi, F. E. Wang, T. Ehalt, J. A. Hammer, and S. S. Miller
Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue.
Invest. Ophthalmol. Vis. Sci., August 1, 2006; 47(8): 3612 - 3624.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
L. Rizzolo and Z. Li
Diffusible, retinal factors stimulate the barrier properties of junctional complexes in the retinal pigment epithelium
J. Cell Sci., January 11, 1993; 106(3): 859 - 867.
[Abstract] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Copyright © 1990 by the Association for Research in Vision and Ophthalmology