A monoclonal antibody against brain calmodulin-dependent protein kinase type II detects putative conformational changes induced by Ca2+-calmodulin.
A mouse monoclonal IgG1 antibody has been generated against the soluble form of the calmodulin-dependent protein kinase type II. This antibody recognizes both the soluble and cytoskeletal forms of the enzyme, requiring Ca2+ (EC50 = 20 microM) for the interaction. Other divalent cations such as Zn2+, Mn2+, Cd2+, Co2+, and Ni2+ will substitute for Ca2+, while Mg2+ and Ba2+ will not. The antibody reacts with both the alpha- and beta-subunits on Western blots in a similar Ca2+-dependent fashion but with a lower sensitivity. The affinity of the antibody for the kinase is 0.13 nM determined by displacement of 125I Bolton-Hunter-labeled kinase with unlabeled enzyme. A variety of other proteins including tubulin do not compete for antibody binding. The Mr 30,000 catalytic fragment obtained by proteolysis of either the soluble or the cytoskeletal form of the kinase fails to react with the antibody. Calmodulin and antibody reciprocally potentiate each other's interaction with the enzyme. This is illustrated both by direct binding studies and by a decrease of the Kmapp for calmodulin and an increase in the Vmax for the autophosphorylation reaction of the enzyme. The antibody thus appears to recognize and stabilize a conformation of the kinase which favors calmodulin binding although it does not itself activate the kinase in the absence of calmodulin. Since the Mr 30,000 catalytic fragment of the kinase is not immunoreactive, either the antibody combining site of the kinase must be present in the noncatalytic portion of the protein along with the calmodulin binding site or proteolysis interferes with the putative Ca2+-dependent conformational change.(ABSTRACT TRUNCATED AT 250 WORDS)
LeVine
H
H
Department of Molecular Biology, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
Su
J L
JL
Sahyoun
N E
NE
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin / Q000494:pharmacology*; D002413:Cations, Divalent; D003599:Cytoskeleton / Q000201:enzymology; D000939:Epitopes / Q000032:analysis*; D007074:Immunoglobulin G; D007700:Kinetics; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D008970:Molecular Weight; D007703:Peptidyl-Dipeptidase A / Q000302:isolation & purification; D011487:Protein Conformation; D011494:Protein Kinases / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002147:Calmodulin; D002413:Cations, Divalent; D000939:Epitopes; D007074:Immunoglobulin G; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D007703:Peptidyl-Dipeptidase A; D002118:Calcium