Lectin-affinity isolation of microvillous membranes from the pigmented epithelium of rat retina.
The pigmented epithelium of the vertebrate retina phagocytizes the discarded tips of photoreceptors and it is likely that a specific cellular recognition process is involved in this phenomenon. The apical surface of retinal pigmented epithelium (RPE) contains microvilli which interdigitate with the outer segment regions of photoreceptor cells and it is this apical microvillous surface that is of particular interest with respect to phagocytosis. The present study is a report of a method to isolate a fraction that is enriched in microvilli from the apical surface of this highly polarized epithelial cell. Wheat germ agglutinin (WGA) conjugated sepharose beads are used to remove the microvillous membranes which are then observed with scanning and transmission electron microscopy. The proteins of this RPE-subfraction are separated through use of SDS-polyacrylamide gel electrophoresis. The relative molecular weights (Mr) and lectin binding properties of glycoproteins are examined in Western blots through the use of lectin-peroxidase conjugates as probes for carbohydrate residues. A preliminary comparison of membranes isolated from Long Evans (normal) and Royal College of Surgeons (dystrophic) rat retina RPE shows that the glycoproteins in these two preparations are different with respect to the binding of Concanavalin-A (Con-A) and WGA. In particular a glycoprotein in the normal RPE preparation with a Mr of 175K binds Con-A and WGA, but in the dystrophic RPE preparation binds little or no WGA. A glycoprotein present in the normal RPE preparation with a Mr of 86K binds Con-A and WGA, but both lectins have reduced binding sites in the dystrophic preparation. Limax flavus agglutinin (specific for sialic acid residues) binds to a high molecular weight glycoprotein with a Mr of 195K-196K which is present in both normal and dystrophic RPE membrane preparations and which also binds Con-A and WGA.
Cooper
N G
NG
Department of Anatomy and Neurobiology, College of Medicine, University of Tennessee, Memphis 38163.
Tarnowski
B I
BI
McLaughlin
B J
BJ
D000818:Animals; D002462:Cell Membrane / Q000648:ultrastructure; D005591:Chemical Fractionation / Q000379:methods*; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D037102:Lectins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D008871:Microvilli / Q000648:ultrastructure; D010857:Pigment Epithelium of Eye / Q000378:metabolism / Q000648:ultrastructure*; D051381:Rats; D011919:Rats, Inbred Strains; D012394:Rosaniline Dyes
D037102:Lectins; D008565:Membrane Proteins; D012394:Rosaniline Dyes; C048139:Coomassie blue