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

摘要: We have attempted to isolate samples of apical and basal-lateral plasma membranes from cultured fetal human RPE. Cells confluent, dome-forming cultures were disrupted with a Dounce apparatus. Nuclei melanin granules sedimented by centrifugation at 2600 g for 10 min. The supernates layered over gradients 17.5-65% sorbitol centrifuged 122,000 5 hr. Fractions grouped into "density windows" on the basis their biochemical marker contents. Na,K-ATPase alkaline phosphatase overlapped but did not precisely parallel one another, suggesting associations two partially separated membrane populations; in density window I, was enriched 4.3-fold, 1.7-fold, whereas II corresponding enrichment factors 7.7 6.7. These markers well resolved mitochondrial marker, they endoplasmic reticulum Golgi markers. Additional gradient centrifugations, performed after had been suspended 55% sorbitol, further phosphatase- Na,K-ATPase-containing membranes, yielding cumulative 6.8 2.5 sample I 9.3 10.9 II. Subsequent phase partitioning analysis an alkalinephosphatase-rich population, which is believed represent RPE membranes. contained populations, both Na,K-ATPase, phosphatase, galactosyltransferase, appear be derived membrane. SDS-PAGE Western blotting confirmed correlation between catalytic activity alpha subunit immunoreactivity. Invest Ophthalmol Vis Sci 31:863-878,1990