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Smooth muscle calponin bound to the biologically active fluorescent calmodulin [2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid-calmodulin] (MIANS.CaM) with a Kd of 80 nM and produced a 3.4-fold fluorescence enhancement. PKC-phosphorylated calponin (1.3 mol of Pi/mol) bound to CaM with approximately 15-fold lower affinity. Calponin inhibited CaM (10 nM) activation of the Ca(2+)-/CaM-activated cyclic nucleotide phosphodiesterase (PDE) with an IC50 of 138 nM. The calponin-CaM interaction was Ca(2+)-dependent: half-maximal binding of calponin to MIANS.CaM occurred at pCa 6.6 with a Hill coefficient of 2.4. Stopped-flow fluorescence kinetic analysis demonstrated that EGTA chelation of Ca2+ from CaM disrupted the MIANS.CaM-calponin complex at a rate of 1 s-1. Calponin bound MIANS.CaM at a rate of (6.0 +/- 1.8) x 10(6) M-1s-1, and melittin and unlabeled brain CaM both disrupted the MIANS.CaM-calponin complex at a rate of 0.3 +/- 0.1 s-1. These studies suggest that calponin binds CaM with 80-fold lower affinity than myosin light-chain kinase and that calponin associates with CaM much slower than it associates with caldesmon or myosin light-chain kinase. The physiological relevance of the CaM-calponin interaction was evaluated by analysis of the effects of Ca(2+)-CaM on (i) the interaction of calponin with actin and (ii) calponin-mediated inhibition of actin-activated myosin MgATPase activity. Ca(2+)-CaM half-maximally inhibited calponin (2 microM) binding to smooth and skeletal muscle actins (9 microM) at 5.4 and 11 microM CaM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

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