Preparation, characterization, and properties of a novel triple-modified derivative of the Ca2+-dependent protein modulator

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Abstract
A novel, chemically modified derivative of the bovine brain Ca2+-dependent protein modulator of cyclic nucleotide phosphodiesterase was prepared by the sequential treatment of the native protein with tetranitromethane, 1,2-cyclohexanedione and diethylpyrocarbonate. Three derivatives were isolated during the synthesis, i.e., a single-modified derivative containing 2 modified tyrosine residues/mol; a double-modified derivative containing 2 modified tyrosines plus 5 modified arginine residues/mol; and a triple-modified derivative containing 2 modified tyrosines, 5 modified arginines and 1 modified histidine residue/mol. Intermolecular cross-linking was observed to occur as a minor side reaction during nitration of the native protein with tetranitromethane. All the derivatives were examined for phosphodiesterase-stimulating activity and troponin C like activities. All derivatives were found to retain the capacity of the native protein to stimulate modulator-deficient phosphodiesterase; furthermore, in each case, the stimulation of phosphodiesterase was a Ca2+-dependent process. Both the double- and triple-modified modulators lost the troponin C like activities, i.e., the Ca2+-dependent change in mobility in urea-polyacrylamide gels and the ability to interact with troponin I in the presence of Ca2+ to form a urea-stable complex, while the nitrotyrosyl modulator retained these properties of the native protein.