Chaperone activity and prodan binding at the self-associating domain of erythroid spectrin.
Spectrin, the major constituent protein of the erythrocyte membrane skeleton, exhibits chaperone activity by preventing the irreversible aggregation of insulin at 25 degrees C and that of alcohol dehydrogenase at 50 degrees C. The dimeric spectrin and the two subunits, alpha-spectrin and beta-spectrin prevent such aggregation appreciably better, 70% in presence of dimeric spectrin at an insulin:spectrin ratio of 1:1, than that in presence of the tetramer of 25%. Our results also show that spectrin binds to denatured enzymes alpha-glucosidase and alkaline phosphatase during refolding and the reactivation yields are increased in the presence of the spectrin derivatives when compared with those refolded in their absence. The unique hydrophobic binding site on spectrin for the fluorescence probe, 6-propionyl-2-(dimethylamino)naphthalene (Prodan) has been established to localize at the self-associating domain with the binding stoichiometry of one Prodan/both dimeric and tetrameric spectrin. The other fluorescence probe, 1-anilinonaphthalene-8-sulfonic acid, does not show such specificity for spectrin, and the binding stoichiometry is between 3 and 5 1-anilinonaphthalene-8-sulfonic acid/dimeric and tetrameric spectrin, respectively. Regions in alpha- and beta-spectrins have been found to have sequence homology with known chaperone proteins. More than 50% similarities in alpha-spectrin near the N terminus with human Hsp90 and in beta-spectrin near the C terminus with human Hsp90 and Escherichia coli DnaJ have been found, indicating a potential chaperone-like sequence to be present near the self-associating domain that is formed by portions of alpha-spectrin near the N terminus and the beta-spectrin near the C terminus. There are other patches of sequences also in both the spectrin polypeptides, at the other termini as well as in the middle of the rod domain having significant homology with well known chaperone proteins.
Bhattacharyya
Malyasri
M
Biophysics Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagr, Kolkata 700064, India.
Ray
Sibnath
S
Bhattacharya
Shekhar
S
Chakrabarti
Abhijit
A
D015081:2-Naphthylamine / Q000031:analogs & derivatives* / Q000737:chemistry*; D000426:Alcohol Dehydrogenase / Q000737:chemistry; D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000737:chemistry; D004912:Erythrocytes / Q000378:metabolism*; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry; D006360:Heat-Shock Proteins / Q000737:chemistry; D006736:Horses; D006358:Hot Temperature; D006801:Humans; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D018832:Molecular Chaperones / Q000737:chemistry*; D008969:Molecular Sequence Data; D009154:Mutation; D010179:Pancreas / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013049:Spectrin / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors; D000520:alpha-Glucosidases / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D007328:Insulin; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D013049:Spectrin; C027132:1-anilino-8-naphthalenesulfonate; C038517:prodan; D004492:Edetic Acid; D015081:2-Naphthylamine; D000426:Alcohol Dehydrogenase; D000469:Alkaline Phosphatase; D000520:alpha-Glucosidases