Proteolytic analysis of the topological arrangement of red cell phosphoproteins

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Abstract
The topology of human erythrocyte membrane phosphoproteins was determined by using protease digestion and selective solubilization. The distribution of 32P among the membrane polypeptides in intact cells differed from the pattern found in isolated ghosts. The following membrane skeleton polypeptides, according to the nomenclature of Steck, were phosphorylated 2, 2.1, 4.1, 4.5b and 4.9, together with 2 polypeptides at 105,000 and 110,000 daltons that were heavily phosphorylated. Among integral proteins, band 3 and glycophorins A and B were phosphorylated. Glycophorin C appeared to have little turnover of phosphate. Autoradiograms of trypsin-treated membranes permitted identification of the transmembrane proteolytic fragment of glycophorin A as a dimer of 38,000 daltons. The band 5 region contained 2 phosphoproteins, the peripheral protein 4.9 (48 kdaltons) and PAS[periodic-acid Schiff]-2. Band 7 resolved into an integral phosphoprotein and a nonphosphorylated protein associated with the membrane skeleton.