DITYROSINE FORMATION IN CALMODULIN - CROSS-LINKING AND POLYMERIZATIONCATALYZED BY ARTHROMYCES PEROXIDASE

We employ bovine brain calmodulin, a protein that is subject to photoa ctivated dityrosine formation [Malencik, D. A., & Anderson, S, R. (198 7) Biochemistry 26, 695; (1994) Biochemistry 33, 13363], as a model fo r the development of an efficient enzyme-catalyzed protein cross-linki ng technique. Key steps in the elaboration of the procedure are (1) id entification of a peroxidase, from Arthromyces ramosus, that catalyzes dityrosine production in proteins that are not acted on by other comm on peroxidases, (2) monitoring of the intrinsic fluorescence of dityro sine to determine optimum reaction conditions, achieved with calmoduli n in solutions containing boric acid-sodium berate (concentration grea ter than or equal to 0.2 M), similar to pH 8.3, similar to 40 degrees C, and (3) quenching of the reaction with reduced glutathione. Arthrom yces,ces peroxidase is the only common peroxidase able to catalyze sig nificant dityrosine production in calmodulin, through a reaction that is largely intermolecular. Gel filtration yields fractions (accounting for similar to 40% of the initial calmodulin) that represent differin g mobility ranges in NaDodSO(4) polyacrylamide gel electrophoresis and contain close to the maximum possible amounts of dityrosine. The vari ous fractions undergo Ca2+-dependent conformational changes detected i n sedimentation velocity and/or fluorescence anisotropy measurements. Most of the samples stimulate the Ca2+-dependent activity of smooth mu scle myosin light chain kinase. In catalytic assays utilizing the synt hetic phosphate acceptor peptide, the average activities range from 50 to 100% of that determined for native calmodulin. However, only the l east polymerized fraction and the photogenerated calmodulin dimers sig nificantly enhance the p-nitrophenylphosphatase activity of calcineuri n. The ability to prepare soluble calmodulin polymers that retain a su bstantial degree of biological activity and exhibit the intense visibl e fluorescence of dityrosine illustrates the potential usefulness of A rthromyces peroxidase in the zero-length cross-linking of proteins.