Extensive interactions between troponins C and I. Zero-length cross-linking of troponin I and acetylated troponin C.
Extensive interactions between troponins C and I. Zero-length cross-linking of troponin I and acetylated troponin C.
复制标题
肌钙蛋白 C 和 I 之间广泛的相互作用。肌钙蛋白 I 和乙酰化肌钙蛋白 C 的零长度交联。
DOI：
10.1021/bi00034a029
复制
发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Collins,JH
中科院分区：
文献类型：
--
作者：
Kobayashi,T;Grabarek,Z;Gergely,J;Collins,JH
1995年6月9日收到的修订版Mandarin pt ®摘要：肌钙蛋白C（TnC）和肌钙蛋白I（TnI）之间的相互作用在脊椎动物横纹肌收缩的Ca 2+依赖性调节中起重要作用。早期的研究已经提出TnI的”抑制区”（残基96-116）结合至TnC的非调节性C末端结构域中的包含残基89-100的螺旋区段。随后，基于零长度交联的结果，我们提出Tnl的抑制区也与TnC的N-末端调节结构域相互作用[Leszyk，J.，Grabarek，Z.，German，J.，&柯林斯，J. H.（1990）Biochemistry 29，299-304]。在本研究中，我们乙酰化TnC的九个赖氨酸的6-NH 2基团，以避免可能由TnC的NH 2和COOH基团之间的分子内交联引起的并发症。然后我们用1-乙基-3-[3-（二甲氨基）丙基]碳二亚胺和N-羟基琥珀酰亚胺活化乙酰化TnC（AcTnC）的COOH基团。活化的AcTnC与TnI结合，并且AcTnC中的COOH基团与TnI中的赖氨酸e-NH 2基团之间形成零长度交联。用溴化氰和蛋白酶切割交联的异源二聚体（AcCxI），并通过HPLC分离所得交联肽，然后测序。我们的研究结果表明，AcTnC和TnI之间广泛的交联，涉及TnC的N-末端和C-末端结构域，以及N-末端，C-末端，和cardiovascular脊椎动物横纹肌收缩是由Ca 2+调节，并需要蛋白肌钙蛋白和原肌球蛋白，位于肌动蛋白containingthinfilaments。肌钙蛋白由三个亚基组成：肌钙蛋白C（TnC），1，其结合Ca 2 +; TnI，其抑制肌动球蛋白ATP酶活性;和TnT，其结合原肌球蛋白。Ca ~（2+）依赖的TnC和TnI相互作用的变化是激活收缩的关键事件。据认为，TnC中的Ca 2+依赖性结构变化减弱了TnI与肌动蛋白的相互作用，因此肌动蛋白激活肌球蛋白ATP酶并使肌肉收缩[综述参见Leavis和Germinal（1984），Zot和Potter（1987），Grabarek et al.（1992）和Chalovich（1992）]。兔快骨骼肌的氨基酸序列分析
Revised Manuscript Received June 9, 1995® abstract: Interactions between troponin C (TnC) and troponin I (Tnl) play an important role in the Ca2+-dependent regulation of vertebrate striated muscle contraction. Earlier studies have led to the proposal that the" inhibitory region"(residues 96—116) of Tnl binds to an-helical segment of TnC comprising residues 89—100 in the nonregulatory, C-terminal domain. Subsequently, on the basis of the results of zero-length cross-linking, we suggested that the inhibitory region of Tnl also interacts with the N-terminal, regulatory domain of TnC [Leszyk, J., Grabarek, Z., Gergely, J., & Collins, J. H.(1990) Biochemistry 29, 299—304], In the present study, we acetylated the 6-NH2 groups of the nine lysines of TnC in order to avoid complications which mayarise from intramolecular cross-linking between NH2 and COOH groups of TnC. We then activated the COOH groups of acetylated TnC (AcTnC) with 1-ethyl-3-[3—(dimethylamino) propyl] carbodiimide and V-hydroxysuccinimide. The activated AcTnC was combined with Tnl, and zero-length cross-links were formed between COOH groups in AcTnC and lysine e-NH2 groups in Tnl. The cross-linked heterodimer (AcCxI) was cleaved with CNBr and proteases, and the resulting cross-linked peptides were separated by HPLC and then sequenced. Our results show extensive cross-linking between AcTnC and Tnl, involving both the N-terminal and C-terminal domains of TnC, as well as the N-terminal, C-terminal, and inhibitoryVertebrate striated muscle contraction is regulated by Ca2+ and requires the proteinstroponin and tropomyosin, located on the actin-containingthin filaments. Troponin is composed of three subunits: troponin C (TnC), 1 which binds Ca2+; Tnl, which inhibits actomyosinATPase activity, and TnT, which binds tropomyosin. Ca2+-dependent changes in the interactions between TnC and Tnl are the key events in the activation of contraction. It is thought that Ca2+-dependent structural changes in TnC weaken the interaction of Tnl with actin, So that actin activates myosin ATPase and muscle contracts,[for reviews, see Leavis and Gergely (1984), Zot and Potter (1987), Grabarek et al.(1992), and Chalovich (1992)]. The amino acid sequences of rabbit fast skeletal