一种提取和纯化肌钙蛋白C的新方法

肌钙蛋白Ⅰ是肌钙蛋白的一个亚基，它有３种异构体：快、慢骨骼肌肌钙蛋白Ⅰ和心肌肌钙蛋白Ⅰ。由于心肌肌钙蛋白Ⅰ具有极强的心脏特异性，因此引起众多学者的兴趣，研究检测患者血清内心肌肌钙蛋白Ⅰ的含量，来诊断急性心肌梗塞。利用肌钙蛋白Ｃ和肌钙蛋白Ⅰ的亲和层析来提取和纯化肌钙蛋白Ⅰ。建立了一种简单的肌钙蛋白Ｃ的提取和纯化方法，从兔肌１００ｇ中可提取肌钙蛋白Ｃ ７５ｍｇ，不仅高于以往研究结果（６０ｍｇ／１００ｇ兔肌），而且使实验周期缩短和实验操作大为简化。     

Structure of co-crystals of tropomyosin and troponin

Troponin, a Ca2+-sensitive complex, regulates the motions of tropomyosin on the thin filaments in many muscles. It has three subunits, each with a different architecture and function: TnC binds Ca2+; TnI binds to actin and inhibits contraction; and TnT binds one complex to each tropomyosin molecule. The troponin complex has an elongated shape with TnC and TnI forming a globular 'head' region and TnT a long (approximately 160 A) 'tail'. TnT binds to two widely separated regions of tropomyosin: the head region of the complex is near Cys 190 of tropomyosin and the tail region is near the overlapping joint that links the tropomyosin molecules into filaments. Here we report the X-ray structure determination at 17 A resolution of glutaraldehyde-treated tropomyosin crystals in which native troponin complex or fragments of TnT have been bound. Our results show that the amino-terminal tail end of TnT spans the head-to-tail joint of the tropomyosin filaments, and that the 'head' region of the whole troponin complex binds approximately 200 A away near residues 150-180 of the tropomyosin molecule.     

Probing the calcium-induced conformational transition of troponin C with site-directed mutants

The contraction of skeletal muscle is regulated by calcium binding to troponin C (TnC). TnC consists of two spatially independent domains, each of which contains two metal ion binding sites. Calcium binding to the regulatory sites of the N-terminal domain triggers muscle contraction by a series of conformational changes. Site-directed mutagenesis offers a means of elucidating the links in this signal path between TnC and actin-myosin crossbridges. Such mapping is possible if the mutants shift the equilibrium between 'on' and 'off' states of the regulatory complex while maintaining the coupling between calcium binding and tension development. Candidate amino-acid residues for yielding this information would be in positions remote from the calcium-binding sites and from the site of development of tension. Analysis of the crystal structure of TnC and of the model of the calcium-activated molecule has enabled us to identify two such residues: Glu 57 and Glu 88. In separate experiments we have replaced each of these residues by lysines. The resulting reduction in calcium affinity indicates that these residues have a long-range effect on calcium binding. This result may reflect the formation of a salt bridge between positions 57 and 88 that is not present in the native molecule. Moreover, the level of tension recovery when the mutants are incorporated into muscle suggests that the interaction between TnC and other muscle components has also been altered. Thus, these residues may participate in the contraction signal transmission.     

Inhibition of mutant troponin C activity by an intra-domain disulphide bond

Triggering of contraction in striated muscles involves a conformational transition in the N-terminal domain of troponin C, the calcium-binding component of thin filaments. We have designed a mutant troponin C in which the key conformational transition and the calcium-regulatory activity are reversibly blocked by the formation of a disulphide bridge. Our results may be applicable to other proteins of the same family of calcium-binding proteins.     

Comparison of amino acid sequence of troponin I from different striated muscles.

The sequence of troponin I from fast and slow skeletal and cardiac muscle shows strong homology in the region which binds to actin and is responsible for inhibition of the actomyosin AT Pase. More differences are found in the N-terminal region which binds to troponin C.     

Distribution of polymorphic forms of troponin components and tropomyosin in skeletal muscle.

Specific antibodies have been used to show that in adult skeletal muscle the slow and fast forms of the components of the troponin complex are located in type I and type II fibres respectively. alpha-Tropomyosin is restricted to type II cells. During development, as a result of changes in innervation and in certain diseased stages, both the slow and fast polymorphic forms of the troponin components are present in the same cell.     

Structure of the core domain of human cardiac troponin in the Ca(2+)-saturated form

Troponin is essential in Ca(2+) regulation of skeletal and cardiac muscle contraction. It consists of three subunits (TnT, TnC and TnI) and, together with tropomyosin, is located on the actin filament. Here we present crystal structures of the core domains (relative molecular mass of 46,000 and 52,000) of human cardiac troponin in the Ca(2+)-saturated form. Analysis of the four-molecule structures reveals that the core domain is further divided into structurally distinct subdomains that are connected by flexible linkers, making the entire molecule highly flexible. The alpha-helical coiled-coil formed between TnT and TnI is integrated in a rigid and asymmetric structure (about 80 angstrom long), the IT arm, which bridges putative tropomyosin-anchoring regions. The structures of the troponin ternary complex imply that Ca(2+) binding to the regulatory site of TnC removes the carboxy-terminal portion of TnI from actin, thereby altering the mobility and/or flexibility of troponin and tropomyosin on the actin filament.     

小儿肺炎并发心肌损伤心肌钙蛋白I的检测意义(附46例分析)

目的:探讨小儿肺炎并发心肌损伤心肌钙蛋白I的检测意义。方法:采用全自动化学免疫系统检测4 6例肺炎患儿心肌钙蛋白I(CTnI)、肌酸激酶同功酶(CKMB) ,并与2 6例正常小儿进行对照比较。结果:肺炎患儿与正常小儿CTnI、CKMB值比较均有非常显著性差异(P <0 0 1) ,且肺炎越重,越易造成心肌损害,二者值越高。两两比较,除重症肺炎组与普通肺炎组CKMB差异无显著性外(P >0 0 5 ) ,其余均有非常显著性差异(P <0 0 1)。结论:CTnI可作为监测小儿肺炎并发心肌损伤的重要标志物。