螫虾腹屈肌的肌球蛋白-副肌球蛋白丝

利用甘油梯度离心方法分离和纯化螯虾腹屈肌粗肌丝,电子显微镜照片显示粗肌丝上有数条纵行条纹,指示其可能由数根亚丝所组成。粗肌丝的 SDS-聚丙烯酰胺凝胶电泳表明其含有肌球蛋白和副肌球蛋白,肌球蛋白仅包含有二种轻链。副肌球蛋白类晶体呈针状,具有14.5nm 和72.5nm 的横纹周期。实验结果表明,螯虾腹屈肌粗肌丝是肌球蛋白-副肌球蛋白丝。     

原肌球蛋白和副肌球蛋白晶体的电子显微镜观察

(一)自猪胃、猪膀胱平滑肌、鵝肫、鴨肫平滑肌、河蟹横紋肌和河蚌閉壳肌及斧足肌制备出原肌球蛋白的針状晶体,自河蚌的两种肌肉制备出副肌球蛋白的針状晶体,分別在电子显微鏡下观察其横紋結构与細节。(二)各种原肌球蛋白的針状晶体与河蚌的副肌球蛋白晶体相类,一般都不是具有三維規律性的真正晶体,而是具有横紋結构和整齐外形的纤維聚集物——类晶体。(三)各种原肌球蛋白类晶体的横紋周期,大都属于n×200A(n=1,2,…)数系,周期或为400A、或为200A。其具有400A周期的,有时伴有200A的細节。这些共同的特征与免原肌球蛋白真正晶体的三維网状周期相同。(四)鴨肫和鹅肫原肌球蛋白的类晶体中,除了400A,200A的横紋外,还出現800A(n=4)的周期,显示其分子长度可能至少不低于800A。(五)河蚌斧足肌和閉壳肌副肌球蛋白的类晶体,同海蚌一样,都具有周期約700A的横紋結构。条件适宜时,还可以在每一周期之間观察到4条横紋,細节間距为140A。(六)河蚌閉壳肌原肌球蛋白的类晶体中,也有接近800A的周期。(七)在高盐条件下所获得的河蚌斧足肌和閉壳肌原肌球蛋白的类晶体,与上述一般低盐条件下的类晶体不同,細节間距与副肌球蛋白的相同,为140A,而非200A。(八)根据电子显微鏡下观察到的类晶体横紋周期和細节間距,可以初步推論:一般而言,原肌球蛋白和副肌球蛋白是两类而非一类的蛋白貭。但在两种蛋白貭共存的同一肌肉,如河蚌閉壳肌或斧足肌之中,它們之間还可能存在着一定的亲属关系。     

对虾原肌球蛋白晶体和纤维的电子显微鏡观察

(1)利用锇酸、磷钨酸染色,复型和超薄切片等技术在电子显微镜下观察了对虾原肌球蛋白长纺锤形晶体的横纹结构。对虾原肌球蛋白在溶解度上与副肌球蛋白接近。(2)对虾原肌球蛋白晶体的横纹周期约400A,与一般的原肌球蛋白相同;而其横纹细节的间距为140A,却与副肌球蛋白相类。因此,对虾原肌球蛋白一如河蚌闭壳肌或斧足肌原肌球蛋白,构成两类蛋白质之同的“中间”类型。(3)在无盐的水溶液中,对虾原肌球蛋白聚合所形成的亚显微纤维,也具有横纹结构,其同距也是140A。(4)讨论了原肌球蛋白,副肌球蛋白和轻酶解肌球蛋白晶体横纹结构的特征与相同之处以及它们与分子结构可能有的内在联系。     

副肌球蛋白长带的细微结构

(1) 用局部酶解方法,发现河蚌白闭壳肌的副肌球蛋白长带系由许多微丝组成,微丝直径约30。(2) 由于这些微丝束在完全松散前,仍呈现145及70横纹结构,而副肌球蛋白类晶体也具有类似的结构,我们推测微丝主要是由副肌球蛋白分子直线聚合而形成的。(3) 用局部酶解方法,可以任意重现副肌球蛋白长带的点阵结构。(4) 在副肌球蛋白长带中,存在有机械的弱点与酶解时易受作用之点,此结构上不均一性的物质基础尚不明了。     

副肌球蛋白长带的细微结构

(1)用局部酶解方法,发现河蚌白闭壳肌的副肌球蛋白长带系由许多微丝组成,微丝直径约30A。(2)由于这些微丝束在完全松散前,仍呈现145A及70A横纹结构,而副肌球蛋白类晶体也具有类似的结构,我们推测微丝主要是由副肌球蛋白分子直线聚合而形成的。(3)用局部酶解方法,可以任意重现副肌球蛋白长带的点阵结构。(4)在副肌球蛋白长带中,存在有机械的弱点与酶解时易受作用之点,此结构上不均一性的物质基础尚不明了。     

栉江珧平滑闭壳肌收缩装置的超微结构

利用电子显微镜观察了栉江珧平滑闭壳肌收缩装置的精细结构,它含有粗肌丝、细肌丝和致密体。分离的天然粗肌丝含肌球蛋白和副肌球蛋白,呈现带状和Bear-Selby网格状图象,其周期为14.5nm和7.2nm。以不含ATP的低离子强度溶液处理粗肌丝,则其近侧集聚大量细肌丝;而以微酸性、中等离子强度溶液处理粗肌丝,可以溶去肌球蛋白,但不破坏其周期性结构。以2M脲处理粗肌丝,它纵向分散成直径约有10nm的长带。     

蜜蜂间接飞翔肌粗肌丝的微丝结构

用能溶解肌球蛋白但不溶解副肌球蛋白的溶液(300 mM KCI,pH6.0)处理分离的蜜蜂间接飞翔肌粗肌丝,经数分钟后可以看到粗肌丝端头散开成为多根微丝,微丝数最多为7根。延长处理时间,可以见到粗肌丝中央部分只剩下直径约为5 nm的徽丝。实验结果支持我们以前提出的蜜蜂间接飞翔肌粗肌丝的结构模式,并指示贯穿肌小节、两端都和Z线相连的内芯至少部分由副肌球蛋白组成。只存在于A带的,由6根微丝形成的外套是由肌球蛋白分子组成。     

微流控芯片电泳分离血清高密度脂蛋白亚类的研究

描述了一种微流控芯片电泳快速分离血清高密度脂蛋白(high density lipoprotein,HDL)亚类的方法.利用自制的微流控芯片,结合激光诱导荧光检测系统,40mmol/L Tricine、50mmol/L甲基葡胺(MEG)、0.2mmol/LSDS(pH8.5)为样品缓冲液,40mmol/L Tricine、50mmol/LMEG、0.01mmol/L SDS(pH8.5)为分离缓冲液,4min内HDL3和HDL2两种亚类得到基线分离.该法操作过程简单,重复性较佳,测试费用低廉,在临床HDL亚类的检测中具有较好的应用前景.     

法罗培南钠片有关物质检查方法的研究

目的:建立法罗培南钠片有关物质检查方法。方法:色谱柱:Agilent-150mm XDB-C18 5μm;流动相:乙腈-磷酸二氢钾缓冲液(20mmol/L磷酸二氢钾水溶液,用10mol/L NaOH溶液调pH值至6.0)(10:90);柱温:30℃;检测波长为256nm;流速:1ml/min;进样量:20μl。结果:辅料不干扰测定,各破坏条件下产生的杂质峰与主峰完全分离;最低检出量:3.175ng;法罗培南钠可与其异构体完全分离。结论:本方法准确、可靠、专属性强,可以更好的控制产品质量。     

小麦线粒体表面存在肌球蛋白

证明了小麦 (TriticumaestivumL .)线粒体上存在肌球蛋白。通过免疫印迹鉴定发现小麦线粒体蛋白重链与抗体进行交叉反应 ,其分子量略高于动物骨骼肌肌球蛋白的重链 ,经计算 ,该蛋白的分子量为 2 10kD。通过电镜观察到溶液中的F_肌动蛋白可以和NEM (N_ethylmaleimide)处理的线粒体结合 ,并发现F_肌动蛋白可以激活线粒体悬浮液的ATP酶活性 ,证明线粒体外膜的外表面存在肌球蛋白     

Immunohistochemistry of chaetognath body wall muscles

Abstract. A light and electron immunohistochemical study was carried out on the body wall muscles of the chaetognath Sagitta friderici for the presence of a variety of contractile proteins (myosin, paramyosin, actin), regulatory proteins (tropomyosin, troponin), and structural proteins (α‐actinin, desmin, vimentin). The primary muscle (～80% of body wall volume) showed the characteristic structure of transversely striated muscles, and was comparable to that of insect asynchronous flight muscles. In addition, the body wall had a secondary muscle with a peculiar structure, displaying two sarcomere types (S1 and S2), which alternated along the myofibrils. S1 sarcomeres were similar to those in the slow striated fibers of many invertebrates. In contrast, S2 sarcomeres did not show a regular sarcomeric pattern, but instead exhibited parallel arrays of 2 filament types. The thickest filaments (～10–15 nm) were arranged to form lamellar structures, surrounded by the thinnest filaments (～6 nm). Immunoreactions to desmin and vimentin were negative in both muscle types. The primary muscle exhibited the classical distribution of muscle proteins: actin, tropomyosin, and troponin were detected along the thin filaments, whereas myosin and paramyosin were localized along the thick filaments; immunolabeling of α‐actinin was found at Z‐bands. Immunoreactions in the S1 sarcomeres of the secondary muscle were very similar to those found in the primary muscle. Interestingly, the S2 sarcomeres of this muscle were labeled with actin and tropomyosin antibodies, and presented no immunore‐actions to both myosin and paramyosin. α‐Actinin in the secondary muscle was only detected at the Z‐lines that separate S1 from S2. These findings suggest that S2 are not true sarcomeres. Although they contain actin and tropomyosin in their thinnest filaments, their thickest filaments do not show myosin or paramyosin, as the striated muscle thick myofilaments do. These peculiar S2 thick filaments might be an uncommon type of intermediate filament, which were labeled neither with desmin or vimentin antibodies.     

Structure of the cross-striated adductor muscle of the scallop

The structure of the cross-striated adductor muscle of the scallop has been studied by electron microscopy and X-ray diffraction using living relaxed, glycerol-extracted (rigor), fixed and dried muscles. The thick filaments are arranged in a hexagonal lattice whose size varies with sarcomere length so as to maintain a constant lattice volume. In the overlap region there are approximately 12 thin filaments about each thick filament and these are arranged in a partially disordered lattice similar to that found in other invertebrate muscles, giving a thin-to-thick filament ratio in this region of 6:1.The thin filaments, which contain actin and tropomyosin, are about 1 μm long and the actin subunits are arranged on a helix of pitch 2 × 38.5 nm. The thick filaments, which contain myosin and paramyosin, are about 1.76 μm long and have a backbone diameter of about 21 nm. We propose that these filaments have a core of paramyosin about 6 nm in diameter, around which the myosin molecules pack. In living relaxed muscle, the projecting myosin heads are symmetrically arranged. The data are consistent with a six-stranded helix, each strand having a pitch of 290 nm. The projections along the strands each correspond to the heads of one or two myosin molecules and occur at alternating intervals of 13 and 16 nm. In rigor muscle these projections move away from the backbone and attach to the thin filaments.In both living and dried muscle, alternate planes of thick filaments are staggered longitudinally relative to each other by about 7.2 nm. This gives rise to a body-centred orthorhombic lattice with a unit cell twice the volume of the basic filament lattice.     

THE FINE STRUCTURE OF THE SHELL MUSCLE OF PATELLID PROSOBRANCH LIMPETS

The structure of the shell muscle of eleven species of patellidlimpet is described from light and transmission electron microscopestudies. Although the muscle has many structural characteristicstypical of molluscan smooth muscle, it also has a number ofunusual features. At the electron microscope level two myofibretypes are distinguishable. Type I cells, present in all species,contain conventional contractile apparatus in the form of thickand thin filaments. Thick filaments contain paramyosin and varyin diameter between 20—180 nm. An axial striation witha repeat of 14.2 nm is calculated from optically diffractedmicrographs of isolated thick filaments. Transverse sectionsof thick filaments reveal bands from which the transverse repeatof the paramyosin crystal lattice is calculated. Type II myofibres,which are present in five species, contain a novel arrangementof thin filaments with electron-dense regions at intervals of80–150 nm. The striated thin filaments are similar inappearance to the microfilament bundles and stress-fibres ofnon-muscle cells. They also have similarities to the leptomericorganelles of some vertebrate muscle tissues. Associated withthe muscle is an unusually large amount of collagen which hasa periodicity of 62 nm calculated from optical diffraction patternsof isolated collagen fibrils. (Received 3 July 1989; accepted 12 October 1989)     

虾夷扇贝过敏原tropomyosin的克隆表达、纯化及免疫学鉴定

从虾夷扇贝(Patinopecten yessoensis)肌肉中提取总RNA,RT-PCR克隆虾夷扇贝中变应原原肌球蛋白的全长基因,根据序列设计带有酶切位点的特异性引物,扩增扇贝tropomyosin的完整开放阅读框,与pET-28a载体连接并转化大肠杆菌Escherichia.coli BL21(DE3),诱导表达后,Ni2+亲和层析柱纯化重组蛋白,Western-blot检测其免疫学活性。经序列测定,该基因含有长度为855bp的开放阅读框,编码284个氨基酸,其在GenBank数据库中的登录号为EU839640。SDS-PAGE检测该重组变应原在大肠杆菌中高效表达36kD的目的蛋白,且重组变应原具有良好的IgE结合活性。研究获得了具有变应原活性的重组虾夷扇贝tropomyosin,为扇贝过敏性疾病的诊断和治疗奠定了基础。       

Fluorescence and the structure of proteins. XXI. Fluorescence of aminotyrosyl residues in peptides and helical proteins.

1. Five peptides containing tyrosine were converted to the 3-aminotyrosyl peptides by nitration with tetranitromethane and subseuqent reduction of the nitro groups to amino groups. The fluorescence of these aminotyrosyl residues was found to be quite similar to that of 3-aminotyrosine and it is concluded that the fluorescence is not sensitive to incorporation of the amino acid into the peptide chain. 2. Fluorescence of 3-aminotyrosine derivatives was sensitive, however, to the nature of the solvent; as the dielectric constant decreased, fluorescence was enhanced ten fold and the emission maximum shifted from the 350-370 nm value in aqueous solution to 320 nm. It is predicted that similar differences might be expected for exposed and buried aminotyrosyl residues in a protein. 3. Exposed tyrosyl residues on the helical protein tropomyosin and a helical segment of paramyosin were aminated in part (39% and 34% of the total tyrosyl residues, respectively). The fluorescence of the aminated tyrosyl residues on these proteins was similar to that of the aminotyrosyl peptides in an aqueous medium. Although the fluorescence efficiency of an aminotyrosyl residue was much lower than that of a tyrosyl residue, it was easy to distinguish the fluorescence of the aminotyrosyl residues (350-355 nm) on the protein from that arising from unmodified tyrosyl residues (305 nm).     

白唇鹿被毛形态学与高寒环境关系的研究

本文对白唇鹿被毛的形态进行了宏观及扫描电镜的观察研究。结果表明,其被毛仅由针毛组成。针毛的毛径特粗;髓质极发达,髓质指数高达96％;髓质形态为蜂窝状网格型;皮质呈退化状态。针毛具有很强的保温性,是适应高寒环境的重要特征之一。     

A method for the production of highly specific polyclonal antibodies

Two polyclonal antibodies directed against paramyosin and tropomyosin from Owenia fusiformis (a marine polychete annelid) were obtained using a new method of immunization. After purification by two-dimensional gel electrophoresis, proteins were transferred onto a nitrocellulose sheet using the Western blot technique. The proteins bound to their cellulose support were injected into rabbits without Freund's adjuvant and without solubilization of nitrocellulose with dimethyl sulfoxide. Highly specific polyclonal antibodies were generated.     

STUDIES ON MUSCLE DIFFERENTIATION. III. IMMUNO-HISTOCHEMICAL IDENTIFICATION OF TROPOMYOSIN IN SKELETAL AND CARDIAC MUSCLES OF DEVELOPING CHICK EMBRYO.*,**

Specific identification of tropomyosin was succeeded in chick embryos by an application of immuno-histochemical techniques with the antisera against frog skeletal muscle tropomyosin. The first appearance of tropomyosin was in the central part of cervical somites of stage 14 embryo, and the area containing tropomyosin extended dorso-ventrally in later stages. In the heart primordium, tropomyosin was first detected in the presumptive epimyocardium at stage 8 embryos and was found to be concentrated in the epimyocardium in later embryos. The stages of the first appearance of tropomyosin in somites and presumptive myocardium corresponded with those of the first appearance of thin filaments in these organ primordia reported by other investigators. Tropomyosin in myogenic cells and in muscle fibers was localized in cell membrane or in cell peripheries. It was also distributed in a striated pattern which seemed to be due to a localization of tropomyosin in the I-bands.     

The paramyosin of insect flight muscle

Paramyosin has been extracted and purified from the flight muscle of the insects Lethocerus cordofanus, Lethocerus maximus (water bugs), Heliocopris japetus (dung beetle) and Pachnoda ephippiata (rosechafer beetle). The subunit molecular weight, estimated by sodium dodecyl sulphate electrophoresis, is 107,000 ± 6000. The intrinsic sedimentation constant is 3.17 S and circular dichroism measurements give about 87 % helix, showing that the molecule is likely to be a two-chain rod.The amino acid composition of insect paramyosins resembles that of molluscan and annelid paramyosins except that the Glu/Asp ratio is higher. The amino acid analysis of insect tropomyosin is also given. Electron microscopy of tactoids shows an axial periodicity of 732 ± 8 Å or 146 Å with fine structure differing from that of molluscan tactoids.The proportion of paramyosin in the myofibrils, estimated by densitometry of stained gels, is 6.3% in L. cordofanus and 9.5% in rosechafer, and the ratio of myosin to paramyosin in L. cordofanus is 8.2. The possibility that paramyosin is a core protein of the myosin filaments is discussed.     

Identification of proteins in excretory/secretory extracts of Echinostoma friedi (Trematoda) from chronic and acute infections

In the present study, we describe the investigation of Echinostoma friedi excretory/secretory products using a proteomic approach combined with the use of heterologous antibodies. We have identified 18 protein spots corresponding to ten proteins, including cytoskeletal proteins like actin, tropomyosin, and paramyosin; glycolytic enzymes like enolase, glyceraldehyde 3P dehydrogenase, and aldolase; detoxifying enzymes like GSTs; and stress proteins like heat shock protein (Hsp) 70. Among these proteins, both actin and, to a lesser extent, Hsp70, exhibited differential expression patterns between chronic and acute infections in the Echinostoma-rodent model, suggesting that these proteins may play a role in the survival within the host.