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Isolation and provisional identification of plasma membrane populations from cultured human retinal pigment epithelium. 1990

A K Mircheff, and S S Miller, and D B Farber, and M E Bradley, and W T 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.

UI MeSH Term Description Entries
D008565 Membrane Proteins Proteins which are found in membranes including cellular and intracellular membranes. They consist of two types, peripheral and integral proteins. They include most membrane-associated enzymes, antigenic proteins, transport proteins, and drug, hormone, and lectin receptors. Cell Membrane Protein,Cell Membrane Proteins,Cell Surface Protein,Cell Surface Proteins,Integral Membrane Proteins,Membrane-Associated Protein,Surface Protein,Surface Proteins,Integral Membrane Protein,Membrane Protein,Membrane-Associated Proteins,Membrane Associated Protein,Membrane Associated Proteins,Membrane Protein, Cell,Membrane Protein, Integral,Membrane Proteins, Integral,Protein, Cell Membrane,Protein, Cell Surface,Protein, Integral Membrane,Protein, Membrane,Protein, Membrane-Associated,Protein, Surface,Proteins, Cell Membrane,Proteins, Cell Surface,Proteins, Integral Membrane,Proteins, Membrane,Proteins, Membrane-Associated,Proteins, Surface,Surface Protein, Cell
D010857 Pigment Epithelium of Eye The layer of pigment-containing epithelial cells in the RETINA; the CILIARY BODY; and the IRIS in the eye. Eye Pigment Epithelium
D002458 Cell Fractionation Techniques to partition various components of the cell into SUBCELLULAR FRACTIONS. Cell Fractionations,Fractionation, Cell,Fractionations, Cell
D002462 Cell Membrane The lipid- and protein-containing, selectively permeable membrane that surrounds the cytoplasm in prokaryotic and eukaryotic cells. Plasma Membrane,Cytoplasmic Membrane,Cell Membranes,Cytoplasmic Membranes,Membrane, Cell,Membrane, Cytoplasmic,Membrane, Plasma,Membranes, Cell,Membranes, Cytoplasmic,Membranes, Plasma,Plasma Membranes
D002478 Cells, Cultured Cells propagated in vitro in special media conducive to their growth. Cultured cells are used to study developmental, morphologic, metabolic, physiologic, and genetic processes, among others. Cultured Cells,Cell, Cultured,Cultured Cell
D002499 Centrifugation, Density Gradient Separation of particles according to density by employing a gradient of varying densities. At equilibrium each particle settles in the gradient at a point equal to its density. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed) Centrifugations, Density Gradient,Density Gradient Centrifugation,Density Gradient Centrifugations,Gradient Centrifugation, Density,Gradient Centrifugations, Density
D004591 Electrophoresis, Polyacrylamide Gel Electrophoresis in which a polyacrylamide gel is used as the diffusion medium. Polyacrylamide Gel Electrophoresis,SDS-PAGE,Sodium Dodecyl Sulfate-PAGE,Gel Electrophoresis, Polyacrylamide,SDS PAGE,Sodium Dodecyl Sulfate PAGE,Sodium Dodecyl Sulfate-PAGEs
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000254 Sodium-Potassium-Exchanging ATPase An enzyme that catalyzes the active transport system of sodium and potassium ions across the cell wall. Sodium and potassium ions are closely coupled with membrane ATPase which undergoes phosphorylation and dephosphorylation, thereby providing energy for transport of these ions against concentration gradients. ATPase, Sodium, Potassium,Adenosinetriphosphatase, Sodium, Potassium,Na(+)-K(+)-Exchanging ATPase,Na(+)-K(+)-Transporting ATPase,Potassium Pump,Sodium Pump,Sodium, Potassium ATPase,Sodium, Potassium Adenosinetriphosphatase,Sodium-Potassium Pump,Adenosine Triphosphatase, Sodium, Potassium,Na(+) K(+)-Transporting ATPase,Sodium, Potassium Adenosine Triphosphatase,ATPase Sodium, Potassium,ATPase, Sodium-Potassium-Exchanging,Adenosinetriphosphatase Sodium, Potassium,Pump, Potassium,Pump, Sodium,Pump, Sodium-Potassium,Sodium Potassium Exchanging ATPase,Sodium Potassium Pump
D000469 Alkaline Phosphatase An enzyme that catalyzes the conversion of an orthophosphoric monoester and water to an alcohol and orthophosphate. EC 3.1.3.1.

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