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1.
《Cell biology international reports》1984,8(12):1065-1068
Application of ATP to cryosections of plasmodial strands from Physarum polycephalum leads to an isotonic contraction of the cytoplasmic actomyosin fibrils: when the fibrils are labelled with NBD-phallacidin, their contraction can be observed in the fluorescence microscope.While performing contraction, the fibrils separate into many small units and the formerly continuous fibrils exhibit the appearance of beaded chains.The possibility of visualizing directly the contraction of cytoplasmic actomyosin fibrils in the fluorescence microscope represents a favourable condition for the study of their physiological contraction mechanism, because this new and convenient cell-free model offers in situ contractile structures that are non-denatured and non-extracted. 相似文献
2.
Cell-free models should offer "in situ conditions" to study the physiology of cytoplasmic actomyosin in its natural environment, while, if possible, still associated with its regulatory control proteins and other cytoplasmic components. Detergents and glycerol as the usual media to permeabilize the plasmalemma and to extract a portion of the cytoplasmic components, are accompanied by several disadvantages. We investigated a cell-free model consisting of cryosections of plasmodial strands that were previously enriched with "stress fibrils" and fluorescently labelled with phallotoxins and that contain the non-denatured structures that are to be reactivated in situ. The contraction reaction can be directly observed in the fluorescence microscope. This procedure allows the study of contraction conditions in the natural environment of the fibrils. The aim of these reactivation experiments was to identify the role of calcium ions. According to our results, a reactivation of cryosections is not Ca++ dependent but is partly inhibited at concentrations of 10(-4) to 5 X 10(-2)M Ca++. Complete inhibition occurs at 10 to 20 mM Ca++. Electron microscopical investigations revealed that the fluorescently labelled contracting structures were identical to actomyosin fibrils. 相似文献
3.
M. Hauser 《Protoplasma》1980,102(1-2):53-62
Summary The addition of 2 mM-3 mM ATP to macronuclei ofParamecium bursaria suspended in a glycerol buffer medium causes a decrease in their volume up to 23% within 3 minutes. The infiltration medium must not only contain Ca2+, but must also be of low ionic strength for ATP to be effective. A slow, careful exchange of the glycerol medium for the contraction solution is also necessary. Ca2+ present alone in the standard contraction buffer can likewise induce a limited volume decrease; in the presence of low concentrations of Ca2+, Mg2+ shows no detectable effect on glycerinated nuclei. When the nuclear volume has been reduced by ATP in the presence of Ca2+, the addition of EGTA induces a reexpansion of the nuclei. Salyrgan, an organic mercurial, either prevents or abolishes the ATP-induced contraction. Other nucleotide triphosphates such as guanosine triphosphate (GTP), inosine triphosphate (ITP) or uridine triphosphate (UTP) likewise induce a volume decrease of the glycerinated macronuclei, but to a distinctly lesser extent than ATP.The results indicate that the volume decrease caused by the ATP-contraction solution is not a passive osmotic process. The resemblance to actomyosin contractions suggests that the volume decrease reported here might also be the result of the reaction of nuclear actomyosin and ATP. 相似文献
4.
A combined application of 5 mM KCN and 19 microM Ca++-ionophore A-23187 leads to pronounced contractures of plasmodial strands of Physarum polycephalum. The appearance of the contractures is independent of the amount of Ca++ in the external medium. Tensiometric registrations of longitudinal contraction activity (isometric regime) reveal an average tension increase of 50 mp compared with the preceding tension level before the addition of KCN and ionophore. This high force output during the contracture coincides with a pronounced increase in the number of cytoplasmic actomyosin fibrils. Their ultrastructure is seen as a high lateral density of strictly parallel arranged F-actin filaments; the state of cytoplasmic actomyosin during this isometric contracture corresponds to the ultrastructure of isometrically contracted fibrils during the normal contraction-relaxation cycle of this organism. A simultaneous impediment of respiration and Ca++ homeostasis strongly favours a shift of the actin equilibrium to the high polymeric side in the form of fibrils and may thus be used as a preparatory step improving the specimens in the context of other investigations, e.g., for immunocytochemical investigations or for the preparation of cell-free models to be reactivated after extraction procedures. 相似文献
5.
Vivianne T. Nachmias 《Protoplasma》1981,109(1-2):13-21
Summary
Physarum myosin is composed of a heavy chain of about 225,000 daltons and two small polypeptides of 17,700 and 16,100 daltons, called light chain one (LC 1) and two (LC 2). Light chain one is shown to belong to the general class of regulating light chains by two independent criteria. After denaturation, purification and renaturation of thePhysarum light chains only LC 1 will combine with scallop myofibrils in which one myosin regulatory light chain has been removed. This LC 1 can restore inhibition of the ATPase activity of the myofibrils at 10–8 M Ca++ just as well as light chains from rabbit skeletal myosin. Secondly, this LC 1 is the only component of the myosin that is significantly phosphorylated by an endogenous kinase present in crude actomyosin. An active phosphatase is also present. Preliminary results could not detect calcium sensitivity for either kinase or phosphatase, nevertheless the importance of phosphorylation in affecting activity of biological systems suggests that LC 1 may serve some regulating function for plasmodial actomyosin. 相似文献
6.
Sham Sunder Arvinder Jit Singh Sukhdeep Gill Balwant Singh 《Molecular and cellular biochemistry》1996,158(2):121-124
The intracellular level of Na+ and K+ of S. cerevisiae strain AB1375 revealed that under KCl as well as sorbitol stress, the cationic level was comparable to the level under no stress conditions. On the other hand, there was a sharp drop in the intracellular K+ content and increase in the Na+ content on addition of NaCl to the medium. However, the total cationic level was close to that under control conditions. In addition to changes in the cationic level, an enhanced production and accumulation of glycerol were also observed under osmotic stress. A regulatory mechanism co-ordinating the intracellular concentration of glycerol as well as Na+, K+ content under osmotic stress conditions has been proposed. 相似文献
7.
Summary The two human homologues of the fission yeast cell cycle protein p13
suc1
displayed structural characteristics consistent with their existing in solution as differently folded monomers despite 81% identity with respect to their primary structures and both being capable of fulfilling the functions of their homologues in fission and budding yeasts. Carboxyfluorescein-labelled p9
CksHs1
and p9
CksHs2
retained their native structures. When microinjected into live stamen hair cells ofTradescantia virginiana, the labelled proteins accumulated in the nuclei of the cells. Markedly different nuclearaccumulation kinetics indicated that the human proteins interact differently with other cellular constituents, which supports the proposition that they may have different roles in cellular regulation.Abbreviations Cdk
cyclin-dependent kinase
- tris
tris(hydroxymethyl)aminomethane
- Hepes
N-(2-hydroxyethyl)piperazine-N-(3-ethanesulphonic acid)
- CF
5(6)-carboxyfluorescein-N-hydroxysuccinamide ester
- SDS-PAGE
sodium dodecyl sulphatepolyacrylamide gel electrophoresis
- IEF
isoelectric focusing
- DEAE
Sephacel diethylaminoethyl Sephacel
- ELISA
enzyme-linked immunosorbent assay
- IgG
immunoglobulin 相似文献
8.
Transfer of 15N between interacting mycelia of a wood-decomposing fungus (Hypholoma fasciculare) and an ectomycorrhizal fungus (Tomentellopsis submollis) was studied in a mature beech (Fagus sylvatica) forest. The amount of 15N transferred from the wood decomposer to the ectomycorrhizal fungus was compared to the amount of 15N released from the wood-decomposing mycelia into the soil solution as 15N-NH4. The study was performed in peat-filled plastic containers placed in forest soil in the field. The wood-decomposing mycelium was growing from an inoculated wood piece and the ectomycorrhizal mycelium from an introduced root from a mature tree. The containers were harvested after 41 weeks when physical contact between the two foraging mycelia was established. At harvest, 15N content was analyzed in the peat (total N and 15NH4
+) and in the mycorrhizal roots. A limited amount of 15N was transferred to the ectomycorrhizal fungus and this transfer could be explained by 15NH4
+ released from the wood-decomposing fungus without involving any antagonistic interactions between the two mycelia. Using our approach, it was possible to study nutritional interactions between basidiomycete mycelia under field conditions and this and earlier studies suggest that the outcomes of such interactions are highly species-specific and depend on environmental conditions such as resource availability. 相似文献
9.
J. Dvořak M. M. Noaman S. Goyal J. Gorham 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1994,87(7):872-877
Durum wheat, Triticum turgidum L. (2n= 4x=28, genome formula AABB) is inferior to bread wheat, T. aestivum L. (2n=6x=42, genome formula AABBDD), in the ability to exclude Na+ under salt strees, in the ratio of the accumulated K+ to Na+ in the leaves under salt stress, and in tolerance of salt stress. Previous work showed that chromosome 4D has a major effect on Na+ and K+ accumulation in the leaves of bread wheat. The 4D chromosome was recombined with chromosome 4B in the genetic background of durum wheat. The recombinants showed that Na+ exclusion and enhanced K+/Na+ ratio in the shoots were controlled by a single locus, Kna1, in the long arm of chromosome 4D. The recombinant families were grown in the field under non-saline conditions and two levels of salinity to determine whether Kna1 confers salt tolerance. Under salt stress, the Kna1 families had higher K+/Na+ ratios in the flag leaves and higher yields of grain and biomass than the Kna1
- families and the parental cultivars. Kna1 is, therefore, one of the factors responsible for the higher salt tolerance of bread wheat relative to durum wheat. The present work provides conceptual evidence that tolerance of salt stress can be transferred between species in the tribe Triticeae. 相似文献
10.
Summary Fragments excised from front regions of thinspread Physarum plasmodia were used to examine a possible correlation between the periodical dynamic activity of such specimens and the spatial organization of actin fibrils. Under isotonic conditions, symmetrical contractions and relaxations of the entire fragment alternate with a period of 1–4 min, whereas under isometric conditions local contractions and relaxations occur simultaneously in different regions of the same specimen. Rapid fixation and phalloidin-staining at distinct stages of the contraction-relaxation cycle demonstrates the permanent existence of cytoplasmic actin fibrils under both isometric and isotonic conditions. During the transition from relaxation to contraction the fibrils shorten in length from 25.5 m to 21.0 m and increase in density from 1.2 fibrils/1000 m2 to 2.3 fibrils/1000 m2. The present results demonstrate that actin fibrils in Physarum plasmodia are not completely decomposed and reformed every contraction-relaxation cycle.Series Studies on microplasmodia of Physarum polycephalum VIII 相似文献
11.
Isolation and characterization of a Na+/H+ antiporter gene from the halophyte Atriplex gmelini 总被引:25,自引:0,他引:25
Hamada Akira Shono Mariko Xia Tao Ohta Masaru Hayashi Yasuyuki Tanaka Akira Hayakawa Takahiko 《Plant molecular biology》2001,46(1):35-42
With a homologous gene region we successfully isolated a Na+/H+ antiporter gene from a halophytic plant, Atriplex gmelini, and named it AgNHX1. The isolated cDNA is 2607 bp in length and contains one open reading frame, which comprises 555 amino acid residues with a predicted molecular mass of 61.9 kDa. The amino acid sequence of the AgNHX1 gene showed more than 75% identity with those of the previously isolated NHX1 genes from glycophytes, Arabidopsis thaliana and Oryza sativa. The migration pattern of AgNHX1 was shown to correlate with H+-pyrophosphatase and not with P-type H+-ATPase, suggesting the localization of AgNHX1 in a vacuolar membrane. Induction of the AgNHX1 gene was observed by salt stress at both mRNA and protein levels. The expression of the AgNHX1 gene in the yeast mutant, which lacks the vacuolar-type Na+/H+ antiporter gene (NHX1) and has poor viability under the high-salt conditions, showed partial complementation of the NHX1 functions. These results suggest the important role of the AgNHX1 products for salt tolerance. 相似文献
12.
A new isolate of Salmonella, strain MR4, reduced Mn(IV)O2 at 2.3 mM under aerobic conditions by about 83% over 24 h. Direct contact of cells to MnO2 was not necessary as the cell-free spent medium produced a similar amount of Mn(II). Pyruvate (1.6 mM) and oxalate (0.8 mM) were identified in the culture medium and presumed to have a role in Mn(II) production in this microorganism. 相似文献
13.
Salt stress is one of the most serious factors limiting the productivity of agricultural crops. Increasing evidence has demonstrated that vacuolar Na+/H+ antiporters play a crucial role in plant salt tolerance. In the present study, we expressed the Suaeda salsa vacuolar Na+/H+ antiporter SsNHX1 in transgenic rice to investigate whether this can increase the salt tolerance of rice, and to study how overexpression of this gene affected other salt-tolerant mechanisms. It was found that transgenic rice plants showed markedly enhanced tolerance to salt stress and to water deprivation compared with non-transgenic controls upon salt stress imposition under outdoor conditions. Measurements of ion levels indicated that K+, Ca2+ and Mg2+ contents were all higher in transgenic plants than in non-transformed controls. Furthermore, shoot V-ATPase hydrolytic activity was dramatically increased in transgenics compared to that of non-transformed controls under salt stress conditions. Physiological analysis also showed that the photosynthetic activity of the transformed plants was higher whereas the same plants had reduced reactive oxygen species generation. In addition, the soluble sugar content increased in the transgenics compared with that in non-transgenics. These results imply that up-regulation of a vacuolar Na+/H+ antiporter gene in transgenic rice might cause pleiotropic up-regulation of other salt-resistance-related mechanisms to improve salt tolerance.Fengyun Zhao and Zenglan Wang contributed equally to this work. 相似文献
14.
【目的】套索肽作为一类核糖体翻译后修饰肽(RiPPs)广泛分布于放线菌中,以其独特的修饰结构和多样的生理活性受到了广泛的关注。为了更好地研究未知的套索肽,期望开发基于链霉菌的无细胞转录翻译平台(下称“无细胞平台”)实现无细胞合成套索肽或其前体肽。【方法】首先尝试以不同的链霉菌构建无细胞合成平台,并以绿色荧光蛋白为报告蛋白对平台产率进行优化;在构建合适稳定的表达体系后,将包含有套索肽生物合成基因的质粒引入体系中以探索套索肽的无细胞合成。【结果】在对基于模式菌株Streptomyces lividans TK24的无细胞体系进行制备工艺、体系组分、反应条件等多个参数进行优化后,该体系最高能达到90μg/mL的荧光蛋白表达量;基于该体系成功表达了目标套索肽的前体肽,并通过融合SUMO标签增加前体肽在该体系中的稳定性。【结论】本研究成功构建了一类链霉菌无细胞平台,为丰富来源的基因表达提供了可能性。尽管该体系在对表达套索肽未知蛋白的适用性上仍有待进一步提升,但无细胞平台在天然产物的探索中将起到越来越重要的作用。 相似文献
15.
Richard Grosse Tom Rapoport Johannes Malur Jrg Fischer Kurt R.H. Repke Ian M. Glynn 《生物化学与生物物理学报:生物膜》1979,550(3)
The controlling effect of ATP, K+ and Na+ on the rate of (Na+ + K+)-ATPase inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is used for the mathematical modelling of the interaction of the effectors with the enzyme under equilibrium conditions.
- 1. 1. Of a series of conceivable interaction models, designed without conceptual restrictions to describe the effector control of inactivation kinetics, only one fits the experimental data described in a preceding paper.
- 2. 2. The model is characterized by the coexistence of two binding sites for ATP and the coexistence of two separate binding sites for K+ and Na+ on the enzyme-ATP complex. On the basis of this model, the effector parameters fitting the experimental data most closely are estimated by means of nonlinear least-squares fits.
- 3. 3. The apparent dissociation constants for ATP of the enzyme-ATP complex or of the enzyme-(ATP)2 complex are computed to lie near 0.0024 mM and 0.34 mM, respectively, irrespective of whether K+ and Na+ were absent or K+ and K+ plus Na+, respectively, were present in the experiments.
- 4. 4. The origin of the high and the low affinity site for binding of ATP to the (Na+ + K+)-ATPase molecule is traced back to the coexistence of two catalytic centres which, although primarily equivalent as to the reactivity of their thiol groups with NBD-Cl, are induced into anticooperative communication by ATP binding and thus show an induced geometric asymmetry.
Keywords: (Na+ + K+)-ATPase; SH-group alkylation; Inactivation kinetics; Mathematical modelling; Substrate affinity 相似文献
16.
Michele Lorusso Tiziana Cocco Michele Minuto Nazzareno Capitanio Sergio Papa 《Journal of bioenergetics and biomembranes》1995,27(1):101-108
The effect of pH and transmembrane pH on the efficiency of the proton pump of the mitochondrialbc
1 complex bothin situ and in the reconstituted state was studied. In both cases the H+/e
– ratio for vectorial proton translocation by thebc
1 complex respiring at the steady state, under conditions in which the transmembrane pH difference (pH) represents the only component of the proton motive force (p), was significantly lower than that measured under level flow conditions. The latter amounts, at neutral pH, to 1 (2 including the scalar H+ release). In the reconstituted system steady-state pH was modulated by changing the intravesicular buffer as well as the intra/extra-liposomal pH. Under these conditions the H+/e– ratio varied inversely with the pH. The data presented show that pH exerts a critical control on the proton pump of thebc
1 complex. Increasing the external pH above neutrality caused a decrease of the level flowH
+/e
– ratio. This effect is explained in terms of proton/electron linkage inb cytochromes. 相似文献
17.
P. R. Levering G. Weenk W. Olijve L. Dijkhuizen W. Harder 《Archives of microbiology》1988,149(6):534-539
Gluconobacter spp. possess the enzymic potential for two pathways of direct glucose oxidation. It has been proposed that the major part of glucose is oxidized to gluconate via NADP-dependent glucose dehydrogenase and that reoxidation of NADPH under these conditions proceeds via recycling of gluconate through ketogluconates. This hypothesis was tested in experiments in which Gluconobacter oxydans ATCC 621-H was grown in glucose-yeast extract medium containing [14C]2-ketogluconate. As expected, glucose was almost quantitatively oxidized to gluconate, without further accumulation of 2- and 5-ketogluconate. Interestingly, the total amount of neither [14C]2-ketogluconate nor [14C]gluconate did change significantly during this oxidation phase, indicating that recycling of gluconate through ketogluconates did not occur. An analysis of enzyme activities in cell-free extracts of glucose-grown cells of G. oxydans ATCC 621-H showed that the membrane-bound glucose dehydrogenase was far more active than the NADP-linked glucose dehydrogenase. The activity of the latter enzyme constituted only 10–15% of that of quinoprotein glucose dehydrogenase and was far too low to match the in vivo rates of gluconate production in batch cultures of G. oxydans. It is concluded that under these conditions glucose is mainly oxidized to gluconate via the membrane-bound glucose dehydrogenase. Implications of these results for the regulation of ketogluconate formation are discussed.Abbreviations DCPIP
2,6-dichlorophenolindophenol
- PMS
phenazine methosulphate
- PQQ
pyrrolo-quinoline quinone 相似文献
18.
Mata Catarina van Vemde Nicole Clarkson David T. Martins-Loução Maria Amélia Lambers Hans 《Plant and Soil》2000,221(1):25-32
Nitrate influx, efflux and net nitrate uptake were measured for the slow-growing Quercus suber L. (cork-oak) to estimate the N-uptake efficiency of its seedlings when grown with free access to nitrate. We hypothesise
that nitrate influx, an energetically costly process, is not very efficiently controlled so as to avoid losses through efflux,
because Q. suber has relatively high respiratory costs for ion uptake. Q. suber seedlings were grown in a growth room in hydroponics with 1 mM NO3
-. Seedlings were labelled with 15NO3
- in nutrient solution for 5 min to measure influx and for 2 h for net uptake. Efflux was calculated as the difference between
influx and net uptake. Measurements were made in the morning, afternoon and night. The site of nitrate reduction was estimated
from the ratio of NO3
- to amino acids in the xylem sap; the observed ratio indicated that nitrate reduction occurred predominantly in the roots.
Nitrate influx was always much higher than net acquisition and both tended to be lower at night. High efflux occurred both
during the day and at night, although the proportion of 15NO3
- taken up that was loss through efflux was proportionally higher during the night. Efflux was a significant fraction of influx.
We concluded that the acquisition system is energetically inefficient under the conditions tested.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
19.
The role of putative Na+/H+ antiporters encoded by nhaS1 (slr1727), nhaS3 (sll0689), nhaS4 (slr1595), and nhaS5 (slr0415) in salt stress response and internal pH regulation of the cyanobacterium Synechocystis PCC 6803 was investigated. For this purpose the mutants (single, double, and triple) impaired in genes coding for Na+/H+ antiporters were constructed using the method of interposon mutagenesis. PCR analyses of DNA demonstrated that mutations in nhaS1, nhaS4, and nhaS5 genes were segregated completely and the mutants contained only inactivated copies of the corresponding genes. Na+/H+ antiporter encoded by nhaS3 was essential for viability of Synechocystis since no completely segregated mutants were obtained. The steady-state intracellular sodium concentration and Na+/H+ antiporter activities were found to be the same in the wild type and all mutants. No differences were found in the growth rates of wild type and mutants during their cultivation in liquid media supplemented with 0.68 M or 0.85 M NaCl as well as in media buffered at pH 7.0, 8.0, or 9.0. The expression of genes coding for Na+/H+ antiporters was studied. No induction of any Na+/H+ antiporter encoding gene expression was found in wild type or single mutant cells grown under high salt or at different pH values. Nevertheless, in cells of double and triple mutants adapted to high salt or alkaline pH some of the remaining Na+/H+ antiporter encoding genes showed induction. These results might indicate that some of Na+/H+ antiporters can functionally replace each other under stress conditions in Synechocystis cells lacking the activity of more than one antiporter. 相似文献
20.
The initial response of coleoptile cells to growth hormones and light is a rapid change in plasma-membrane polarization. We have isolated protoplasts from the cortex of maize (Zea mays L.) coleoptiles to study the electrical properties of their plasma membrane by the patch-clamp techniqueUsing the whole-cell configuration and cell-free membrane patches we could identify an H+-ATPase, hyperpolarizing the membrane potential often more negative than -150 mV, and a voltage-dependent, inward-rectifying K+ channel (unit conductance 5–7 pS) as the major membrane conductan-ces Potassium currents through this channel named CKC1in (for Coleoptile K
+ Channel inward rectifier) were elicited upon voltage steps negative to -80 mV, characterized by a half-activation potential of -112 mV. The kinetics of activation, well described by a double-exponential process, were strongly dependent on the degree of hyperpolarization and the cytoplasmic Ca2+ level. Whereas at nanomolar Ca2+ concentrations K+ currents increased with a t1/2=16 ms (at -180 mV), higher calcium levels slowed the activation process about fourto fivefoldUpon changes in the extracellular K+ concentration the reversal potential of the K+ channel followed the Nernst potential for potassium with a 56-mV shift for a tenfold increaseThe absence of a measurable conductance for Na+, Rb+, Cs+ and a permeability ratio PNH
4
+
/PK+ around 0.25 underlines the high selectivity of CKC1in for K+In contrast to Cs+, which at submillimolar concentration blocks the channel in a voltage-dependent manner, Rb+, often used as a tracer for K+, does not permeate this type of K+ channelThe lack of Rb+ permeability is unique with respect to other K+ transporters. Therefore, future molecular analysis of CKC1in, considered as a unique variation of plant inward rectifiers, might help to understand the permeation properties of K+ channels in general.Abbreviations CKC1in
Coleoptile K
+ Channel inward rectifier
- U
membrane voltage
- Iss
steady-state currents
- Itail
tail currents
Experiments were conducted in the laboratory of F.G. during the stay of RHas a guest professor sponsored by Special Project RAISA, subproject N2.1, paper N2155. 相似文献