CALMODULIN-DEPENDENT ENZYMES UNDERGO A PROTON-INDUCED CONFORMATIONAL CHANGE THAT IS ASSOCIATED WITH THEIR INTERACTIONS WITH CALMODULIN

The anionic hydrophobic (amphipathic) fluorescent probe 2-(p-toluidiny l)-naphthalene-6-sulfonate was used to investigate the surface hydroph obic properties of calmodulin (caM)-dependent enzymes as follows: calc ineurin, myosin light chain kinase, cyclic nucleotide phosphodiesteras e, CaM-dependent protein kinase II, and the gamma-subunit of phosphory lase kinase. We found that certain domains of these enzymes that inter acted with 2-(p-toluidinyl)-naphthalene-6 sulfonate were exposed by a transient proton (H+) increase within the neutral pH range. This H+-in duced exposure, which could be caused either by direct addition of Hor by the release of H+ from metal chelators upon their binding of Ca2 +, seemed to be more closely linked with a change in pH value (i.e. tr ansient H+ increase) than with the actual equilibrium pH value of the system. Unlike the case with CaM-dependent enzymes, the H+-induced con formational change was uncommon in caM-independent enzymes. When CaM-b inding domains were removed from calcineurin and smooth muscle myosin light chain kinase, the resultant enzymes no longer exposed new do mai ns in response to H+ increase. Using dansylated CaM to monitor the for mation of CaM-enzyme complexes, we found that complex formation occurr ed with an uptake of H+ from solution. When caM-dependent enzymes were evaluated at suboptimal concentrations of Ca2+, addition of H+ enhanc ed both the formation of CaM-enzyme complexes and the caM-dependent ca talytic activities, but this synergistic H+ effect occurred within onl y a narrow range of Ca2+ concentrations. These findings suggest that t he H+-exposed domains in CaM-dependent enzymes are involved in the bin ding of CaM and that both conformational changes in caM and its enzyme targets are necessary for complex formation. Further, the findings ar e consistent with the notion that CaM-binding domains are masked in th e nonactivated (uncomplexed) conformations of CaM-dependent enzymes. T he interplay between H+ and Ca2+ is discussed in relation to other sys tems that display interdependent effects of these two ions.