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1.
Gelsolin can be purified from horse blood plasma by treating the plasma sequentially with an anion-exchange medium in the presence and then the absence of free Ca2+. The purified gelsolin migrates as a 90-kilodalton protein on electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulfate. It has an absorption coefficient of 1.4 mL/(mg.cm) and is similar in amino acid composition to other plasma gelsolins. Horse plasma gelsolin has an intrinsic sedimentation coefficient of 4.8S and a Stokes' radius of 3.8 nm. Hydrodynamic calculations suggest it to be a rather globular protein of 75,000 relative mass, a value similar to those calculated for human and pig plasma gelsolins from their amino acid sequences. Horse plasma gelsolin is able to nucleate actin polymerization, i.e., to abolish the lag observed between the initiation of polymerization of monomeric actin by the addition of salts and the rapid elongation phase of actin filament growth. This nucleation activity also results in lower final viscosities of F-actin solutions, as the existence of a larger number of filaments in samples that contain gelsolin requires that their average length be shorter. 相似文献
2.
Interaction of plasma gelsolin with ADP-actin 总被引:3,自引:0,他引:3
In the presence of Ca2+, gelsolin forms a very tight, stoichiometric complex with 2 molecules of ADP-G-actin. Removal of free Ca2+ causes the 1:2 complex to dissociate to a 1:1 complex. Gelsolin accelerates the very slow polymerization of ADP-actin, apparently by accelerating the rate of nucleation, but the number concentration of filaments formed is probably less than the gelsolin concentration, indicating that the GA2 complex is not a true nucleus. These results are similar to those obtained for the interaction of gelsolin with ATP-G-actin. Both kinetic and equilibrium measurements demonstrate that the critical concentration of gelsolin-capped ADP-actin filaments (8 microM in 1 mM MgCl2 and 0.2 mM ADP) is the same as for the uncapped filaments, proving that the critical concentration is the same at both ends of the equilibrium polymer in ADP as predicted by theory. The association and dissociation rate constants for the addition of ADP-G-actin at the pointed end of an ADP-F-actin filament are estimated to be 4.6 X 10(4) M-1 s-1 and 0.4 s-1, respectively, about 15-fold lower than the rate constants at the barbed end. 相似文献
3.
H Yamamoto H Ito H Nakamura E Hayashi S Kishimoto T Hashimoto K Tagawa 《Journal of biochemistry》1990,108(4):505-506
Binding studies of human plasma gelsolin with ATP were done by equilibrium dialysis. Analysis of the binding data showed that plasma gelsolin had one class of ATP binding site with Kd = 2.8 x 10(-7) M, which saturated at an ATP/gelsolin ratio of 0.6. The bioluminescent assay for ATP with luciferin and firefly luciferase confirmed that the protein contained a nucleotide as ATP. 相似文献
4.
Severin is a gelsolin prototype 总被引:2,自引:0,他引:2
A number of Ca2(+)-activated actin filament severing proteins have been identified in eukaryotic cells of diverse lineages. Gelsolin and villin, with molecular mass of about 80-90 kDa, and severin and fragmin, with molecular mass of about 40 kDa, have been isolated from vertebrates and invertebrates, respectively. We report here a direct comparison of the functional properties of gelsolin and severin, and the finding that the actin filament severing activity of severin, like that of gelsolin, is inhibited by polyphosphoinositides. However, severin does not nucleate actin filament assembly as well as gelsolin. These characteristics are very similar to those ascribed to the NH2-terminal half of gelsolin, supporting the idea that they are evolutionarily related. Regulation of severin by polyphospholipids raises the possibility that it may participate in agonist-stimulated regulation of the actin cytoskeleton in Dictyostelium discoideum. 相似文献
5.
Inactivation of endotoxin by human plasma gelsolin 总被引:7,自引:0,他引:7
Bucki R Georges PC Espinassous Q Funaki M Pastore JJ Chaby R Janmey PA 《Biochemistry》2005,44(28):9590-9597
Septic shock from bacterial endotoxin, triggered by the release of lipopolysaccharide (LPS) molecules from the outer wall of Gram-negative bacteria, is a major cause of human death for which there is no effective treatment once the complex inflammatory pathways stimulated by these small amphipathic molecules are activated. Here we report that plasma gelsolin, a highly conserved human protein, binds LPS from various bacteria with high affinity. Solid-phase binding assays, fluorescence measurements, and functional assays of actin depolymerizing effects show that gelsolin binds more tightly to LPS than it does to its other known lipid ligands, phosphatidylinositol 4,5-bisphosphate and lysophosphatidic acid. Gelsolin also competes with LPS-binding protein (LBP), a high-affinity carrier for LPS. One result of gelsolin-LPS binding is inhibition of the actin binding activity of gelsolin as well as the actin depolymerizing activity of blood serum. Simultaneously, effects of LPS on cellular functions, including cytoskeletal actin remodeling, and collagen-induced platelet activation by pathways independent of toll-like receptors (TLRs) are neutralized by gelsolin and by a peptide based on gelsolin residues 160-169 (GSN160-169) which comprise part of gelsolin's phosphoinositide binding site. Additionally, TLR-dependent NF-kappaB translocation in astrocytes appears to be blocked by gelsolin. These results show a strong effect of LPS on plasma gelsolin function and suggest that some effects of endotoxin in vivo may be mediated or inhibited by plasma gelsolin. 相似文献
6.
Human plasma gelsolin binds to fibronectin 总被引:3,自引:0,他引:3
Human plasma gelsolin, a 93,000-dalton actin-binding protein binds to human plasma fibronectin. Qualitative data obtained from experiments employing quasi-elastic light scattering, sucrose gradient sedimentation, gel filtration chromatography, and fibronectin polymerization indicate that gelsolin and fibronectin form a complex in solution. Solid-phase binding studies show that both human plasma and rabbit macrophage gelsolin bind to immobilized fibronectin with a Kd of about 1 microM in a 1:1 complex. The ability of gelsolin to interact with actin was not affected by the presence of fibronectin. Fibronectin also increased the amount of gelsolin binding to fibrin clots. Binding of gelsolin to fibronectin may serve to localize plasma gelsolin in regions where fibronectin is deposited, such as inflammatory sites. 相似文献
7.
IgG3 is the major source of cryoglobulins in mice 总被引:8,自引:0,他引:8
M Abdelmoula F Spertini T Shibata Y Gyotoku S Luzuy P H Lambert S Izui 《Journal of immunology (Baltimore, Md. : 1950)》1989,143(2):526-532
A total of 20 of 23 IgG3 mAb derived from unmanipulated autoimmune MRL/MpJ-lpr/lpr mice was shown to generate cryoglobulins which were composed exclusively of IgG3. Although three IgG3 mAb failed to develop cryoglobulins, they were able to bind nonspecifically to any IgG3 molecules as efficiently as cryoprecipitable IgG did. The direct role of the gamma 3 constant region for the generation of cryoglobulins was demonstrated by the following findings: 1) the cryoglobulin activity was independent of the specificity of the IgG3 mAb, 2) no mAb other than those of the IgG3 subclass, including IgM rheumatoid factors (RF), generated cryoglobulins, and 3) the cryoglobulin activity was gained after the Ig class switch of mAb from IgM to IgG3. Analysis of Ig components in three different sources of cryoglobulins, either induced by the injection of bacterial LPS or by the infection with Plasmodium yoelii in BALB/c mice or developed spontaneously in MRL/MpJ-lpr/lpr mice, revealed the selective concentration of IgG3 in these cryoglobulins; greater than 99%, 73% and 58% of IgG recoverable from these three cryoglobulins, respectively, were IgG3. This further attests to the major role of IgG3 in the generation of cryoglobulins in mice. In addition, the enhanced formation and even induction of IgG3 cryoglobulins in the presence of IgM anti-IgG3 RF mAb, and the enrichment of IgM RF in LPS- or malaria-induced cryoglobulins indicated that IgM RF can be involved in the generation of cryoglobulins by interacting with noncryoprecipitable IgG3 as well as cryoprecipitable IgG3. 相似文献
8.
Horse plasma gelsolin labelled with benzophenone-4-isothiocyanate can be photochemically cross-linked to rabbit cardiac tropomyosin. The cross-linking proceeds with greater efficiency in calcium-containing buffers. Further evidence for interaction between these proteins is provided by retention of fluorescently labelled gelsolin on tropomyosin-agarose affinity columns and by the ability of tropomyosin to cause an increase in the fluorescence intensity of gelsolin labelled with fluorescein-5-isothiocyanate. Both of these effects require the presence of calcium ions. 相似文献
9.
Martín-Romero FJ Gutiérrez-Martín Y Henao F Gutiérrez-Merino C 《Journal of neurochemistry》2002,82(3):604-614
Plasma membrane vesicles from adult rat brain synaptosomes (PMV) have an ascorbate-dependent NADH oxidase activity of 35-40 nmol/min/(mg protein) at saturation by NADH. NADPH is a much less efficient substrate of this oxidase activity, with a Vmax 10-fold lower than that measured for NADH. Ascorbate-dependent NADH oxidase activity accounts for more than 90% of the total NADH oxidase activity of PMV and, in the absence of NADH and in the presence of 1 mm ascorbate, PMV produce ascorbate free radical (AFR) at a rate of 4.0 +/- 0.5 nmol AFR/min/(mg protein). NADH-dependent *O2- production by PMV occurs with a rate of 35 +/- 3 nmol/min/(mg protein), and is a coreaction product of the NADH oxidase activity, because: (i) it is inhibited by more than 90% by addition of ascorbate oxidase, (ii) it is inhibited by 1 micro g/mL wheat germ agglutinin (a potent inhibitor of the plasma membrane AFR reductase activity), and (iii) the KM(NADH) of the plasma membrane NADH oxidase activity and of NADH-dependent *O2- production are identical. Treatment of PMV with repetitive micromolar ONOO- pulses produced almost complete inhibition of the ascorbate-dependent NADH oxidase and *O2- production, and at 50% inhibition addition of coenzyme Q10 almost completely reverts this inhibition. Cytochrome c stimulated 2.5-fold the plasma membrane NADH oxidase, and pretreatment of PMV with repetitive 10 microm ONOO- pulses lowers the K0.5 for cytochrome c stimulation from 6 +/- 1 (control) to 1.5 +/- 0.5 microm. Thus, the ascorbate-dependent plasma membrane NADH oxidase activity can act as a source of neuronal.O2-, which is up-regulated by cytosolic cytochrome c and down-regulated under chronic oxidative stress conditions producing ONOO-. 相似文献
10.
Gelsolin, a multifunctional actin-modulating protein, has two actin-binding sites which may interact cooperatively. Native gelsolin requires micromolar Ca2+ for optimal binding of actin to both sites, and for expression of its actin filament-severing function. Recent work has shown that an NH2-terminal chymotryptic 17-kD fragment of human plasma gelsolin contains one of the actin-binding sites, and that this fragment binds to and severs actin filaments weakly irrespective of whether Ca2+ is present. The other binding site is Ca2+ sensitive, and is found in a chymotryptic peptide derived from the COOH-terminal two-thirds of plasma gelsolin; this fragment does not sever F-actin or accelerate the polymerization of actin. This paper documents that larger thermolysin-derived fragments encompassing the NH2-terminal half of gelsolin sever actin filaments as effectively as native plasma gelsolin, although in a Ca2+-insensitive manner. This result indicates that the NH2-terminal half of gelsolin is the actin-severing domain. The stringent Ca2+ requirement for actin severing found in intact gelsolin is not due to a direct effect of Ca2+ on the severing domain, but indirectly through an effect on domains in the COOH-terminal half of the molecule to allow exposure of both actin-binding sites. 相似文献
11.
M Hiyoshi Y Terano N Tatsumi K Okuda 《Japanese journal of medical science & biology》1992,45(2):59-73
Human plasma gelsolin was purified by column chromatography. The method yielded a protein of high purity and activity. Using this protein, we produced monoclonal antibody (Mab H6B11) against human plasma gelsolin by somatic cell fusion. This monoclonal antibody reacted in a dose-dependent manner with gelsolin derived from human plasma and platelets and neutralized depolymerizing activity to F-actin. It differed from the commercially available substance (Mab G4896; Sigma) in that the time required for the reaction between the antigen and antibody in the enzyme-linked immunosorbent assay could be shortened by one-third. The antibody was judged to be useful in assays for elucidating the physiological role of plasma gelsolin. 相似文献
12.
Loss of calcium sensitivity of plasma gelsolin is associated with the presence of calcium ions during preparation 总被引:1,自引:0,他引:1
Gelsolin is a calcium-dependent actin severing and capping protein. Calcium 'opens' the molecule to make actin binding sites accessible, but removal of calcium from the medium does not necessarily fully reverse this process. The calcium sensitivity of actin monomer binding and actin filament severing is here shown to vary considerably with the source of gelsolin and conditions of preparation. Plasma gelsolin undergoes irreversible loss of calcium sensitivity when prepared in the presence of calcium ions. This is not due solely to effects of bound calcium, because purified human plasma gelsolin expressed in E. coli and stored in calcium shows no comparable loss of calcium sensitivity when prepared or stored in calcium. These results suggest the presence of factors in plasma which, in the presence of calcium, promote an irreversible structural change in gelsolin resulting in permanent loss of calcium sensitivity. 相似文献
13.
Calcium binding of swine plasma gelsolin was examined. When applied to ion-exchange chromatography, its elution volume was drastically altered depending on the free Ca2+ concentration of the medium. The presence of two classes of Ca2+ binding sites, high-affinity sites (Kd = 7 microM) and low-affinity sites (Kd = 1 mM), was suggested from the concentration dependence of the elution volume. The tight binding sites were specific for Ca2+. The weakly bound Ca2+ could be replaced by Mg2+ once the tight binding sites were occupied with Ca2+. The binding of metal ions was totally reversible. Circular dichroism measurement of plasma gelsolin indicated that most change in secondary structure was associated with Ca2+ binding to the high-affinity sites. Binding of Mg2+ to the low-affinity sites caused a secondary structural change different from that caused by Ca2+ bound to the high-affinity sites. Gel permeation chromatography exhibited a small change in Stokes radius with and without Ca2+. Microheterogeneity revealed by isoelectric focusing did not relate to the presence of two classes of Ca2+ binding sites. These results indicated that plasma gelsolin drastically altered its surface charge property due to binding of Ca2+ or Ca2+, Mg2+ with a concomitant conformational change. 相似文献
14.
Affinity separation of human plasma gelsolin on Affi-Gel Blue 总被引:1,自引:0,他引:1
H Yamamoto M Terabayashi T Egawa E Hayashi H Nakamura S Kishimoto 《Journal of biochemistry》1989,105(5):799-802
Human plasma gelsolin was specifically eluted with 1 mM adenosine 5'-triphosphate from an Affi-Gel Blue column. Since the ionic strength of sodium chloride required to elute the protein from the dye column was much higher than that of 1 mM adenosine 5'-triphosphate, the binding of plasma gelsolin with the dye-ligand appeared to be biospecific. Taking advantage of this affinity interaction, we have developed a revised purification method of human plasma gelsolin. The purification included ammonium sulfate precipitation, diethylaminoethyl-Sepharose chromatography, Affi-Gel Blue chromatography, and Phenyl-Sepharose chromatography. The method allowed a reproducible purification of the protein to apparent homogeneity, producing a 331-fold purification with a yield of 6%. 相似文献
15.
16.
Proteins regulating actin assembly in oogenesis and early embryogenesis of Xenopus laevis: gelsolin is the major cytoplasmic actin-binding protein 总被引:1,自引:1,他引:0
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T Ankenbauer J A Kleinschmidt J Vandekerckhove W W Franke 《The Journal of cell biology》1988,107(4):1489-1498
Oocytes, notably those of amphibia, accumulate large pools of nonfilamentous ("soluble") actin, both in the cytoplasm and in the nucleoplasm, which coexist with extensive actin filament arrays in the cytoplasmic cortex. Because the regulation of oogenically accumulated actin is important in various processes of oogenesis, egg formation, fertilization and early embryogenesis, we have purified and characterized the major actin-binding proteins present in oocytes of Xenopus laevis. Here we report that the major actin-binding component in the ooplasm, but not in the nucleus, is a polypeptide of Mr approximately 93,000 on SDS-PAGE that reduces actin polymerization in vitro in a Ca2+-dependent manner but promotes nucleation events, and also reduces the viscosity of actin polymers, indicative of severing activity. We have raised antibodies against the purified oocyte protein and show that it is different from villin, is also prominent in unfertilized eggs and early embryos and is very similar to a corresponding protein present in various tissues and in cultured cells, and appears to be spread over the cytoplasm. Using these antibodies we have isolated a cDNA clone from a lambda gt11 expression library of ovarian poly(A)+-RNA. Determination of the amino acid sequence derived from the nucleotide sequence, together with the directly determined sequence of the amino terminus of the native protein, has shown that this clone encodes the carboxy-terminal half of gelsolin. We conclude that gelsolin is the major actin-modulating protein in oogenesis and early embryogenesis of amphibia, and probably also of other species, that probably also plays an important role in the various Ca2+-dependent gelation and contractility processes characteristic of these development stages. 相似文献
17.
It is widely accepted that the DNA, RNA and protein content of Enterobacteriaceae is regulated as a function of exponential growth rates; macromolecular content increases with faster growth regardless of specific composition of the growth medium. This phenomenon, called growth rate control, primarily involves regulation of ribosomal RNA and ribosomal protein synthesis. However, it was uncertain whether the global regulator ppGpp is the major determinant for growth rate control. Therefore, here we re-evaluate the effect of ppGpp on macromolecular content for different balanced growth rates in defined media. We find that when ppGpp is absent, RNA/protein and RNA/DNA ratios are equivalent in fast and slow growing cells. Moreover, slow growing ppGpp-deficient cells with increased RNA content, display a normal ribosomal subunit composition although polysome content is reduced when compared with fast growing wild-type cells. From this we conclude that growth rate control does not occur in the absence of ppGpp. Also, artificial elevation of ppGpp or introduction of stringent RNA polymerase mutants in ppGpp-deficient cells restores this control. We believe these findings strongly argue in favour of ppGpp and against redundant regulation of growth rate control by other factors in Escherichia coli and other enteric bacteria. 相似文献
18.
19.
H E Harris 《FEBS letters》1985,190(1):81-83
G-Actin-ATP bound to plasma gelsolin to form a 2:1 complex. The complex contained approximately equivalent amounts of nucleotide and actin. More than 84% of this nucleotide was ATP. Half of the bound nucleotide was displaced by cold chase and the remainder did not exchange, implying that the two actins in the complex are not equivalent. 相似文献
20.
Osborn TM Verdrengh M Stossel TP Tarkowski A Bokarewa M 《Arthritis research & therapy》2008,10(5):R117