Mapping the domain structure of human erythrocyte adducin.
Adducin is a 200-kDa heterodimeric protein associated with the erythrocyte membrane skeleton which binds to Ca2+/calmodulin, promotes binding of spectrin to actin, and is a substrate for protein kinases C and A. Adducin polypeptides can be structurally and functionally divided into two distinct regions. The amino-terminal 39-kDa domain of each subunit is more basic and resistant to proteases than the C-terminal 60-64-kDa domain, which is very sensitive to proteolytic degradation. Two-dimensional peptide map analysis revealed that the 39-kDa protease-resistant domains represent a portion of adducin which is highly conserved between the alpha and beta subunits whereas the protease-sensitive regions are different in each subunit. Comparison of the structural and functional properties of purified 39-kDa domains with intact adducin showed that the 39-kDa domains were not phosphorylated by protein kinases C or A and did not bind to Ca2+/calmodulin or interact with spectrin and actin. This suggests that the protease-sensitive domains may perform the various functions of adducin since these activities were all lacking from the protease-resistant domains. It is also possible that the conserved and variable domains are both required for one or more activities of adducin or that the 39-kDa domains play a role in maintaining the oligomeric state of adducin necessary for interaction of the variable domains with spectrin-actin complexes.
Joshi
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R
Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710.
Bennett
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D001798:Blood Proteins / Q000235:genetics*; D002148:Calmodulin-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification; D002918:Chymotrypsin; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D046911:Macromolecular Substances; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D011487:Protein Conformation; D014357:Trypsin; D000099291:Adducins
D001798:Blood Proteins; D002148:Calmodulin-Binding Proteins; D002918:Chymotrypsin; D046911:Macromolecular Substances; D010446:Peptide Fragments; D014357:Trypsin; D000099291:Adducins