PROTEOLYTIC CLEAVAGE SITES OF BAND-3 PROTEIN IN ALKALI-TREATED MEMBRANES - FIDELITY OF HYDROPATHY PREDICTION FOR BAND-3 PROTEIN

To assess the fidelity of hydropathy prediction for band 3 protein, we determined the cleavage sites of the protein and the portions of the protein tightly bound to the membrane lipid bilayer by means of in. si tu proteolytic digestion. For the removal of all anticipated hydrophil ic connector loops from membranes, we had to denature the band 3 prote in molecule in situ by alkali treatment. When the alkali-treated membr anes were digested with trypsin, chymotrypsin, and pepsin, the majorit y of the anticipated transmembrane portions remained in the membrane f raction. However, five anticipated transmembrane portions were release d into the supernatant fraction. Thus, the first, second, third, sixth and tenth anticipated transmembrane portions, in accordance with the hydropathy prediction, were released into the supernatant with the pro teolytic digestion method. This indicates that these anticipated trans membrane portions are not bound with the boundary lipids although the hydrophobicity of these portions is comparable to that of the portions experimentally remaining in the membrane fraction. It is conceivable that the membrane peptide portions of band 3 protein could be classifi ed into at least two categories, i.e. one bound to the boundary lipids and the other free from the boundary lipids, Approximately 90% of the transmembrane domain of the band 3 protein are recovered in either th e supernatant fraction or the membrane fraction, The fidelity of hydro pathy prediction for polytopic membrane proteins and the nature of the membrane embedded peptide portions are discussed.