蜜蜂飞翔肌收缩蛋白的电子显微镜鉴定

自五十年代肌丝滑行模型建立以来,关于脊椎动物骨胳肌的蛋白质成分,肌丝排列以及肌肉收缩时结构变化的研究取得了很大的进展。骨胳肌肌原纤维由粗、细肌丝有规律地排列所组成。对于肌肉收缩蛋白的选择性抽提,专一性抗体标记以及重组肌丝的研究,证实肌球蛋白存在于粗肌丝;肌动蛋白、原肌球蛋白和原宁蛋白存在于细肌丝(Huxley,A.F.,1957;Huxley,H.E;,1972)。昆虫间接飞翔肌的结构和生理特性有许多不同于脊椎动物骨胳肌的特点。蜜蜂飞翔肌肌原纤维虽然也包含有粗、细两     

横纹肌肌原纤维的第三肌丝──肌联蛋白

实验研究证明,在动物横纹肌肌原纤维中,除包含有粗肌丝、细肌丝外,还有纤肌丝的存在,肌联蛋白（肌巨蛋白）是具有挠性的线状蛋白质,分子量为3000 000,长度约为0.9μm,跨越肌原纤维的M－线和Z－线,形成纤肌丝.其生理功能是在粗肌丝装配中具有分子模板作用,并将粗肌丝稳定于肌原纤维肌小节中央以及可参与肌球蛋白活性的调节.     

骨骼肌内质网Ca2+泵转运Ca2+的结构基础

Ca²⁺泵(Ca²⁺-ATPase)是调节细胞内Ca²⁺浓度的重要蛋白质之一. Ca²⁺泵在转运Ca²⁺的过程中经历一系列构象变化. 其中,E1状态为外向的Ca²⁺高亲和状态,E2状态则为内向的Ca²⁺低亲和状态. 目前,骨骼肌内质网Ca²⁺泵转运Ca²⁺过程中的几个中间状态,包括E1-2Ca²⁺,E1-ATP,E1-P-ADP,E2-Pi和E2状态的三维晶体结构已经解析. 介绍这几种状态的晶体结构,并分析Ca²⁺泵在执行功能过程中结构与功能的关系.     

螯虾腹屈肌的超微结构

用电子显微镜观察,发现螯虾(Procambarus clarkii)腹屈肌浅层(慢肌,tonic fiber)肌纤维和深层(快肌,twitch fiber)肌纤维的超微结构存在显著差异。浅层腹屈肌肌原纤维有相对长的肌节(5—10μm),肌原纤维直径较大,每根粗肌丝周围有9—12根细肌丝环绕,细肌丝与粗肌丝数量比约为6∶1;深层腹屈肌有相对短的肌节(3—4.5μm),肌原纤维直径较小,每根粗肌丝周围有6根细肌丝环绕,细肌丝与粗肌丝数量比为3∶1。以上结果提示,在决定螯虾腹屈肌收缩速度方面,可能肌原纤维直径大小比肌节长度更为重要。细、粗肌丝排列方式也可能与收缩速度有关。这与脊椎动物骨骼肌的情况是不同的。     

钙调蛋白结合蛋白,类肌钙蛋白和脊椎动物平滑肌收缩的调节

平滑肌细胞包含有粗肌丝、细肌丝和中等纤维。一般认为Ｃａ＾２＋／ＣａＭ依赖性的肌球蛋白磷酸化作用是平滑肌收缩的重要调控环节。然而,新近在平滑肌中发现钙调蛋白结合蛋白和类肌钙蛋白,它们也参与平滑肌收缩的调节。     

钙的光释放技术及其在细胞研究中的应用

Ca²⁺的光释放技术通过光解作用使预先引入细胞内的光敏感性螯合剂对Ca²⁺的亲和性改变,从而实现对细胞内游离钙离子浓度的调控,有助于阐明Ca²⁺作为第二信使对电兴奋性、肌肉收缩、神经分泌等细胞功能的调制作用.     

脉冲电场对粒细胞内Ca2+浓度影响的动态变化

利用激光共聚焦扫描显微镜和装有CCD系统的荧光显微镜 ,研究在单脉冲电场作用下经fluo 3/AM标记的鸡胚小脑粒细胞内自由Ca²⁺浓度 ( [Ca ²⁺]_i)的动态变化过程 .结果表明 :在单个电脉冲作用下 ,细胞内Ca²⁺浓度立刻升高并达到其最大值 .Ca²⁺浓度升高的幅度以及升高的速率具有电场强度的依赖性 .当细胞外Ca²⁺被过量的EGTA络合或细胞膜上的Ca²⁺通道被La ³⁺堵塞后 ,细胞内的Ca²⁺浓度仍然升高 .细胞内不同区域的Ca²⁺浓度同时升高 ;两极内的Ca²⁺浓度早于胞体的Ca²⁺浓度达到最大值并迅速恢复 .     

豚鼠精子质膜Ca2+ -ATPase活性与精子顶体反应关系的研究

用生化测定法首次证实豚鼠精子质膜Ca²⁺-ATPase活性在精子获能和顶体反应过程中显著下降.Ca²⁺-ATPase抑制剂利尿酸(ethacrynic acid)抑制质膜Ca²⁺-ATPase活性,但钙调素(50μg/mL)的拮抗剂三氟拉嗪(TFP,200～500μmol/L)对该酶活性没有影响,说明钙调素不直接参与精子依赖于ATP的Ca²⁺的主动泵出.但钙调素与精子的Ca²⁺内流有关,钙调素拮抗剂TFP显著促进精子顶体反应和精子对Ca²⁺的摄入.Ca²⁺-ATPase抑制剂栎皮酮(quercetin)、原钒酸钠(sodiumorthovandate)、利尿磺胺(furosemide)和利尿酸均显著促进豚鼠精子的顶体反应,但却抑制精子对Ca²⁺的摄入,这无法用它们对质膜Ca²⁺-ATPase活性的抑制作用解释.推测这可能是由于Ca²⁺-ATPase抑制剂在抑制质膜Ca²⁺-ATPase活性的同时也抑制了顶体外膜或线粒体外膜上的该酶的活性,导致Ca²⁺在细胞质内的积累,进而通过负反馈机制抑制Ca²⁺进一步内流所致.另外,Ca²⁺-ATPase抑制剂对糖酵解的抑制作用也可能是Ca²⁺在细胞质中积累和抑制精子Ca²⁺摄入的原因.     

猪脑突触体质膜(Ca2+-Mg2+)-ATPase的分离纯化与性质

以猪脑为材料,经匀浆、差速离心、蔗糖密度梯度离心分离突触体. 低渗破膜得到突触体膜. Triton X-100增溶后,经钙调蛋白亲和层析可得去脂的质膜Ca²⁺-ATPase. 用大体积亲和柱和大体积低Ca²⁺淋洗液淋洗,可得产率、纯度和活性均较高的质膜Ca²⁺-ATPase. 与大豆磷脂保温后,去脂的Ca²⁺-ATPase的水解活力可恢复达3.32 μmol/(mg·min).SDS-聚丙烯酰胺凝胶电泳银染显示单一蛋白质带,分子质量约为140 ku,纯度在90%以上. 不同Ca²⁺浓度明显影响酶的活力.     

钙调素结合蛋白参与调控植物逆境胁迫的研究进展

Ca²⁺是植物体内重要的第二信使,当植物受到各种环境刺激时,细胞内的Ca²⁺浓度瞬间产生变化,并被Ca²⁺信号效应器识别,通过与下游的靶蛋白结合并调节其活性,参与调控植物各种生理活动。钙调素结合蛋白以依赖Ca²⁺或不依赖Ca²⁺的方式结合钙调素。对目前已经鉴定的植物钙调素结合蛋白结构特点进行了综述,并着重介绍了钙调素结合蛋白是如何参与调节植物对生物胁迫和非生物胁迫的反应,为提高作物抗病抗逆能力研究提供理论基础。     

Structural proteins in the myofilaments and regulation of contraction in vertebrate smooth muscle.

The contractile systems of vertebrate smooth and striated muscles are compared. Smooth muscles contain relatively large amounts of actin and tropomyosin organized into thin filaments, and smaller amounts of myosin in the form of thick filaments. The protein contents are consistent with observed thin:thick filament ratios of about 15-18:1 in smooth compared to 2:1 in striated muscle. The basic characteristics of both types of contractile proteins are similar; but there are a variety of quantitative differences in protein structures, enzymatic activities and filament stabilities. Biochemical and X-ray diffraction data generally support recent ultrastructural evidence concerning the organization of the myofilaments in smooth muscle, although a basic contractile unit comparable to the sarcomere in striated muscle has not been discerned. Myofilament interactions and contraction in smooth muscle are controlled by changes in the Ca2+ concentration. Recent evidence suggests the Ca2+-binding regulatory site is associated with the myosin in vertebrate smooth muscle (as in a variety of invertebrate muscles), rather than with troponin which is the regulatory protein associated with the thin filament in vertebrate striated muscle.     

Signal transduction and protein phosphorylation in smooth muscle contraction

Smooth muscles are important constituents of vertebrate organisms that provide for contractile activity of internal organs and blood vessels. Basic molecular mechanism of both smooth and striated muscle contractility is the force-producing ATP-dependent interaction of the major contractile proteins, actin and myosin II molecular motor, activated upon elevation of the free intracellular Ca²⁺ concentration ([Ca²⁺]_i). However, whereas striated muscles display a proportionality of generated force to the [Ca²⁺]_i level, smooth muscles feature molecular mechanisms that modulate sensitivity of contractile machinery to [Ca²⁺]_i. Phosphorylation of proteins that regulate functional activity of actomyosin plays an essential role in these modulatory mechanisms. This provides an ability for smooth muscle to contract and maintain tension within a broad range of [Ca²⁺]_i and with a low energy cost, unavailable to a striated muscle. Detailed exploration of these mechanisms is required to understand the molecular organization and functioning of vertebrate contractile systems and for development of novel advances for treating cardiovascular and many other disorders. This review summarizes the currently known and hypothetical mechanisms involved in regulation of smooth muscle Ca²⁺-sensitivity with a special reference to phosphorylation of regulatory proteins of the contractile machinery as a means to modulate their activity.     

Preserved Close Linkage Between the Genes Encoding Troponin I and Troponin T, Reflecting an Evolution of Adapter Proteins Coupling the Ca2+ Signaling of Contractility

Ca²⁺-regulated motility is essential to numerous cellular functions, including muscle contraction. Systems with troponin C, myosin light chain, or calmodulin as the Ca²⁺ receptor have evolved in striated muscle and other types of cells to transduce the cytoplasm Ca²⁺ signals into allosteric conformational changes of contractile proteins. While these Ca²⁺ receptors are homologous proteins, their coupling to the responding elements is quite different in various cell types. The Ca²⁺ regulatory system in vertebrate striated muscle represents a highly specialized such signal transduction pathway consisting of the troponin complex and tropomyosin associated with the actin filament. To understand the molecular mechanism in the Ca²⁺ regulation of muscle contraction and cell motility, we have revealed a preserved ancestral close linkage between the genes encoding two of the troponin subunits, troponin I and troponin T, in the genome of mouse. The data suggest that the troponin I and troponin T genes may have originated from a single locus and evolved in parallel to encode a striated muscle-specific adapter to couple the Ca²⁺ receptor, troponin C, to the actin–myosin contractile machinery. This hypothesis views the three troponin subunits as two structure–function domains: the Ca²⁺ receptor and the signal transducing adapter. This model may help to further our understanding of the Ca²⁺ regulation of muscle contraction and the structure–function relationship of other potential adapter proteins which are converged to constitute the Ca²⁺ signal transduction pathways governing nonmuscle cell motility. Received: 15 April 1999 / Accepted: 15 July 1999     

Ultrastructural diversity in motor units of crustacean stomach muscles

The physiological and ultrastructural properties of muscle fiber.s comprising three motor units in the gastric mill of blue crabs are described. In their contractile properties muscle fibers in all motor units are similar and resemble the slow type fibers in crustacean limb muscles. The majority of fibers generate large excitatory post-synaptic potentials which do not facilitate strongly. Structurally two types of fibers are found. The one type has long sarcomeres (greater than 6 mum), thin to thick myofilament ratios of 5-6:1 and diads located near the ends of the A-band. The other type has shorter sarcomeres (less than 6 mum), thin to thick myofilament ratios of 3:1 and diads located at mid sarcomere level. Both types of fibers occur within a single motor unit and this differs from the vertebrate situation. Furthermore, the finding of fibers with a low thin to thick myofilament ratio of 3:1 demonstrates that they are not exclusive to fast type crustacean muscle but also occur in slow stomach muscles.     

Interaction of cardiac troponin with cardiotonic drugs: a structural perspective

Over the 40 years since its discovery, many studies have focused on understanding the role of troponin as a myofilament based molecular switch in regulating the Ca²⁺-dependent activation of striated muscle contraction. Recently, studies have explored the role of cardiac troponin as a target for cardiotonic agents. These drugs are clinically useful for treating heart failure, a condition in which the heart is no longer able to pump enough blood to other organs. These agents act via a mechanism that modulates the Ca²⁺-sensitivity of troponin; such a mode of action is therapeutically desirable because intracellular Ca²⁺ concentration is not perturbed, preserving the regulation of other Ca²⁺-based signaling pathways. This review describes molecular details of the interaction of cardiac troponin with a variety of cardiotonic drugs. We present recent structural work that has identified the docking sites of several cardiotonic drugs in the troponin C-troponin I interface and discuss their relevance in the design of troponin based drugs for the treatment of heart disease.     

Role of regulatory proteins (troponin-tropomyosin) in pathologic states

In vertebrate striated muscle, troponon-tropomyosin is responsible, in part, not only for transducing the effect of calcium on contractile protein activation, but also for inhibiting actin and myosin interaction when calcium is absent. The regulatory troponin (Tn) complex displays several molecular and calcium binding variations in cardiac muscles of different species and undergoes genetic changes with development and in various pathologic states.Extensive reviews on the role of tropomyosin (Tm) and Tn in the regulation of striated muscle contraction have been published describing the molecular mechanisms involved in contractile protein regulation. In our studies, we have found an increase in Mg²⁺ ATPase activity in cardiac myofibrils from dystrophic hamsters and in rats with chronic coronary artery narrowing. The abnormalities in myofibrillar ATPase activity from cardiomyopathic hamsters were largely corrected by recombining the preparations with a TnTm, complex isolated from normal hamsters indicating that the TnTm, may play a major role in altered myocardial function. We have also observed down regulation of Ca²⁺ Mg²⁺ ATPase of myofibrils from hypertrophic guinea pig hearts, myocardial infarcted rats and diabetic-hypertensive rat hearts. In myosin from diabetic rats, this abnormality was substantially corrected by adding troponin-tropomyosin complex from control hearts. All of these disease models are associated with decreased ATPase activities of pure myosin and in the case of rat and hamster models, shifts of myosin, heavy chain from alpha to beta predominate.In summary, there are three main troponin subunit components which might alter myofibrillar function however, very few direct links of molecular alterations in the regulatory proteins to physiologic and pathologic function have been demonstrated so far.     

Troponin T isoforms and posttranscriptional modifications: Evolution, regulation and function

Troponin-mediated Ca²⁺-regulation governs the actin-activated myosin motor function which powers striated (skeletal and cardiac) muscle contraction. This review focuses on the structure–function relationship of troponin T, one of the three protein subunits of the troponin complex. Molecular evolution, gene regulation, alternative RNA splicing, and posttranslational modifications of troponin T isoforms in skeletal and cardiac muscles are summarized with emphases on recent research progresses. The physiological and pathophysiological significances of the structural diversity and regulation of troponin T are discussed for impacts on striated muscle function and adaptation in health and diseases.     

Sarcomere lattice geometry influences cooperative myosin binding in muscle

In muscle, force emerges from myosin binding with actin (forming a cross-bridge). This actomyosin binding depends upon myofilament geometry, kinetics of thin-filament Ca²⁺ activation, and kinetics of cross-bridge cycling. Binding occurs within a compliant network of protein filaments where there is mechanical coupling between myosins along the thick-filament backbone and between actin monomers along the thin filament. Such mechanical coupling precludes using ordinary differential equation models when examining the effects of lattice geometry, kinetics, or compliance on force production. This study uses two stochastically driven, spatially explicit models to predict levels of cross-bridge binding, force, thin-filament Ca²⁺ activation, and ATP utilization. One model incorporates the 2-to-1 ratio of thin to thick filaments of vertebrate striated muscle (multi-filament model), while the other comprises only one thick and one thin filament (two-filament model). Simulations comparing these models show that the multi-filament predictions of force, fractional cross-bridge binding, and cross-bridge turnover are more consistent with published experimental values. Furthermore, the values predicted by the multi-filament model are greater than those values predicted by the two-filament model. These increases are larger than the relative increase of potential inter-filament interactions in the multi-filament model versus the two-filament model. This amplification of coordinated cross-bridge binding and cycling indicates a mechanism of cooperativity that depends on sarcomere lattice geometry, specifically the ratio and arrangement of myofilaments.     

Cardiomyopathic troponin mutations predominantly occur at its interface with actin and tropomyosin

Reversible Ca²⁺ binding to troponin is the primary on-off switch of the contractile apparatus of striated muscles, including the heart. Dominant missense mutations in human cardiac troponin genes are among the causes of hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy. Structural understanding of troponin action has recently advanced considerably via electron microscopy and molecular dynamics studies of the thin filament. As a result, it is now possible to examine cardiomyopathy-inducing troponin mutations in thin-filament structural context, and from that to seek new insight into pathogenesis and into the troponin regulatory mechanism. We compiled from consortium reports a representative set of troponin mutation sites whose pathogenicity was determined using standardized clinical genetics criteria. Another set of sites, apparently tolerant of amino acid substitutions, was compiled from the gnomAD v2 database. Pathogenic substitutions occurred predominantly in the areas of troponin that contact actin or tropomyosin, including, but not limited to, two regions of newly proposed structure and long-known implication in cardiomyopathy: the C-terminal third of troponin I and a part of the troponin T N terminus. The pathogenic mutations were located in troponin regions that prevent contraction under low Ca²⁺ concentration conditions. These regions contribute to Ca²⁺-regulated steric hindrance of myosin by the combined effects of troponin and tropomyosin. Loss-of-function mutations within these parts of troponin result in loss of inhibition, consistent with the hypercontractile phenotype characteristic of HCM. Notably, pathogenic mutations are absent in our dataset from the Ca²⁺-binding, activation-producing troponin C (TnC) N-lobe, which controls contraction by a multi-faceted mechanism. Apparently benign mutations are also diminished in the TnC N-lobe, suggesting mutations are poorly tolerated in that critical domain.     

螯虾横纹肌粗肌丝超微结构的计算机图象分析

本文在微机ＡＳＴ／３８６和真彩色图形采集卡ＣＡ－５４０构成的趁科象处理系统上开发了一个软件,包括用于对动物横纹肌粗肌丝的超微结构进行分析的专用模块,一般图象处理系统的通用模块和文件管理模块三大部分。本系统对动物横纹肌粗肌丝的亚结构从旋转功率谱的角度分析了其对称性的存在,并运用图象的迭加,旋转平衡和旋转滤波等手段来处理,显示其对称性;同时,对存在的不对称结构也作了相应的分析处理。利用本系统,对螯虾的