Quantitative analysis of the interaction between S-100 proteins and brain tubulin.
S-100 was shown to regulate the in vitro assembly of brain microtubule proteins (MTPs) in a Ca2+-mediated way by acting on both the nucleation and the elongation of microtubules (MTs). Here data will be shown suggesting that S-100 binds to tubulin. The binding is time-, temperature-, Ca2+-, and pH-dependent, and saturable with respect to S-100. At pH 6.75, the saturation curve is biphasic, displaying a high affinity component (dissociation constant, Kd1, approximately 0.1 microM) and a low affinity component (Kd2 approximately 3.8 microM). At pH 6.75, as the free Ca2+ concentration raises from 0 to 100 microM, the overall binding capacity increases from 0.065 to 0.66 mol S-100/mol tubulin dimer. This finding, together with the observation that the S-100 effect on MTP assembly is Ca2+-dependent at that pH, suggests that the S-100-induced inhibition of MTP assembly depends on S-100 binding to the low affinity sites on the tubulin molecule. The S-100 binding to tubulin is pH-dependent; as the pH raises from 6.75 to 8.3, both binding components are affected, the major changes consisting of an increase in the binding capacity and a decrease in the overall affinity. Moreover, as the pH raises, Ca2+ is no longer required for S-100 to bind to tubulin. S-100 also interacts with a component of whole MTPs (probably tubulin, on the basis of the above results). No S-100 binding to microtubule-associated proteins (MAPs) could be evidenced by the techniques employed in this study. On the contrary, some competition between S-100 and MAPs for binding sites or tubulin seems to occur.
D000645:Ammonium Sulfate; D000818:Animals; D001923:Brain Chemistry; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008868:Microtubule Proteins / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D009418:S100 Proteins / Q000378:metabolism*; D013696:Temperature; D014404:Tubulin / Q000378:metabolism*
D046911:Macromolecular Substances; D008868:Microtubule Proteins; D008869:Microtubule-Associated Proteins; D009418:S100 Proteins; D014404:Tubulin; D000645:Ammonium Sulfate; D002118:Calcium