Fluorescence polarization assay for calmodulin binding to plasma membrane Ca2+-ATPase: dependence on enzyme and Ca2+ concentrations.
Calmodulin (CaM) is a Ca2+ signaling protein that binds to a wide variety of target proteins, and it is important to establish methods for rapid characterization of these interactions. Here we report the use of fluorescence polarization (FP) to measure the Kd for the interaction of CaM with the plasma membrane Ca2+-ATPase (PMCA), a Ca2+ pump regulated by binding of CaM. Previous assays of PMCA-CaM interactions were indirect, based on activity or kinetics measurements. We also investigated the Ca2+ dependence of CaM binding to PMCA. FP assays directly detect CaM-target interactions and are rapid, sensitive, and suitable for high-throughput screening assay formats. Values for the dissociation constant K(d) in the nanomolar range are readily measured. We measured the changes in anisotropy of CaM labeled with Oregon Green 488 on titration with PMCA, yielding a K(d) value of CaM with PMCA (5.8 +/- 0.5 nM) consistent with previous indirect measurements. We also report the binding affinity of CaM with oxidatively modified PMCA (K(d) = 9.8 +/- 2.0 nM), indicating that the previously reported loss in CaM-stimulated activity for oxidatively modified PMCA is not a result of reduced CaM binding. The Ca2+ dependence follows a simple Hill plot demonstrating cooperative binding of Ca2+ to the binding sites in CaM.
Liyanage
Mangala Roshan
MR
Department of Chemistry, University of Kansas, Lawrence, KS 66045, USA.
Zaidi
Asma
A
Johnson
Carey K
CK
D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000737:chemistry*; D002264:Carboxylic Acids / Q000737:chemistry; D005454:Fluorescence Polarization / Q000379:methods*; D007700:Kinetics; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects
D002147:Calmodulin; D002264:Carboxylic Acids; C436864:Oregon Green 488 carboxylic acid; D053499:Plasma Membrane Calcium-Transporting ATPases; D002118:Calcium