Cameron L C, Carvalho R N, Araujo J R, Santos A C, Tauhata S B, Larson R E, Sorenson M M
Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-590 RJ, Brazil.
Arch Biochem Biophys. 1998 Jul 1;355(1):35-42. doi: 10.1006/abbi.1998.0700.
Myosin V isolated from chick brain (BM V) is a multimeric protein of about 640 kDa consisting of two intertwined heavy chains of 212 kDa and multiple light chains of 10 to 20 kDa. A distinctive feature of the heavy chain is an extended neck region with six consensus IQ sites for the binding of calmodulin (CaM) and myosin light chains. The actin-activated MgATPase has been shown to require >/=1 microM Ca2+ for full activity, and evidence points to a myosin-linked regulatory system where the CaM light chains participate as modulators for the Ca2+ signal. Still, the precise mechanism of Ca2+ regulation remains unknown. In the present study we have used the intrinsic tryptophan fluorescence of native BM V to monitor conformational changes of BM V induced by Ca2+, and we relate these changes to CaM dissociation from the BM V molecule. The fluorescence intensity decreases approximately 17% upon addition of sub-micromolar concentrations of Ca2+ (K0.5 = 0.038 microM). This decrease in fluorescence, which is dominated by a conformational change in the heavy chain, can be reversed by addition of 1, 2-di(2-aminoethoxy)ethane-N,N,N',N'tetraacetic acid (EGTA) followed by an excess of CaM, but not by addition of EGTA alone. Gel filtration of native BM V using HPLC shows that CaM is partially dissociated from the heavy chain in EGTA and dissociates further upon addition of sub-micromolar concentrations of Ca2+. These observations suggest that the affinity of CaM for at least one of the IQ sites on the BM V heavy chain decreases with Ca2+ and that the Ca2+ concentration required for this effect is lower than that needed to activate acto-BM V. Using a cosedimentation assay in the presence of actin, we also observe partial dissociation of CaM when Ca2+ is absent, but now the addition of Ca2+ has a biphasic effect: sub-micromolar Ca2+ concentrations lead to reassociation of CaM with the heavy chain, followed by dissociation when Ca2+ exceeds 5-10 microM. Thus, the binding of CaM to BM V is affected by both actin and Ca2+.
从鸡脑中分离出的肌球蛋白V（BM V）是一种约640 kDa的多聚体蛋白，由两条212 kDa的相互缠绕的重链和多条10至20 kDa的轻链组成。重链的一个显著特征是有一个延伸的颈部区域，带有六个用于结合钙调蛋白（CaM）和肌球蛋白轻链的共有IQ基序。已表明肌动蛋白激活的MgATP酶需要≥1 microM的Ca2+才能达到完全活性，并且有证据表明存在一种肌球蛋白相关的调节系统，其中CaM轻链作为Ca2+信号的调节剂发挥作用。然而，Ca2+调节的确切机制仍然未知。在本研究中，我们利用天然BM V的内在色氨酸荧光来监测Ca2+诱导的BM V构象变化，并将这些变化与CaM从BM V分子上的解离联系起来。加入亚微摩尔浓度的Ca2+（K0.5 = 0.038 microM）后，荧光强度大约降低17%。这种荧光强度的降低主要由重链的构象变化引起，加入1, 2 - 二（2 - 氨基乙氧基）乙烷 - N, N, N', N' - 四乙酸（EGTA），随后加入过量的CaM可以使其逆转，但仅加入EGTA则不能。使用高效液相色谱对天然BM V进行凝胶过滤显示，在EGTA中CaM会从重链上部分解离，加入亚微摩尔浓度的Ca2+后会进一步解离。这些观察结果表明，Ca2+会降低CaM对BM V重链上至少一个IQ位点的亲和力，并且产生这种效应所需的Ca2+浓度低于激活肌动蛋白 - BM V所需的浓度。在有肌动蛋白存在的情况下使用共沉降分析，我们还观察到在没有Ca2+时CaM会部分解离，但此时加入Ca2+会产生双相效应：亚微摩尔浓度的Ca2+会导致CaM与重链重新结合，当Ca2+超过5 - 10 microM时则会解离。因此，CaM与BM V的结合受到肌动蛋白和Ca2+两者的影响。
