Isolation and initial characterization of a lymphocyte cap structure.
A method for isolating the cap structure induced by polycationized ferritin on the surface of mouse T-lymphoma cells is described. The procedure, based on the 'density perturbation' approach designed by Wallach and co-workers (Wallach, D.F.H., Kranz, B., Ferber, E. and Fischer, H. (1972) FEBS Lett. 21, 29-33), involves a simple, one-step density gradient centrifugation using metrizamide as the gradient material. The isolated polycationized ferritin cap fraction is approx. 20-fold enriched in plasma membrane relative to the whole cell homogenate and is apparently free of all uncapped membrane. Our initial analysis of the protein composition of the isolated cap structure indicates that there are approx. 30 membrane-bound polypeptides specifically associated with the polycationized ferritin cap fraction. Interestingly, there are at least four phosphorylated membrane-bound polypeptides (mol.wt. approximately 130 000, 100 000, 30 000 and 20 000) which are preferrentially accumulated in the cap fraction. These findings provide further evidence for the selective redistribution of certain surface membrane proteins during lymphocyte capping.
D000373:Agglutinins / Q000302:isolation & purification*; D000818:Animals; D002499:Centrifugation, Density Gradient; D005293:Ferritins / Q000276:immunology*; D033862:Iron-Binding Proteins; D008214:Lymphocytes / Q000276:immunology*; D008793:Metrizamide; D051379:Mice; D008854:Microscopy, Electron; D008970:Molecular Weight; D011956:Receptors, Cell Surface; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013499:Surface Properties
D000373:Agglutinins; D033862:Iron-Binding Proteins; D011956:Receptors, Cell Surface; C027293:ferritin receptor; D005293:Ferritins; D000254:Sodium-Potassium-Exchanging ATPase; D008793:Metrizamide