Purification and characterization of myosin from wheat mitochondria

Myosin was purified from wheat mitochondria using DE-52 anion exchange chromatography and Sephacryl S-300 gel filtration. The molecular weight of its heavy chain is about 210 ku, similar to that of muscle myosin Ⅱ(205 ku), and it could be recognized by the polyclonal antibodies against human skeletal muscle myosin Ⅱ. The ATPase activity of the mitochondrial myosin stimulated by F-actin from chicken muscle is 202.5 nmoles Pi/min·mg. The mitochondrial myosin could be activated by Ca²⁺ and was not inhibited by Ca²⁺ at high concentration. The results demonstrate that the myosin of wheat mitochondria shares some similarities with the skeletal muscle myosin Ⅱ.     

Structure of the actin-myosin interface

The topography of the rigor complex between F-actin and myosin heads (S1) has been investigated by carbodiimide zero-length cross-linking. The results demonstrate for the first time that the 95,000-molecular weight (95K) heavy chain of the myosin head enters into van der Waals contact with two neighbouring actin monomers; one is bound to the 50K domain and the other to the 20K domain of the myosin chain. The covalent F-actin-S1 complex can be isolated; it shows a vastly elevated Mg2+-ATPase. Each pair of actin subunits in the thin filament seems to act as a functional unit for specific binding of a myosin head and stimulation of its Mg2+-ATPase activity.     

H-ATPase in the plasma membrane of Arabidopsis pollen cells is involved in extracellular calmodulin-promoted pollen germination

In this paper, the role of the plasma membrane (PM) H⁺-ATPase in extracellular calmodulin (CaM)-promoted pollen germination and in tube growth of Arabidopsis thaliana was investigated. Pollen germination, pollen tube growth rate, free cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) and Ca²⁺ channel activity in the PM of pollen cells were measured. In response to fusicoccin or CaM treatment, in vitro pollen germination and pollen tube growth rate, [Ca²⁺]_cyt and activity of a hyperpolarization-activated Ca²⁺-permeable channel increased. Sodium vanadate inhibited the promotion of these parameters by extracellular CaM. The results suggest that H⁺-ATPase may be involved in extracellular CaM-regulated pollen germination and in tube growth by modulation of the hyperpolarization-activated Ca²⁺ channel in the PM of A. thaliana pollen cells.     

Immunochemical identification of gelsolin in maize pollen

Gelsolin is a representative of a type of actin-binding proteins (ABPs) universally found in eukaryotes. It plays role in nucleation, capping and severing of actin filamentsin vitro. In our experiment, gelsolin was purified from pig plasma and the polyclonal antibodies against it were prepared. The crude extracts of maize pollen were immunodetected by Western-blotting with polyclonal antibody and monoclonal antibody respectively. The immunodetection results show that gelsolin exists in maize pollen and its molecular weight is about 91 ku, similar to that of gelsolin found in animal tissues.     

Stimulation of Acanthamoeba actomyosin ATPase activity by myosin-II polymerization

Phosphorylation of the regulatory light chains of vertebrate smooth muscle or cytoplasmic myosins alters the structure of myosin monomers, favours myosin filament formation and stimulates the actin-activated Mg2+-ATPase of myosin. Similarly, in Dictyostelium and Acanthamoeba phosphorylation of the myosin heavy chains exhibits both polymerization and actin-activated Mg2+ATPase. Unfortunately, the relationships between phosphorylation, myosin assembly and activation of ATP hydrolysis are not fully understood in any of these systems, as there has been no way of varying the extent of polymerization of intact myosin without changing solution conditions or the level of myosin phosphorylation, parameters that may have independent effects on ATPase activity. Taking an entirely new approach, we have used monoclonal antibodies against the tail of Acanthamoeba myosin-II that cause filament disassembly to show that myosin polymerization itself stimulates actomyosin ATPase activity. With a fixed level of myosin-II phosphorylation and constant solution conditions, depolymerization of myosin-II filaments by antibodies causes a concomitant loss of actin-activated ATPase activity.     

Identification of globular mechanochemical heads of kinesin

Kinesin is a mechanoenzyme which uses energy liberated from ATP hydrolysis to transport particles towards the 'plus ends' of microtubules. The enzyme consists of two polypeptide heavy chains of relative molecular mass (Mr) approximately 110,000-140,000 (110K-140K) plus copurifying light chains; these polypeptides are arranged in a structure consisting of two globular heads attached to a fibrous stalk which terminates in a 'feathered' tail. Here we report that a function-disrupting monoclonal antikinesin, which binds to the 45K fragment of the kinesin heavy chain, recognizes an epitope located towards the N-terminal end of the heavy chain, and decorates the two globular heads lying at one end of the intact molecules (one antibody per head). The results show that the two heavy chains of native kinesin are arranged in parallel, and that the 45K fragments, which display nucleotide-sensitive interactions with microtubules, represent mechanochemical 'heads' located at the N-terminal regions of the heavy chains. Thus, it is likely that the kinesin heads are analogous to the subfragment-1 domains of myosin.     

Site-directed mutagenesis of the regulatory light-chain Ca2+/Mg2+ binding site and its role in hybrid myosins

The regulatory light chains, small polypeptides located on the myosin head, regulate the interaction of myosin with actin in response to either Ca2+ or phosphorylation. The demonstration that the regulatory light chains on scallop myosin can be replaced by light chains from other myosins has allowed us to compare the functional capabilities of different light chains, but has not enabled us to probe the role of features, such as the Ca2+/Mg2+ binding site, that are common to all of them. Here, we describe the use of site-directed mutagenesis to study the function of that site. We synthesized the chicken skeletal myosin light chain in Escherichia coli and constructed mutants with substitutions within the Ca2+/Mg2+ binding site. When the aspartate residues at the first and sixth Ca2+ coordination positions are replaced by uncharged alanines, the light chains have a reduced Ca2+ binding capacity but still bind to scallop myosin with high affinity. Unlike the wild-type skeletal light chain which inhibits myosin interaction with actin, the mutants activate it. Thus, an intact Ca2+/Mg2+ binding site in the N-terminal region of the light chain is essential for regulating the interaction of myosin with actin.     

Drosophila muscle myosin heavy chain encoded by a single gene in a cluster of muscle mutations

Drosophila muscle myosin heavy chain is encoded by a single-copy gene which is transcribed during both larval and adult development. This myosin gene maps to a chromosomal locus distant from any of the actin genes, but is within a cluster of flight muscle mutations.     

Role of Ubisch bodies secreted by tapetum in Ca2+ transprot

The distribution of Ca²⁺ in the anthers of wheat was observed using cytochemical method of potassium antimonite. At the later tetrad stage, Ubisch bodies carrying Ca²⁺ were observed on the inner surface of tapetum, in anther locule and on pollen surface. The Ubisch bodies contacted with pollen, and Ca²⁺ began to accumulate on pollen surface. At the uninucleate pollen stage, abundant Ubisch bodies were distributed in anther locule, and the amount of Ca²⁺ on pollen surface increased. At the mature pollen stage a large amount of Ca²⁺ ions were localized on the inner surface of tapetum, the surface of pollen and Ubisch bodies. In the pollen wall, Ca²⁺ precipitates arranged in radial lines. These results demonstrated that Ubisch bodies were involved in Ca²⁺ transport from anther wall to pollen surface at some developmental stages of anther.     

Peptide mimetics of an actin-binding site on myosin span two functional domains on actin

The sites on the myosin heavy chain that interact with actin and are responsible for force generation are ill-defined: crosslinking and experiments with isolated domains of the myosin head implicate regions in both the 50K and 20K (molecular weights in thousands) domains of the myosin head (subfragment 1, S1) in this process. We have synthesized peptides from the sequence around the fast-reacting SH1 thiol residue in the 20K domain of S1 in order to delineate precisely an actin-binding site. We used a combination of 1H-NMR and enzyme inhibition assay and also assessed the effects of peptides on skinned rabbit psoas muscle fibres to show that the region of amino acids 690-725 contains an actin-binding site. Peptides from this region bind to actin, act as mixed inhibitors of the actin-stimulated S1 Mg2(+)-ATPase, and influence the contractile force developed in skinned fibres, whereas peptides flanking this sequence are without effect in our test systems. Remarkably, peptides from the N-terminal half of this segment 690-725 increase force development in skinned fibres at submaximal activating concentrations of Ca2+, that is, they behave as calcium-sensitizers; C-terminal peptides, however, inhibit force development without effecting sensitivity to calcium. These different responses indicate that this region is probably binding at two functionally distinct sites on actin.     

胚胎干细胞向心肌细胞分化研究进展(综述)

胚胎干细胞(embryonic stem cells,ES)在体外分化培养条件下可以分化出各种组织细胞，其中包括心肌细胞。ES细胞在体外向心肌细胞分化与体内完整胚胎心肌发育过程相符合。该细胞在体外分化过程中顺序表达心肌细胞特有结构蛋白和离子通道，如肌球蛋白轻链和重链、特异性肌动蛋白、电压依赖性Ca^2 通道、K^ 通道等。ES细胞分化来源的心肌细胞具有体内心肌细胞的生理学特点，如产生的动作电位、表现自发性收缩等。因此，ES细胞是研究心肌细胞发育分化机制及鉴定其关键基因的理想模型。     

Effects of nitrogen ion implantation on Ca2+ concentration and membrane potential of pollen cell

The effects of low energy nitrogen ion implantation on Ca²⁺ concentration and membrane potential of lily (lilium davidii Duch) pollen cell have been studied. The results showed that the Ca²⁺ concentration was increased when pollen grain was implanted by nitrogen ion with energy 100 keV and dose 10¹³ ions/cm². However, the increase of Ca²⁺ concentration was partly inhibited by the addition of Ca²⁺ channel inhibitor depending on dose. And nitrogen ion implantation caused depolarization of pollen cell membrane potential. In other words, membrane potential was increased, but the effect decreased by adding Ca²⁺ channel inhibitor. However, it was still significantly higher than the membrane potential of control cells. It was indicated that the depolarization of cell membrane potential opened the calcium channel on the membrane that caused the increasing of intracellular calcium concentration. This might be an earlier step of the effect of low energy nitrogen ion implantation on pollen germination.     

Low Ca2+ impedes cross-bridge detachment in chemically skinned Taenia coli

Muscle force is generated by cycling cross-bridges between actin and myosin filaments. In smooth muscle, cyclic attachment and detachment of cross-bridges is thought to be induced by a Ca2+- and calmodulin-dependent myosin light chain kinase which phosphorylates myosin. The relaxation that occurs after Ca2+ removal is usually ascribed to dephosphorylation of myosin by a phosphatase as non-phosphorylated myosin is unable to form force-generating criss-bridges. Recently, Dillon et al. claimed, however, that dephosphorylation of attached cross-bridges may impede cross-bridge detachment, thus forming so-called 'latch bridges'. Here we present evidence that after a Ca2+- and calmodulin-induced contraction of chemically skinned guinea pig Taenia coli, the rapid removal of Ca2+ impedes the detachment of the myosin cross-bridges from the actin filament; force can then be maintained without energy consumption. The extremely slowly detaching cross-bridges which maintain the force after Ca2+ removal may indeed correspond to the 'latch bridges' mentioned above.     

Polymerization of fluorescent analogue of plant actinin vitro andin vivo

Maize pollen actin has been labeled with Oregon Green 488 iodoacetamide. A yield of 3 mg fluorescent actin analogue has been obtained from 10 mg of maize pollen actin, which is 99% in purity and the dye/protein ratio is 72%. In the presence of Mg²⁺ and K⁺, the fluorescent actin analogue polymerized into filamentsin vitro. Green fluorescent filaments were observed when the fluorescent actin was introduced into living plant cells by microinjection, indicating that the fluorescent actin analogue functions similarly to the native actin.     

Cardiac protective role of a novel erythrocyte-derived depressing factor on rats and its Ca^2＋ mechanism

The cardiac protective role of a novel erythro-cyte-derived depressing factor (EDDF) on spontaneous hy-pertensive rats (SHR), calcium overload (CaO) rats and Wistar rats and its mechanism was evaluated. Mean artery pressure (MAP), heart rate (HR) and LVdp/dtmax were measured by physiological recorder. The effect of EDDF on the Ca2+-ATPase activity in myocardial sarcoplasmic reticu-lum (SR) of CaO rats was determined by inorganic phos-phate assay. Calcium transport in myocytes was measured by 45Ca2+ radioactive isotope measurement. The phosphoryla-tion levels of extracellular signal-regulated protein kinases (ERK1/2) in myocardial tissue of SHR and CaO rats were measured by Western blot method. And the ultrastructures of cardiac muscle cells were observed with the transmission electron microscope. The results indicated that EDDF could significantly decrease MAP, HR and LVdp/dtmax in a dose dependent manner (P < 0.05). It seems that the mechanism might relate with activating the Ca2+-APTase, enhancing the uptake and release of Ca2+ from SR (P < 0.05), decreasing the phosphorylation levels of ERK1/2 of myocytes (P < 0.01) and lightening the ultrastructural lesion of cardiac muscle cells. In CaO rats, the Ca2+-ATPase activity decreased clearly com-pared to control (64.99 7.16 vs 94.48 7.68 nmol·min-1 ·mg-1 protein, P < 0.01), while EDDF (100 mg/mL) could significantly increase the activity (87.93 ?9.54 vs 64.99 ?7.16, P < 0.05, n = 7). Both uptake and release rate of Ca2+ (祄ol 45Ca2+/g protein/min) from myocardial SR of CaO rats re-markably decreased compared to control (32.40 ?2.70 and 15.46 ?1.49 vs 61.09 ?10.89 and 25.47 ?4.29, P < 0.05); EDDF (100 mg/mL) could significantly stimulate their activi-ties (50.48 6.76 and 21.76 2.75 vs 32.40 2.70 and 15.46 1.49, P < 0.05). EDDF could evidently down-regulate the phosphorylation of ERK1/2 in myocardial tissue from SHR and CaO rats (P < 0.01), lighten the ultrastructural lesion of cardiac muscle cells of SHR as well. It is concluded that EDDF seems to play protective roles on both structure and function of heart, which closely related with amelioration of Ca2+ transport and inhibition of Ca2+-MAP kinase pathway.     

Activities and properties of calcineurin catalytic domain

Calcineurin (CN) is the only protein phosphatase known to be under the control of calcium (Ca²⁺) and calmodulin (CaM). The enzyme consists of two subunits, the catalytic A subunit of 61 ku (CNA) and a regulatory B subunit of 19 ku (CNB). In this study, we used PCR amplication to construct a truncation consisting of only the CNA catalytic domain. The truncation was induced by IPTG and expressed inE. coli. PNPP was used as a substrate to study the phosphatase activity of the CNA catalytic domain. The findings show that its activity is 20 times greater than CNA in the presence of CNB and CaM. The optimum reaction temperature for the CNA catalytic domain protein is 40°C, and the optimum reaction pH value is 8.0. Mn²⁺ is still an effective activator for the CNA catalytic domain, but its activity is not controlled by Ca²⁺. In the presence of 6 mmol/L Mg²⁺, adding either Ca²⁺ or EGTA did not change the activity of the CNA catalytic domain.     

Regulation of non-muscle myosin assembly by calmodulin-dependent light chain kinase

The presence of actin and myosin in non-muscle cells suggests that they may be involved in a wide range of cellular contractile activities. The generally accepted view is that interaction between actin and myosin in these cells and in vertebrate smooth muscle, is regulated by the level of phosphorylation of the 20,000-molecular weight (MW) light chain. In the absence of calcium, this light chain is not phosphorylated and the myosin cannot interact with actin. Calcium activates a specific calmodulin-dependent kinase which phosphorylates the light chain, initiating actin-myosin interaction. Although most studies on the role of phosphorylation have concentration on the regulation of actin-activated myosin Mg-ATPase activity, phosphorylation of the light chain also seems to control the assembly of smooth muscle myosin into filaments. Using purified smooth muscle light chain kinase, we have confirmed this observation. We report here studies of myosins isolated from the two non-muscle sources, thymus cells and platelets. We observed that these myosins are assembled into filaments at physiological ionic strength and Mg-ATP concentrations, only when the 20,000-MW light chain is phosphorylated.     

Redistribution of intermediate filaments during capping of lymphocyte surface molecules

Intermediate filaments (IF) constitute a major cytoplasmic filamentous network of higher eukaryotic cells that is distinct from actin and myosin microfilaments or microtubules. Although structurally similar, these filaments are formed by chemically and antigenically different proteins. Vimentin is the major IF polypeptide of mesenchymal cells and cultured non-mesenchymal cell lines. Recently, we have characterized a monoclonal IgM antibody from a patient with Waldenstr?m's macroglobulinaemia which is directed against vimentin. Using this monoclonal antibody, we have shown by direct immunofluorescence that intermediate filaments of human B and T lymphocytes consist of vimentin. In cells exposed to colcemid, the intermediate filaments retracted into a juxtanuclear aggregate ('coli') characteristic of vimentin filaments. As most components of the cytoskeleton, especially actin and myosin, have been implicated in the capping phenomenon, we investigated the effect of capping of either beta 2-microglobulin or membrane immunoglobulins on the organization of the intermediate filament network. We report that capping of these surface molecules induced the redistribution of vimentin just beneath the cap. When colcemid-treated cells were allowed to cap, the location of the cap always coincided with the coil, suggesting that the anchorage point of intermediate filaments is situated within the uropod.     

The essential light chains constitute part of the active site of smooth muscle myosin

Myosin, a major contractile protein, characteristically possesses a long coiled-coil alpha-helical tail and two heads. Each head contains both an actin binding site and an ATPase site and is formed from the NH2-terminal half of one of the two heavy chains (relative molecular mass, Mr, 200,000) and a pair of light chains; the so-called regulatory and essential light chains of approximately Mr 20,000 each. Recently we have identified Trp 130 of the myosin heavy chain from rabbit skeletal muscle as an active-site amino-acid residue after labelling with a new photoaffinity analogue of ADP, N-(4-azido-2-nitrophenyl)-2-aminoethyl diphosphate (NANDP). Nonspecific labelling was eliminated by first trapping NANDP at the active site with thiol crosslinking agents. Exclusive labelling of the heavy chains with no labelling of the light chains agreed with previous findings that the heavy chains alone contain the actin-activated Mg-ATPase activity of rabbit skeletal myosin. Here we report similar photolabelling experiments with smooth muscle myosin (chicken gizzard) in which 3H-NANDP is trapped at the active site with vanadate and which show that both the heavy chains and the essential light chains are labelled. The results indicate that both chains contribute to the ATP binding site and represent the first direct evidence for participation of the essential light chains in the active site of any type of myosin.     

Identification of a DNase activated inXenopus egg extracts undergoing apoptosis

A cell-free apoptosis system was established by adding dATP and cytochrome c toXenopus laevis egg extracts S-150. Accompanied by an incubation process, an apoptosis-specific DNase was activated in egg extracts which depended on Mg²⁺ and inhibited by Zn²⁺. Two nucleases existing in egg extracts were revealed by in-gel nuclease assay. Further experiments showed that 27 ku nuclease which was different from other Ca²⁺ /Mg²⁺ -dependent nucleases was a possible candidate involved in apoptosis.