Differential recognition of calmodulin-enzyme complexes by a conformation-specific anti-calmodulin monoclonal antibody

Journal Article · Wed Nov 05 04:00:00 EST 1986 · J. Biol. Chem.; (United States)

OSTI ID:6782097

Hansen, R S; Beavo, J A

An anti-calmodulin monoclonal antibody having an absolute requirement for Ca/sup 2 +/ has been produced from mice immunized with a mixture of calmodulin and calmodulin-binding proteins. Radioimmune assays were developed for the determination of its specificity. The epitope for this antibody resides on the COOH-terminal half of the mammalian protein. Plant calmodulin or toponin C had little reactivity. The apparent affinity of the antibody for calmodulin was increased approximately 60-fold in the presence of heart calmodulin-dependent phosphodiesterase. The presence of heart phosphodiesterase in the radioimmune assay greatly enhanced the sensitivity for calmodulin. The intrinsic calmodulin subunit of phosphorylase kinase and calmodulin which was bound to brain phosphodiesterases was also recognized with high affinity by the antibody. In direct binding experiments, most of the calmodulin-binding proteins studied were unreactive with the antibody. This selectivity allowed purification of heart and two brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes on immobilized antibody affinity columns. The data suggest that the binding of ligands to Ca/sup 2 +//calmodulin induce conformation changes in calmodulin which alter reactivity with the anti-calmodulin monoclonal antibody. The differential antibody reactivity toward calmodulin-enzyme complexes indicates that target proteins either induce very different conformations in calmodulin and/or interact with different geometries relative to the antibody binding site. The anti-calmodulin monoclonal antibody should be useful for the purification of other calmodulin-dependent phosphodiesterases as well as isozymes of phosphorylase kinase.

• Research Organization: Univ. of Washington, Seattle
• OSTI ID: 6782097
• Journal Information: J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 261:31; ISSN JBCHA
• Country of Publication: United States
• Language: English

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59 BASIC BIOLOGICAL SCIENCES
62 RADIOLOGY AND NUCLEAR MEDICINE
ALCOHOLS
ANIMALS
ANTIBODIES
BIOCHEMISTRY
BODY
BRAIN
CARBOXYLIC ACIDS
CARDIOVASCULAR SYSTEM
CENTRAL NERVOUS SYSTEM
CHELATING AGENTS
CHEMISTRY
CHROMATOGRAPHY
COMPLEXES
CONFORMATIONAL CHANGES
EGTA
ENZYMES
ESTERASES
GLYCOLS
HEART
HYDROLASES
HYDROXY COMPOUNDS
IMMUNOASSAY
IMMUNOLOGY
ION EXCHANGE CHROMATOGRAPHY
ISOENZYMES
ISOTOPE APPLICATIONS
MAMMALS
MEMBRANE PROTEINS
MICE
MONOCLONAL ANTIBODIES
NERVOUS SYSTEM
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANS
PHOSPHODIESTERASES
PROTEINS
RADIOASSAY
RADIOIMMUNOASSAY
RADIOIMMUNOLOGY
RECEPTORS
RODENTS
SEPARATION PROCESSES
TRACER TECHNIQUES
VERTEBRATES