肌原纤维上蛋白质定位问题的研究 Ⅰ.标记抗体的电子显微镜观察在副肌球蛋白定位问题上的初步应用

(一)应用铁蛋白标记抗体的技术对尖蚌闭壳肌白色部分的粗丝作了副肌球蛋白的定位研究。结果支持了许多作者根据小角度X-光衍射和电子显微镜分别从副肌球蛋白肌丝和晶体反映出来的共同特征周期所作的推测,即副肌球蛋白是粗丝的组成部分。从免疫化学反应的结果来看,副肌球蛋白分布于粗丝的表面。(二)粗丝与标记的副肌球蛋白抗体反应后,再经反染,周期为360A的横纹结构更为清晰。此数值恰为一般在副肌球蛋白天然及人工纤维和晶体中所见到的720A周期的一半。这个观察和平行的物化研究以及对副肌球蛋白分子的电子显微镜观察,都证明副肌球蛋白可能具有亚基结构,亚基的长度为360A。(三)观察到粗丝在与标记抗体反应后,有侧向偶联现象。     

肌肉收缩Hill特性式的理论研究

从肌球蛋白工作循环的机械化学偶联模型出发,利用化学动力学方法和生物化学热力学原理,结合肌球蛋白单分子实验结果,从能量转化的观点给出了肌肉收缩的Hill特性式,加深了对Hill特性式及肌肉收缩过程中能量转化的理解,在整合肌球蛋白单分子性质与肌肉收缩宏观性质的信息方面做了尝试。     

非肌肉Ⅱ型肌球蛋白在细胞骨架网络中动力学行为的图像分析(已撤稿2016-06-15)

贴壁细胞的形状和弹性(硬度)与细胞骨架网络的形态以及纤维的交联方式密切相关．而细胞骨架网络的形态和组成与细胞中的二型肌球蛋白的活动,尤其是肌球蛋白组装成的肌球蛋白粗丝(minifilament)的活动有关．细胞通过与其外部环境的机械传感(mechanosensing)来调节二型肌球蛋白的活动和肌球蛋白粗丝的相互作用,从而实现对细胞骨架网络重组(remodeling)的控制．当前对活体细胞内二型肌球蛋白的研究从实验测量到理论模型的建立之间还有不小距离,主要是因为直接测量会对细胞结构和生理活动产生影响,而间接测量不能得到肌球蛋白在细胞内活动的准确数据．因此本文提出利用新的免疫荧光显微图像分析技术,例如免疫荧光蛋白图像追踪和局部图像相关函数分析技术,分析HeLa细胞体内肌球蛋白在细胞骨架网络中的动态分布,总结出肌球蛋白主导的细胞骨架和张力纤维组装与分解过程中的基本动力学规律．图像分析结果说明：肌球蛋白纤维在细胞骨架网络构建过程中依次动态处于组装与分解状态,通过其粗丝相对旋转对齐与收缩产生张力以维持纤维束稳定,并形成有不同肌球蛋白和粘着斑数量与分布形态的三类稳定性肌动球蛋白网络,其稳定性和收缩力大小呈正相关、与所结合肌球蛋白数量密度成正比．     

丝瓜肌球蛋白的电子显微学研究

从丝瓜 (Luffacylindrica (L .)Roem .)卷须中纯化得到分子量为 174kD的肌球蛋白 ,并对其进行了酶学与电子显微学的研究。这种肌球蛋白具有肌动蛋白激活的MgATPase活性 ,能够被抗动物肌肉的肌球蛋白的单克隆抗体识别。电子显微学研究表明 :它有两个头部 (大小和形状与动物肌肉的肌球蛋白相似 )和一条相对较短的尾部。还对丝瓜卷须的肌动蛋白进行了观测 ,偶尔发现一些尾部有球状结构的肌球蛋白。该肌球蛋白的免疫特性和超微结构证明了它由 2条重链组成 ,并与传统的肌球蛋白相似。然而 ,这种 174kD的肌球蛋白是否参与了丝瓜的接触卷曲有待于进一步研究。     

非肌肉Ⅱ型肌球蛋白在细胞骨架网络中动力学行为的图像分析

贴壁细胞的形状和弹性(硬度)与细胞骨架网络的形态以及纤维的交联方式密切相关.而细胞骨架网络的形态和组成与细胞中的二型肌球蛋白的活动,尤其是肌球蛋白组装成的肌球蛋白粗丝(minifilament)的活动有关.细胞通过与其外部环境的机械传感(mechanosensing)来调节二型肌球蛋白的活动和肌球蛋白粗丝的相互作用,从而实现对细胞骨架网络重组(remodeling)的控制.当前对活体细胞内二型肌球蛋白的研究从实验测量到理论模型的建立之间还有不小距离,主要是因为直接测量会对细胞结构和生理活动产生影响,而间接测量不能得到肌球蛋白在细胞内活动的准确数据.因此本文提出利用新的免疫荧光显微图像分析技术,例如免疫荧光蛋白图像追踪和局部图像相关函数分析技术,分析He La细胞体内肌球蛋白在细胞骨架网络中的动态分布,总结出肌球蛋白主导的细胞骨架和张力纤维组装与分解过程中的基本动力学规律.图像分析结果说明:肌球蛋白纤维在细胞骨架网络构建过程中依次动态处于组装与分解状态,通过其粗丝相对旋转对齐与收缩产生张力以维持纤维束稳定,并形成有不同肌球蛋白和粘着斑数量与分布形态的三类稳定性肌动球蛋白网络,其稳定性和收缩力大小呈正相关、与所结合肌球蛋白数量密度成正比.     

平滑肌肌球蛋白磷酸化与肌动球蛋白功能调节

在有Ｃａ２＋和钙调蛋白存在时,肌球蛋白轻链激酶催化肌球蛋白磷酸化,促使肌动蛋白激活的肌球蛋白（肌动球蛋白）Ｍｇ２＋－ＡＴＰ酶活性显著增加．然而,肌球蛋白磷酸化水平与Ｍｇ２＋－ＡＴＰ酶之间的关系是非线性的,原肌球蛋白可以进一步增加Ｍｇ２＋－ＡＴＰ酶的活性,但仍不改变它们之间的非线性关系．肌球蛋白轻链激酶的合成肽抑制剂抑制了肌球蛋白磷酸化和Ｍｇ２＋－ＡＴＰ酶活性,并导致平滑肌去膜肌纤维的等长收缩张力与速度的降低．结果提示肌球蛋白轻链激酶参与脊椎动物平滑肌收缩的调节过程,肌球蛋白轻链磷酸化作用会引起平滑肌收缩     

鸭肫平滑肌肌球蛋白经α－糜蛋白酶轻微酶解及其Mg~（2＋）－ATP酶性质的改变

用α－糜蛋白酶对鸭肫平滑肌肌球蛋白进行有限酶解,使肌球蛋白重链的电泳双带变成单带。酶解后肌球蛋白与正常肌球蛋白相比,肌动蛋白激活的磷酸化肌球蛋白的Ｍｇ２＋－ＡＴＰ酶活力在低Ｍｇ２＋离子浓度时反而较高。从酶解后肌球蛋白溶液中只分离到１个４．２ｋｄ的小肽段。氨基酸组成测定及荧光胺末端标记实验提示,该小肽段酶切自平滑肌肌球蛋白重链ＳＭ１的Ｃ末端。     

丝瓜肌球蛋白的电子显微学研究

从丝瓜(Luffa cylindrica (L.) Roem.)卷须中纯化得到分子量为174kD的肌球蛋白,并对其进行了酶学与电子显微学的研究.这种肌球蛋白具有肌动蛋白激活的MgATPase活性,能够被抗动物肌肉的肌球蛋白的单克隆抗体识别.电子显微学研究表明:它有两个头部(大小和形状与动物肌肉的肌球蛋白相似)和一条相对较短的尾部.还对丝瓜卷须的肌动蛋白进行了观测,偶尔发现一些尾部有球状结构的肌球蛋白.该肌球蛋白的免疫特性和超微结构证明了它由2条重链组成,并与传统的肌球蛋白相似.然而,这种174 kD的肌球蛋白是否参与了丝瓜的接触卷曲有待于进一步研究.     

小檗碱对平滑肌肌球蛋白功能及胃肠平滑肌收缩性的影响

目的:探讨小檗碱对平滑肌肌球蛋白功能及胃肠平滑肌收缩性的影响.方法:以平滑肌肌球蛋白Mg2+-ATPase活性、肌球蛋白磷酸化以及胃与肠道平滑肌的收缩振幅为指标,考察小檗碱对平滑肌肌球蛋白Mg2+-ATPase活性和肌球蛋白磷酸化程度的影响,及其对离体小肠与胃平滑肌条收缩性的影响.结果:(1)在肌球蛋白轻链的Ca2+依赖性磷酸化反应中.小檗碱能抑制磷酸化肌球蛋白Mg2+-ATPase活性;(2)在肌球蛋白轻链的Ca2+依赖性磷酸化反应中,小檗碱可显著抑制磷酸化肌球蛋白轻链磷酸化程度;(3)小檗碱对大鼠离体小肠及胃平滑肌条收缩性均具有抑制作用.且均呈剂量依赖性.结论:小檗碱可通过抑制平滑肌肌球蛋白的功能,抑制胃肠道平滑肌的收缩性.     

鸭肫平滑肌肌球蛋白经α—糜蛋白酶轻微酶解及其Mg^2＋—ATP酶…

用α－糜蛋白酶对鸭肫平滑肌肌球蛋白进行有限酶解,使肌球蛋白重链的电泳双带变成单带。酶解后肌球蛋白与正常肌球蛋白相比,肌动蛋白激活的磷酸化肌球蛋白的Ｍｇ＾２＋－ＡＴＰ活力在低Ｍｇ＾２＋离子浓度时反而较高。从酶解后肌球蛋白溶液中只分离到１个４．２ｋｄ的小肽段。氨基酶组成测定及荧光胺末端标记实验提示,该小肽段酶氏自平滑肌肌球蛋白重链ＳＭ１的Ｃ末端。     

Heat-induced Gelation of Myosin from Leg and Breast Muscles of Chicken

Heat-induced gelation of myosin from leg and breast muscles of chicken was studied in 0.6 m KC1. Gel strength of breast myosin was higher than that of leg myosin between pH 5.2 and 6.0. Turbidity of breast myosin increased below pH 6.0 but that of leg myosin did not increase at pH 5.7. Turbidity of leg myosin was higher than that of breast myosin below pH 5.6. Viscosity of breast myosin increased between pH 5.5 and 6.0 as the pH decreases, although that of leg myosin decreased. The breast myosin assembled to form long filaments at pH 5.7, but leg myosin failed to form long filaments. At pH 5.4, breast myosin filaments became longer and leg myosin assembled into filaments though they were shorter than breast myosin filaments. The strength of heat-induced gel formed from the filamentous leg and breast myosins at acidic region was not influenced by F-actin. These results indicate that the strength of heat-induced gel of both myosins is closely related to their morphological properties.     

Biochemical properties of ordinary and dark muscle myosin from carp skeletal muscle

Two types of myosin isolated from ordinary (fast) and dark (slow) muscles of carp were examined by ATPase and in vitro motility assays. Vmax of the ATPase activity and sliding velocity of ordinary myosin showed 1.6 and 1.5 times higher activities than those of dark myosin, whereas those of mammalian fast myosin were much higher, 3 to 10 times, than those of slow myosin. Although ordinary myosin had almost identical activities to those of mammalian fast myosin, activities of dark myosin was twice of those of mammalian slow myosin. This high motile activity of dark myosin can account for the physiological role of dark muscle in cruising of fish. By comparing Km of the actin-activated ATPase activity, ordinary myosin was appeared to have higher affinity to F-actin than dark myosin, and this was confirmed by the binding assay of HMM or S-1 of carp myosin to F-actin. Investigation of myosin assembly by electron microscopy and the centrifugation assay revealed that ordinary myosin assembled much poorly than dark myosin or mammalian fast myosin. This phenomenon may reflect characteristic cellular function of fish skeletal muscle.     

Correlation of conformation and phosphorylation and dephosphorylation of smooth muscle myosin

The rate of phosphorylation and dephosphorylation of smooth muscle myosin by myosin light chain kinase and by two myosin light chain phosphatases (gizzard phosphatase IV and aorta phosphatase) are measured in various conditions; the relationship between the rate of phosphorylation and dephosphorylation of myosin and the myosin conformation is also studied. The rate of dephosphorylation of myosin was completely inhibited in the presence of 1 mM MgCl2 and ATP at low ionic strength where phosphorylated myosin forms a folded conformation. The inhibition was released when myosin formed either an extended monomer or filaments. The rate of phosphorylation of myosin was also affected by the conformation of myosin. The rate for a folded myosin was slower than those for an extended monomer and filamentous myosin. The phosphorylation and dephosphorylation of heavy meromyosin, subfragment-1, and the isolated 20,000-dalton light chain are not inhibited at low ionic strength, and the rate of phosphorylation and dephosphorylation was decreased with increasing ionic strength. KCl dependence of the rate of phosphorylation and dephosphorylation of myosin was normalized by using KCl dependence of subfragment-1, and it was found that the marked inhibition of the rate of phosphorylation and dephosphorylation of myosin is closely related to the change from an extended to a folded conformation of myosin.     

Conformational Change of Chymotryptic Myosin Rod

Myosin rod was prepared from hen myosin by chymotryptic digestion. The indigested myosin was successfully removed by ultracentrifugation following alcohol treatment. No significant difference in UV absorption and CD spectra was observed between pH 7.0 and pH 10.5 for both myosin rod and myosin. When pH was raised to 11.7, the phenolic groups of the tyrosyl residues were ionized, and the helical configuration of the myosin rod and myosin could not withstand the electrostatic repulsion. When pH was further raised to 13.6, “abnormal” tyrosyl residues were ionized, resulting in decreased helix content. However, the myosin rod was stabler and less flexible against pH change than myosin, because of the lower content of tyrosyl residues in myosin rod.     

Myosin from fast and slow skeletal and cardiac muscles of mammals of different size.

The ATPase activity of myosin and contraction time in extensor digitorum longus muscle, soleus muscle and cardiac muscle was compared in mammals differing in size. It was shown that the myosin ATPase activity of homologous muscles decreases and contraction time increases with increasing size of animals. The rate of tryptic digestion of myosin, the electrophoretic pattern of light chains of myosin and the effect of p-chloromercuribenzoate on ATPase activity of myosin were also studied. All these three myosin properties are very characteristic when the myosin from a fast muscle is compared with the myosin from a slow muscle of the same animal, but no relationship between these three myosin properties and ATPase activity of myosin was found, when homologous muscles of various mammals were compared.     

Macrophage myosin. Regulation of actin-activated ATPase, activity by phosphorylation of the 20,000-dalton light chain.

Myosin was purified from rabbit alveolar macrophages in a form that could not be activated by actin. This myosin could be phosphorylated by an endogenous myosin light chain kinase, up to 2 mol of phosphate being incorporated/mol of myosin. The site phosphorylated was located on the 20,000-dalton myosin light chain. Phosphorylation of macrophage myosin was found to be necessary for actin activation of myosin ATPase activity. Moreover, the actin-activated ATPase activity was found to vary directly with the extent of myosin phosphorylation, maximal phosphorylation (2 mol of Pi/mol of myosin) resulting in an actin-activated MgATPase activity of approximately 200 nmol of Pi/mg of myosin/min at 37 degrees C. These results establish that phosphyoyration of the 20,000-dalton light chain of myosin is sufficient to regulate the actin-activated ATPase activity of macrophage myosin.     

Properties of porcine platelet myosin. I. Similarity between vertebrate smooth muscle and nonmuscle myosins in their binding properties with F-actin

The mechanism of the ATPase [EC 3.6.1.3] reaction of porcine platelet myosin and the binding properties of platelet myosin with rabbit skeletal muscle F-actin were investigated. The kinetic properties of the platelet myosin ATPase reaction, that is, the rate, the extent of fluorescence enhancement of myosin, the size of the initial P1 burst of myosin, and the amount of nucleotides bound to myosin during the ATPase reaction, were very similar to those found for other myosins. Strong binding of platelet myosin with rabbit skeletal muscle F-actin, as found for smooth muscle myosin, was suggested by the following results. The rate of the ATP-induced dissociation of hybrid actomyosin, reconstituted from platelet myosin and skeletal muscle F-actin, was very slow. The amount of ATP necessary for complete dissociation of hybrid actomyosin was 2 mol/mol of myosin, although skeletal muscle actomyosin is known to dissociate completely upon addition of 1 mol ATP per mol of myosin. Unlike skeletal muscle myosin, the EDTA(K+)-ATPase activity of platelet myosin was inhibited by skeletal muscle F-actin. These observations indicate that ATP hydrolysis by vertebrate nonmuscle myosin follows the same mechanism as with other myosins and that the binding properties of nonmuscle myosin with F-actin are similar to those of smooth muscle myosin but not to those of skeletal muscle myosin.     

The effect of time, 2-mercaptoethanol and inhibitors of proteases on isolation of cardiac myosin and its properties

Myosin was isolated from the ventricular myocardium of adult rats and the effect of time, 2-mercaptoethanol and inhibitors of proteases was investigated on its properties. It was found that the storage of cardiac muscle up to 4 hours does not influence the myosin ATPase, the electrophoretic pattern of light chains of myosin or the pattern of peptides produced by digestion of myosin with chymotrypsin. Neither does the presence of pepstatin and phenylmethyl sulfonylfluoride during myosin preparation influence the activity of myosin ATPase. It was found that the presence of 2-mercaptoethanol during myosin preparation enhances myosin ATPase of the product. This myosin was more stable when kept at 4 degrees C for four days.     

The regulatory light chain is required for folding of smooth muscle myosin

Light chain phosphorylation causes the folded monomeric form of myosin to extend and assemble into filaments. This observation established the involvement of the 20-kDa regulatory light chain (LC20) in conformational transitions of smooth muscle myosin. To further assess the role of this subunit in the intramolecular folding of myosin, LC20 was removed from turkey gizzard myosin at elevated temperatures in the presence of EDTA through the use of an antibody affinity column. Metal-shadowed images showed that LC20-deficient myosin had a tendency to aggregate through the neck region. When MgATP was added to filaments formed from this myosin, less than 10% of the myosin was solubilized, indicating that myosin could not fold in the absence of light chain. Readdition of native regulatory light chain restored the myosin to its original solubility properties, thus establishing reversibility. Addition of foreign light chains from skeletal muscle myosin or a chymotryptic-cleaved gizzard light chain produced the same amount of monomeric myosin in high salt that was obtained by recombination with the homologous light chain. However, the ability of the hybrid myosins to assume the folded conformation was impaired, and only a partially folded species was obtained. Single-headed myosin, like rod and light chain-deficient myosin, remained filamentous in the presence of MgATP. These results are consistent with the hypothesis that the regulatory light chain in the neck region of myosin contributes to a binding site for the myosin tail.     

BIG1 is a binding partner of myosin IXb and regulates its Rho-GTPase activating protein activity

Myosin IXb, a member of the myosin superfamily, is a molecular motor that possesses a GTPase activating protein (GAP) for Rho. Through the yeast two-hybrid screening using the tail domain of myosin IXb as bait we found BIG1, a guanine nucleotide exchange factor for ADP-ribosylation factor (Arf1), as a potential binding partner for myosin IXb. The interaction between myosin IXb and BIG1 was demonstrated by co-immunoprecipitation of endogenous myosin IXb and BIG1 with anti-BIG1 antibodies in normal rat kidney cells. Using the isolated proteins, it was demonstrated that myosin IXb and BIG1 directly bind to each other. Various truncation mutants of the myosin IXb tail domain were produced, and it was revealed that the binding region of myosin IXb to BIG1 is the zinc finger/GAP domain. Interestingly, the GAP activity of myosin IXb was significantly inhibited by the addition of BIG1 with IC(50) of 0.06 microm. The RhoA binding to myosin IXb was inhibited by the addition of BIG1 with the concentration similar to the inhibition of the GAP activity. Likewise, RhoA inhibited the BIG1 binding of myosin IXb. These results suggest that BIG1 and RhoA compete with each other for the binding to myosin IXb, thus resulting in the inhibition of the GAP activity by BIG1. The present study identified BIG1, the Arf guanine nucleotide exchange factor, as a new binding partner for myosin IXb, which inhibited the GAP activity of myosin IXb. The findings raise a concept that the myosin transports the signaling molecule as a cargo that functions as a regulator for the myosin molecule.