Preparation of azidocalmodulin: a photoaffinity label for calmodulin-binding proteins.

Abstract

A photoaffinity label for calmodulin-binding proteins was prepared from 125I-labeled calmodulin (125I-calmodulin) and methyl-4-azidobenzimidate. Azidocalmodulin containing 1 azido group per calmodulin retained its ability to stimulate the Ca2+-sensitive phosphodiesterase purified from bovine heart muscle. The concentrations of calmodulin and azidocalmodulin required for half-maximal stimulation of phosphodiesterase activity were 170 and 230 pm, respectively. Azido-125I-calmodulin was used to photoaffinity label [rabbit skeletal muscle] troponin I, myosin L chain kinase and the Ca2+-sensitive phosphodiesterase. Formation of cross-linked complexes required the presence of Ca2+ or Mn2+ and was inhibited by excess unmodified calmodulin. The calmodulin-binding subunits all formed 1:1 complexes with calmodulin, and the MW of the cross-linked products obtained with troponin I, the phosphodiesterase and myosin L chain kinase were 43,000, 79,000 and 116,000, respectively. Photolysis experiments using azido-125I-calmodulin and bovine cerebral cortex membranes or detergent-solubilized membranes resulted in formation of a limited number of specifically labeled polypeptides. Azido-calmodulin appears to be an appropriate photoaffinity label for the identification and characterization of calmodulin-binding subunits.