DITYROSINE FORMATION IN CALMODULIN - CONDITIONS FOR INTERMOLECULAR CROSS-LINKING

The pattern and extent of photoactivated dityrosine formation in bovine brain calmodulin are strongly affected by the presence of superoxide dismutase during UV irradiation. The addition of the enzyme to Ca2+-containing solutions of calmodulin results in an altered distribution of the dityrosine-containing photoproducts, from a predominance of cross-linked monomer to a mixture of products with inter- and intramolecular cross-linking. When Ca2+ is absent, significant dityrosine formation occurs only in the presence of superoxide dismutase. Fractionation of the latter reaction mixture yields a dimer of calmodulin, corresponding to a single-component in sedimentation equilibrium, a smaller amount of a more highly polymerized material, and virtually no cross-linked monomer-as was found in the absence of the enzyme [Malencik, D. A., and Anderson, S. R. (1987) Biochemistry 26, 695]. Although it is homogeneous in terms of molecular weight, the purified dimer contains three electrophoretic components. Chemical characterization demonstrates intermolecular cross-linking of Tyr-99 to Tyr-138 and probably of Tyr-99 to Tyr-99. On the average, 85-90% of the dimeric calmodulin molecules bind two molecules of smooth muscle myosin light chain kinase. Catalytic activity determinations with this enzyme detect no difference between the dimer and the native protein. Fluorescence anisotropy measurements of Ca2+ binding give a Hill coefficient of less than or equal to 0.73 and a free Ca2+ concentration of approximate to 11 mu M at 50% saturation. The average pK(a) of the dityrosine cross-link in the dimeric calmodulin is 8.5-8.6 (+/-Ca2+). Sedimentation velocity experiments show that the average Ca2+-liganded dimer has an elongated structure, with a relative frictional ratio similar to 30% greater than that of the native monomer. In terms of functional properties, the dimeric calmodulin preparation is more like the native protein than it is like the previously reported crosslinked monomer.