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 situ proteolytic digestion. For the removal of all anticipated hydrophilic connector loops from membranes, we had to denature the band 3 protein molecule in situ by alkali treatment. When the alkali-treated membranes were digested with trypsin, chymotrypsin, and pepsin, the majority of the anticipated transmembrane portions remained in the membrane fraction. However, five anticipated transmembrane portions were released 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 proteolytic digestion method. This indicates that these anticipated transmembrane 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 classified 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 the supernatant fraction or the membrane fraction. The fidelity of hydropathy prediction for polytopic membrane proteins and the nature of the membrane embedded peptide portions are discussed.
Hamasaki
N
N
Department of Clinical Chemistry and Laboratory Medicine, Faculty of Medicine, Kyushu University, Fukuoka. hamasaki@cclm.med.kyushu-u.ac.jp
Okubo
K
K
Kuma
H
H
Kang
D
D
Yae
Y
Y
D000595:Amino Acid Sequence; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D011237:Predictive Value of Tests; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D001457:Anion Exchange Protein 1, Erythrocyte; D008051:Lipid Bilayers; D008565:Membrane Proteins; D010446:Peptide Fragments; D010447:Peptide Hydrolases