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1.
VDAC forms the major pathway for metabolites across the mitochondrial outer membrane. The regulation of the gating of VDAC channels is an effective way to control the flow of metabolites into and out of mitochondria. Here we present evidence that actin can modulate the gating process of Neurospora crassa VDAC reconstituted into membranes made with phosphatidylcholine. An actin concentration as low as 50 nm caused the VDAC-mediated membrane conductance to drop by as much as 85% at elevated membrane potentials. Actin's effect could be quickly reversed by adding pronase to digest the protein. α-Actin, from mammalian muscle, has a stronger effect than β- and γ-actin from human platelets. The monomeric form of actin, G-actin, is effective. Stabilization of the fibrous form, F-actin, with the mushroom toxin, phalloidin, blocks the effect of actin on VDAC, indicating that F-actin might be ineffective. Cytochalasin B did not interfere with the ability of actin to favor VDAC closure. DNase-I did effectively block actin's effect on VDAC, and VDAC decreased actin's inhibitory effect on DNase-I activity, indicating that N. crassa VDAC competes with DNase-I for the same binding site on actin. The actin-VDAC interaction might be a mechanism by which actin regulates energy metabolism. Received: 28 August 2000/Revised: 1 December 2000  相似文献   

2.
Previous conflicting reports suggest that DNase-I binds F-actin with either equal or drastically different K(D) values compared to G-actin. We developed a high-throughput DNase-I inhibition assay to determine the K(D) of DNase-I for F-actin. We confirmed that phalloidin-stabilized F-actin is protected from depolymerization by DNase-I and that the critical concentration at the pointed end of phalloidin-F-actin is 45.5+/-13.9 nM. We found that DNase-I inhibition by actin follows ultrasensitive mechanics. Using varying lengths of gelsolin-capped phalloidin-F-actin, we concluded that the affinities of DNase-I for G- and the pointed end subunits of F-actin are almost indistinguishable, such that DNase-I may not distinguish between G- and F-actin conformations.  相似文献   

3.
A biochemical assay employing DNase-I affinity chromatography, two- dimensional peptide analysis and SDS polyacrylamide gel electrophoresis was used to isolate, identify, and assess the amount of actin from gonial cells of the crane fly, Nephrotoma suturalis. Based on the analysis of cell homogenates under conditions in which all cellular actin is converted to the monomeric DNase-binding form, actin comprises approximately 1% of the total protein in homogenates of spermatocytes and spermatids. SDS gel analysis of mature sperm reveals no polypeptides with a molecular weight similar to that of actin. Under conditions that preserve native supramolecular states of actin, approximately 80% of the spermatocyte actin is in a sedimentable form whereas only approximately 30% of the spermatid actin is sedimentable. These differences could be meaningful with regard to strutural changes that occur during spermiogenesis. A comparative analysis of two- dimensional peptide maps of several radioiodinated actins reveals similarities among spermatocyte, spermatid, and human erythrocyte actins. The results suggest the general applicability of this approach to other cell types that contain limited amounts of actin.  相似文献   

4.
Crude protein extracts of Uromyces appendiculatus contain a polypeptide that resembles actin in several ways. This protein eluates from DEAE-cellulose with concentrations of KCl known to release actin of other species from the cation. The polypeptide is recognized by polyclonal antibodies directed to sodium dodecyl sulfate-denatured actin of chicken gizzard as well as by a monoclonal antibody also made to gizzard actin from chicken, but not by antibodies made against rabbit skeletal muscle actin. Western blot analysis after electrophoresis of the protein on polyacrylamide revealed that the protein has an electrophoretic mobility very similar to that of rabbit skeletal muscle actin. We were unable either to isolate actin by affinity chromatography using immobilized DNase-I, or to identify bean rust actin using DNase-I inhibition assays. Nevertheless, large quantities of the protein sedimented by high speed centrifugation. The sedimented protein resisted attempts to solubilize it under conditions normally used to depolymerize actin filaments. Both of the latter findings indicate unusual features of bean rust actin. Immunocytochemical studies of actin localization in germlings of the fungus using two chicken gizzard actin antibodies revealed actin-containing sites which were similar to those previously observed with fluorescently tagged phallotoxin derivatives.  相似文献   

5.
Actin and nonmuscle myosin heavy chain (myosin-II) have been identified and localized in the cortex of unfertilized zebrafish eggs using techniques of SDS-polyacrylamide gel electrophoresis, immunoblotting, and fluorescence microscopy. Whole egg mounts, egg fragments, cryosections, and cortical membrane patches probed with rhodamine phalloidin, fluorescent DNase-I, or anti-actin antibody showed the cortical cytoskeleton to contain two domains of actin: filamentous and nonfilamentous. Filamentous actin was restricted to microplicae and the cytoplasmic face of the plasma membrane where it was organized as an extensive meshwork of interconnecting filaments. The cortical cytoplasm deep to the plasma membrane contained cortical granules and sequestered actin in nonfilamentous form. The cytoplasmic surface (membrane?) of cortical granules displayed an enrichment of nonfilamentous actin. An antibody against human platelet myosin was used to detect myosin-II in whole mounts and egg fragments. Myosin-II colocalized with both filamentous and nonfilamentous actin domains of the cortical cytoskeleton. It was not determined if egg myosin was organized into filaments. Similar to nonfilamentous actin, myosin-II appeared to be concentrated over the surface of cortical granules where staining was in the form of patches and punctate foci. The identification of organized and interconnected domains of filamentous actin, nonfilamentous actin, and myosin-II provides insight into possible functions of these proteins before and after fertilization. © 1996 Wiley-Liss, Inc.  相似文献   

6.
Overexpression of cardiac actin with baculovirus is promoter dependent   总被引:1,自引:0,他引:1  
The influence of the promoter and an N-terminal hexahistidine tag on human cardiac actin (ACTC) expression and function was investigated using four baculovirus constructs. It was found that both non-tagged ACTC and hisACTC expression from the p10 promoter was higher than from the polh promoter. Characterization showed that an N-terminal hexahistidine tag has a negative effect on ACTC. Recombinant ACTC inhibits DNase-I and binds myosin S1, indicative of proper folding. Our data support the hypothesis that the actin protein down-regulates the polh promoter.  相似文献   

7.
Talin binds to actin and promotes filament nucleation   总被引:11,自引:0,他引:11  
Platelet talin binds to actin in vitro and hence is an actin binding protein. By four different non-interfering assay conditions (fluorescence, fluorescence recovery after photobleaching, (FRAP), dynamic light scattering and DNase-I inhibition) we show that talin promotes filament nucleation, raises the filament number concentration and increases the net rate of actin polymerization but has no inhibitory effect on filament elongation. Binding of talin to actin occurs at a maximal molar ratio of 1:3 as determined by fluorescencetitration under G-buffer conditions. The overall binding constant was approximately 0.25 microM.  相似文献   

8.
S Ohta  G Schatz 《The EMBO journal》1984,3(3):651-657
The beta-subunit of mitochondrial ATPase is coded by a nuclear gene, synthesized outside the mitochondria as a larger precursor and imported into mitochondria. The beta-subunit precursor was purified from yeast, both as a homogeneous, unlabeled polypeptide and in radiochemically pure form. Both precursor preparations were cleaved to the mature beta-subunit by partially purified processing protease from the mitochondrial matrix. However, import of the radiochemically pure precursor into isolated yeast mitochondria required a cytosolic fraction from yeast or reticulocytes. The cytosolic factor was non-dialyzable and trypsin-sensitive; its apparent mol. wt. was approximately 40 000 as judged by gel filtration. Import of some proteins into mitochondria thus requires proteins of the 'soluble' cytoplasm.  相似文献   

9.
Adenosine 5'-triphosphate or ATP is the primary energy source within the cell, releasing its energy via hydrolysis into adenosine 5'-diphosphate or ADP. Actin is an important ATPase involved in many aspects of cellular function, and the binding and hydrolysis of ATP regulates its polymerization into actin filaments as well as its interaction with a host of actin-associated proteins. Here we study the dynamics of monomeric actin in ATP, ADP-Pi, and ADP states via molecular dynamics simulations. As observed in some crystal structures we see that the DNase-I loop is an alpha-helix in the ADP state but forms an unstructured coil domain in the ADP-Pi and ATP states. We also find that this secondary structure change is reversible, and by mimicking nucleotide exchange we can observe the transition between the helical and coil states. Apart from the DNase-I loop, we also see several key structural differences in the nucleotide binding cleft as well as in the hydrophobic cleft between subdomains 1 and 3 where WH2-containing proteins have been shown to interact. These differences provide a structural basis for understanding the observed differences between the various nucleotide states of actin and provide some insight into how ATP regulates the interaction of actin with itself and other proteins.  相似文献   

10.
Determining the molecular mechanisms that lead to the development of heart failure will help us gain better insight into the most costly health problem in the Western world. To understand the roles that the actin protein plays in the development of heart failure, we have taken a systematic approach toward characterizing human cardiac actin mutants that have been associated with either hypertrophic or dilated cardiomyopathy. Seven known cardiac actin mutants were expressed in a baculovirus system, and their intrinsic properties were studied. In general, the changes to the properties of the actin proteins themselves were subtle. The R312H variant exhibited reduced stability, with a T(m) of 53.6 °C compared to 56.8 °C for WT actin, accompanied with increased polymerization critical concentration and Pi release rate, and a marked increase in nucleotide release rates. Substitution of methionine for leucine at amino acid 305 showed no impact on the stability, nucleotide release rates, or DNase-I inhibition ability of the actin monomer; however, during polymerization, a 2-fold increase in Pi release was observed. Increases to both the T(m) and DNase-I inhibition activity suggested interactions between E99K actin molecules under monomer-promoting conditions. Y166C actin had a higher critical concentration resulting in a lower Pi release rate due to reduced filament-forming potential. The locations of mutations on the ACTC protein correlated with the molecular effects; in general, mutations in subdomain 3 affected the stability of the ACTC protein or affect the polymerization of actin filaments, while mutations in subdomains 1 and 4 more likely affect protein-protein interactions.  相似文献   

11.
Three N-terminal double mutants of beta-actin expressed in the yeast Saccharomyces cerevisiae have been characterized with respect to DNase-I interaction, N-terminal post-translational modification, polymerizability and myosin subfragment-1 binding. The results strongly support earlier suggestions that the acidic residues at the N-terminus of actin are part of the myosin-binding site, while they seem to be of no importance for the other aspects of actin biochemistry tested. The suitability of this expression system for production of recombinant actin in general is discussed.  相似文献   

12.
To study the relation between expression of the putative myofibroblast marker α-smooth muscle actin and the remodelling of extracellular matrix, immunocytochemical, gel electrophoresis, and collagen gel contraction studies were performed on two human fibroblast subtypes. Double immunolabelling for total actins and α-smooth muscle (sm) actin as well as affinity labelling of filamentous and monomeric actins in gingival fibroblasts demonstrated that α-sm was colocalized in stress fibres and in regions with high levels of monomeric actin throughout the cytoplasm. α-sm comprised up to 14% of total cellular actin as assessed by 2D gel electrophoresis. Thirteen different gingival and seven different periodontal ligament fibroblast lines constitutively expressed on α-sm actin. These cells exhibited up to 60% inter-line variations of fluorescence due to α-sm actin and up to 70% and 45% inter-line variation in the rate of collagen gel contraction. Quantitative, single cell fluorimetry of α-sm actin immunoreactivity demonstrated a linear relation between gel contraction and α-sm actin (correlation coefficients of 0.71 for gingival and 0.61 for periodontal ligament cells), but there was no detectable relationship between total actin content and gel contraction. In contrast, flow cytometry demonstrated that 99% of the total gated cells from cell lines exhibiting rapid gel contraction showed α-sm actin staining above background fluorescence as compared to only 35% of cells with slow rates of gel contraction. Contracting collagen gels stained with FITC-phalloidin showed cells with well-developed stress fibres that were progressively more compact and elongated during the time of maximal gel contraction. To examine the dependence of gel contraction on assembly of monomeric actin into actin filaments, cells were electroporated in the presence of phalloidin or cytochalasin D. Collagen gels exhibited up to 100% inhibition of gel contraction that was dose-dependent. Gel contraction was inhibited 93% by electroinjection of cells with α-sm actin antibody prior to incubation, but the antibody did not inhibit actin assembly after attachment and spreading on substrates. These data indicate that gel contraction is dependent on α-sm actin expression and that α-sm actin is a functional marker for a fibroblast subtype that rapidly remodels the extracellular matrix. © 1994 wiley-Liss, Inc.  相似文献   

13.
The authors analyzed changes in mitochondrial activity of boar semen during a standard cryopreservation protocol. For this purpose, mitochondrial activity was evaluated simultaneously with the rhythm of mitochondrial formation of reactive oxygen species (mROS) through a double MitoTracker Red/proxylfluorescamine stain. Moreover, we analyzed changes in the expression and location of two key regulatory elements of mitochondrial function, namely mitofusin-2 (Mfn2) and actin, during the freezing-thawing protocol. Our results indicate that mitochondrial activity and mROS formation decreased during cyropreservation, with an initial decrease during the cooling phase of the protocol. This decrease was accompanied by an increase in the amount of solubilized Mfn2, which was concomitant with a progressive extension of Mfn2 location from the apical zone of the midpiece to the whole midpiece. Simultaneously, cryopreservation induced a decrease in solubilized actin, which was concurrent with significant changes in the midpiece actin location. The observed changes in the expression and location of both Mfn2 and actin were already present after the cooling phase of the cryopreservation protocol. Our results suggest that freezing-thawing impaired mitochondrial function. This impairment was concomitant with a decrease in the mitochondrial capacity to synthesize mROS. This impairment is attributed to changes in mitochondrial volume as a result of alterations in the expression and location of both Mfn-2 and the actin network. Finally, the alterations of mitochondrial function induced by the cryopreservation protocol were already apparent at the cooling phase. This observation indicates that the cooling phase is a crucial stage in which mitochondrial alterations occur during cryopreservation.  相似文献   

14.
We have identified a yeast protein that resembles actins from other eucaryotes in its tight binding to pancreatic deoxyribonuclease I, its copolymerizaton with purified muscle actin, its one-dimensional peptide map, and its apparent polymerization into 7-nm filaments. The yeast actin-like protein yielded a single spot on two-dimensional polyacrylamide gel electrophoresis, suggesting that a single protein species was present. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the actin-like protein had an apparent molecular weight of 45,000 compared with 42,000 for muscle actin. In an attempt to identify the messenger ribonucleic acid coding for the actin-like protein, yeast polyadenylic acid-rich ribonucleic acid was translated in wheat germ and reticulocyte cell-free protein-synthesizing systems. The actin-like protein was identified among the translation products of the reticulocyte system by its tight binding to deoxyribonuclease I, its comigration with the in vivo-synthesized actin-like protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an the similarity of its peptide map to that of the in vivo-synthesized protein. A yeast protein synthesized in the wheat-germ system was also found to bind to deoxyribonuclease I and to copolymerize with muscle actin. However, its apparent molecular weight was about 35,000, suggesting that it was a product either of incomplete translation or of proteolytic cleavage of the actin-like protein.  相似文献   

15.
Mitochondrial preparations isolated from bovine kidney and brain as well as the liver and the brain of rat show thiamine triphosphatase (ThTPase) activity. The activity was determined from the particles by freezing-thawing suggesting that a soluble enzyme is involved. The liberation patterns of ThTPase and marker enzyme activities from mitochondria under osmotic shock or treatment with increasing Triton X-100 concentrations indicate the presence of ThTPase both in the matrix and intermembrane space. It was found, basing on gel filtration behavior, that the mitochondrial ThTPase has the same molecular mass as specific cytosolic ThTPase (EC 3.6.1.28). The enzymes, however, were clearly distinguishable in Km values, the mitochondrial one showing a higher apparent affinity for substrate. These results imply the existence of ThTPase multiple forms in mammalian cells.  相似文献   

16.
Vertebrate striated muscle contraction is regulated in a Ca(2+)-dependent fashion by tropomyosin (Tm) and troponin (Tn). This regulation involves shifts in the position of Tm and Tn on actin filaments and may include conformational changes in actin that are then communicated to myosin subfragment 1 (S1). To determine whether subdomain 2 of actin plays a role in this regulation, the DNase-I loop 38-52 of this subdomain was cleaved by subtilisin between residues Met(47) and Gly(48). Despite impaired unregulated function, the potentiation and regulation of cleaved actin movement in the in vitro motility assay was not significantly different from that of uncleaved actin. Stopped-flow measurements of ADP release from regulated and unregulated cleaved acto-S1 showed a marked increase in ADP release from acto-S1 in the presence of the regulatory complex. The enhancement of the actin affinity for S1 in the presence of regulatory proteins was greater for uncleaved than for cleaved F-actin. Finally, both cleaved and uncleaved actins protect myosin loop 1 from papain cleavage equally well. Our results suggest that the potentiation of actin function in the in vitro motility assay by regulatory proteins stems from changes in cross-bridge cycle kinetics. In addition, the unimpaired calcium-sensitive regulation of cleaved actin indicates that subdomain 2 conformation does not play an essential role in the regulation process.  相似文献   

17.
The status of platelet actin has been studied analytically by the DNase-I inhibition assay. Exposure of platelets to diamide, which oxidizes sulphydryl groups, results in an increase of filamentous actin. Neutralization of the effect of diamide by addition of 2-mercaptopropionylglycine (2-MPG) or washing out the excess of diamide is associated with a normalization of the cellular actin status. These findings strongly suggest that the redox state of platelets is somehow involved in the process of actin polymerization. Alterations of the redox state in metabolically altered platelets could therefore account for changes in the G- to F-actin equilibrium. In the thrombin-induced actin polymerization the redox state of the cell seems to be not involved, since 2-MPG preincubation of platelets (1.25 mM, 30 min) does not inhibit the filament assembly.  相似文献   

18.
Extraction of Nicotiana tabacum cell cultures, chromatography on DEAE-cellulose and gel filtration resulted in a homogeneous protein (Mr = 14500), which strongly reduces the hydrolysis of Escherichia coli DNA by DNase I. DNA degradation by micrococcal nuclease is not inhibited. The inhibitor protein interacts with DNase I in the absence of DNA, as determined by the partial quenching of protein intrinsic fluorescence; a 1:1 stoichiometry is deduced. From the reduction of DNase I activity with increasing inhibitor concentration apparent equilibrium constants for the inhibitor X DNase-I complex have been calculated. This interaction is strongly temperature-dependent; at 20 degrees C and 26 degrees C dissociation constants of 5 nM and 110 nM, respectively, were determined. As a consequence a rather high enthalpy of interaction can be estimated.  相似文献   

19.
Actin Degradation in the Metamorphosing Bullfrog Tadpole Tail   总被引:1,自引:1,他引:0  
Degradation of tail muscle proteins was investigated during metamorphosis of Rana catesbeiana , tadpole. Regressing tail muscle contained actomyosin which was comparable to that of non-regressing tail muscle in its physico-chemical character, althouth the actomyosin content of the former tissue decreased as compared to the latter. However, when muscle proteins were extracted in the SDS-containing medium (TSM) and analyzed by SDS-polyacrylamide gel electrophoresis, we found that the protein band corresponding to actin disappeared completely during the late climax stage of metamorphosis. Detailed studies on this phenomenon showed that the apparent absence of actin on SDS-polyacrylamide gel electrophoresis was dependent upon the metamorphic stages of the tadpoles investigated. When TSM extract from the premetamorphic tadpole tail muscles which contained actin was incubated with the same extract from tadpoles of the climax stage, actin derived from premetamorphic tadpole disappeared on gel electrophoresis, indicating that tail muscle tissues of the climax stages contain the actin-degrading enzyme. Characterization of the enzyme was performed with a crude extract using actin prepared from rabbit thigh muscle as a substrate. Actin degrading activity showed incubation time- and temperature-dependency and the activity decreased gradually when the extract was preheated at increasing temperatures with the complete inactivation at 100°C. The major degradation products of actin hydrolysis by the enzyme had a Mr=28,000 and 14,000 which indicated the enzyme splits actin at a specific point. The activity had an optimum pH of 7.5 and was inhibited by leupeptin and iodoacetate and required the presence of a thiol reagent.  相似文献   

20.
Several lines of evidence indicate that a component of axonemes from Chlamydomonas flagella is similar to actin from rabbit skeletal muscle. The polypeptide has an apparent molecular weight of 42,000 and in a pH gradient has the electrophoretic behavior of beta-actin. It was co-polymerized with rabbit actin and purified by affinity chromatography on DNase I-Sepharose. Incomplete proteolysis of mixed 35S-labeled axonemal protein and cold rabbit actin formed similar sets of peptides as analyzed by one-dimensional gel electrophoresis. The actin-like protein and the tubulin appear to be present in the axoneme in the molar ratio 1:60.  相似文献   

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