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Isolation and provisional identification of plasma membrane populations from cultured human retinal pigment
A K Mircheff1, S S Miller, D B Farber
1Department of Physiology & Biophysics, University of Southern California School of Medicine, Los Angeles 90033.
Abstract:
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.
Insights
This study successfully isolated human RPE apical and basal-lateral plasma membranes using density gradient centrifugation. Key membrane proteins like Na,K-ATPase were identified in distinct fractions, aiding in cell polarity research.
Area of Science:
- Cell Biology
- Membrane Biology
- Biochemistry
Background:
- Human retinal pigment epithelium (RPE) plays a crucial role in vision.
- Understanding the distinct apical and basal-lateral membrane compositions of RPE cells is vital for studying their function.
Purpose of the Study:
- To develop a method for isolating and distinguishing apical and basal-lateral plasma membranes from cultured fetal human RPE cells.
- To characterize the protein markers associated with these separated membrane fractions.
Main Methods:
- Disruption of cultured human RPE cells using a Dounce apparatus.
- Isolation of membrane fractions via differential centrifugation and density gradient centrifugation in sorbitol.
- Analysis of membrane protein enrichment using biochemical markers (Na,K-ATPase, alkaline phosphatase, mitochondrial, endoplasmic reticulum, Golgi markers).
- Further separation and characterization using phase partitioning and SDS-PAGE/Western blotting.
Main Results:
- Density gradient centrifugation resolved membrane populations enriched in specific markers.
- Alkaline phosphatase and Na,K-ATPase showed distinct enrichment patterns in different density windows.
- Further purification steps successfully separated RPE basal-lateral membranes (enriched in alkaline phosphatase) and apical plasma membranes (enriched in Na,K-ATPase, alkaline phosphatase, and galactosyltransferase).
- SDS-PAGE and Western blotting confirmed the correlation between Na,K-ATPase activity and its alpha subunit.
Conclusions:
- A robust method was established for isolating apical and basal-lateral plasma membranes from human RPE.
- The distinct enrichment of specific proteins in different fractions provides insights into RPE cell polarity and membrane protein localization.
- This technique facilitates further investigation into the specialized functions of RPE membrane domains.