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 Ca(2+)-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., & 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 < or = 0.73 and a free Ca2+ concentration of approximately 11 microM at 50% saturation. The average pKa of the dityrosine cross-link in the dimeric calmodulin is 8.5-8.6 (+/- Ca2+). Sedimentation velocity experiments show that the average Ca(2+)-liganded dimer has an elongated structure, with a relative frictional ratio approximately 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 cross-linked monomer.
Malencik
D A
DA
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331-7305.
Anderson
S R
SR
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D016880:Anisotropy; D001923:Brain Chemistry; D002118:Calcium / Q000737:chemistry; D002147:Calmodulin / Q000737:chemistry*; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry*; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry; D011485:Protein Binding; D013482:Superoxide Dismutase / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry*
D000596:Amino Acids; D002147:Calmodulin; D003432:Cross-Linking Reagents; D014443:Tyrosine; D013482:Superoxide Dismutase; D009219:Myosin-Light-Chain Kinase; D002118:Calcium