Winder S J, Walsh M P, Vasulka C, Johnson J D
MRC Signal Transduction Group, University of Calgary, Alberta, Canada.
Biochemistry. 1993 Dec 7;32(48):13327-33. doi: 10.1021/bi00211a046.
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)
平滑肌钙调蛋白与具有生物活性的荧光钙调蛋白2-(4'-马来酰亚胺基苯胺基)萘-6-磺酸-钙调蛋白结合，解离常数为80 nM，并使荧光增强3.4倍。蛋白激酶C磷酸化的钙调蛋白(1.3摩尔磷酸根/摩尔)与钙调蛋白结合的亲和力约低15倍。钙调蛋白抑制钙调蛋白(10 nM)对Ca(2+)/钙调蛋白激活的环核苷酸磷酸二酯酶(PDE)的激活作用，半数抑制浓度为138 nM。钙调蛋白与钙调蛋白的相互作用依赖于Ca(2+)：钙调蛋白与MIANS.CaM的半数最大结合发生在pCa 6.6，希尔系数为2.4。停流荧光动力学分析表明，EGTA从钙调蛋白中螯合Ca2+以1 s-1的速率破坏MIANS.CaM-钙调蛋白复合物。钙调蛋白以(6.0±1.8)×10(6) M-1s-1的速率结合MIANS.CaM，蜂毒肽和未标记的脑钙调蛋白均以0.3±0.1 s-1的速率破坏MIANS.CaM-钙调蛋白复合物。这些研究表明，钙调蛋白与钙调蛋白结合的亲和力比肌球蛋白轻链激酶低80倍，且钙调蛋白与钙调蛋白结合的速度比与钙结合蛋白或肌球蛋白轻链激酶结合的速度慢得多。通过分析Ca(2+)-钙调蛋白对(i)钙调蛋白与肌动蛋白的相互作用和(ii)钙调蛋白介导的对肌动蛋白激活的肌球蛋白MgATPase活性的抑制作用，评估了钙调蛋白-钙调蛋白相互作用的生理相关性。Ca(2+)-钙调蛋白分别在5.4和11 microM钙调蛋白时半数最大程度地抑制钙调蛋白(2 microM)与平滑肌和骨骼肌肌动蛋白(9 microM)的结合。(摘要截短于250字)
