title: Calponin-calmodulin interaction: properties and effects on smooth and skeletal muscle actin binding and actomyosin ATPases.
abstract: 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)
authors.affiliation: MRC Signal Transduction Group, University of Calgary, Alberta, Canada.
authors.forename: S J
authors.initials: SJ
authors.lastname: Winder
authors.forename: M P
authors.initials: MP
authors.lastname: Walsh
authors.forename: C
authors.initials: C
authors.lastname: Vasulka
authors.forename: J D
authors.initials: JD
authors.lastname: Johnson
mesh_terms: D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000205:Actomyosin / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D002417:Cattle; D002645:Chickens; D004533:Egtazic Acid / Q000737:chemistry; D066298:In Vitro Techniques; D007700:Kinetics; D008555:Melitten / Q000494:pharmacology; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D009119:Muscle Contraction; D009130:Muscle, Smooth / Q000378:metabolism; D009132:Muscles / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D011817:Rabbits; D000096985:Calponins
chemical_list: D015106:3',5'-Cyclic-GMP Phosphodiesterases; D000199:Actins; D000205:Actomyosin; D002118:Calcium; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D004533:Egtazic Acid; D008555:Melitten; D008840:Microfilament Proteins; D009219:Myosin-Light-Chain Kinase; D009218:Myosins; D000096985:Calponins