TY - JOUR
T1 - Preparation of azidocalmodulin
T2 - a photoaffinity label for calmodulin-binding proteins.
AU - Andreasen, T. J.
AU - Keller, C. H.
AU - LaPorte, D. C.
AU - Edelman, A. M.
AU - Storm, D. R.
PY - 1981/5
Y1 - 1981/5
N2 - A photoaffinity label for calmodulin-binding proteins was prepared from 125I-labeled calmodulin (125I-calmodulin) and methyl-4-azidobenzimidate. Azidocalmodulin containing one 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 troponin I, myosin light chain kinase, and the Ca2+-sensitive phosphodiesterase. Formation of crosslinked 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 molecular weights of the crosslinked products obtained with troponin I, the phosphodiesterase, and myosin light 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.
AB - A photoaffinity label for calmodulin-binding proteins was prepared from 125I-labeled calmodulin (125I-calmodulin) and methyl-4-azidobenzimidate. Azidocalmodulin containing one 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 troponin I, myosin light chain kinase, and the Ca2+-sensitive phosphodiesterase. Formation of crosslinked 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 molecular weights of the crosslinked products obtained with troponin I, the phosphodiesterase, and myosin light 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.
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U2 - 10.1073/pnas.78.5.2782
DO - 10.1073/pnas.78.5.2782
M3 - Article
C2 - 6265911
AN - SCOPUS:0019571979
SN - 0027-8424
VL - 78
SP - 2782
EP - 2785
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 5
ER -