Effects of calmodulin antagonists on antibody binding to calmodulin. Distinct conformers of calmodulin induced by the binding of drugs.
An indirect enzyme-linked immunosorbent assay has been used to study the interactions between calmodulin and two calmodulin antagonists, trifluoperazine and a neuropeptide isolated from the hypothalamus. The binding of a monospecific anti-calmodulin antibody, raised in rabbit against dinitrophenylated calmodulin, to calmodulin was tested at various concentrations of these drugs under equilibrium conditions. Trifluoperazine at low concentrations stimulated, but at relatively high concentrations inhibited, immunocomplex formation. The neuropeptide displaced the antibody from calmodulin at nanomolar concentrations. Enzyme-linked immunosorbent assays were also carried out with the large tryptic fragments of calmodulin. The results suggest that (i) the C-terminal fragment binds the antibody with an affinity which is comparable with that of intact calmodulin; (ii) the neuropeptide can form complexes with both N- and C-terminal fragments, but with two orders of magnitude less activity in case of the C-terminal fragment; and (iii) trifluorperazine does not stimulate antibody binding to the C-terminal fragment. Therefore the tertiary structure of calmodulin must be intact to ensure long-distance interactions between the binding sites of trifluoperazine, the neuropeptide and the antibody. These interactions may produce distinct conformers of calmodulin which may exhibit altered potency, not only for antibody binding but also for stimulation/inhibition of target enzymes.
Orosz
F
F
Institute of Enzymology, Hungarian Academy of Sciences, Budapest.
Liliom
K
K
Barkhudaryan
N A
NA
Horváth
L
L
Ovádi
J
J
D000818:Animals; D001666:Binding Sites, Antibody; D001921:Brain / Q000378:metabolism; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D007031:Hypothalamus / Q000378:metabolism; D007700:Kinetics; D009479:Neuropeptides / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D014268:Trifluoperazine / Q000378:metabolism*
D002147:Calmodulin; D009479:Neuropeptides; D010446:Peptide Fragments; D014268:Trifluoperazine