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1.
We investigated the mechanical properties of two abundant, cytoplasmic proteins from Acanthamoeba, profilin and actin, and found that while both profilin and nonfilamentous actin alone behaved as solids, mixtures of the two proteins were viscoelastic liquids. When allowed to equilibrate, profilin formed a viscoelastic solid with mechanical properties similar to filamentous and nonfilamentous actin. Consequently, profilin itself may contribute significantly to the elasticity and viscosity of cytoplasm. The addition of profilin to nonfilamentous actin caused a phase transition from gel (viscoelastic solid) to sol (viscoelastic liquid) when the concentration of free actin became too low to form a gel. In contrast, profilin had little effect on the rigidity and viscosity of actin filaments. We speculate that nonfilamentous actin and profilin, both of which form shear-sensitive structures, can be modeled as flocculant materials. We conclude that profilin may regulate the rigidity (elasticity) of the cytoplasm not only by inhibiting polymerization of actin, but also by modulating the mechanical properties of nonfilamentous actin. 相似文献
2.
The amino acid sequence of Acanthamoeba profilin 总被引:15,自引:0,他引:15
C Ampe J Vandekerckhove S L Brenner L Tobacman E D Korn 《The Journal of biological chemistry》1985,260(2):834-840
The complete amino acid sequence of Acanthamoeba profilin was determined by aligning tryptic, chymotryptic, thermolysin, and Staphylococcus aureus V8 protease peptides together with the partial NH2-terminal sequences of the tryptophan-cleavage products. Acanthamoeba profilin contains 125 amino acid residues, is NH2-terminally blocked, and has trimethyllysine at position 103. At five positions in the sequence two amino acids were identified indicating that the amoebae express at least two slightly different profilins. Charged residues are unevenly distributed, the NH2-terminal half being very hydrophobic and the COOH-terminal half being especially rich in basic residues. Comparison of the Acanthamoeba profilin sequence with that of calf spleen profilin (Nystrom, L. E., Lindberg, U., Kendrick-Jones, J., and Jakes, R. (1979) FEBS Lett. 101, 161-165) reveals homology in the NH2-terminal region. We suggest, therefore, that this region participates in the actin-binding activity. 相似文献
3.
Assembly of Acanthamoeba actin, of which trace quantities had been labeled with 5-(iodoacetamido)-fluorescein, was quantified using the modulation detection method of fluorescence photobleaching recovery (FPR). This technique permits explicit determination of the fraction of labeled actin incorporated into filaments and the translational diffusion coefficients of the filaments, from which filament length can be calculated. Addition of Acanthamoeba profilin in molar ratios to actin of about 1.1:1 and 2.3:1 retarded the initial kinetics of assembly (induced by addition of 2mM Mg+2) and reduced the fraction of actin incorporated into filaments. The diffusion coefficients of filaments formed were greatly changed by the presence of profilin at short times, but the differences became increasingly smaller at longer times. After 26 hr. the filaments formed in 1.1:1 profilin were about 12% shorter and in 2.3:1 profilin were about 20% shorter than filaments formed by actin alone under the same conditions. 相似文献
4.
The regulation of actin polymerization and the inhibition of monomeric actin ATPase activity by Acanthamoeba profilin 总被引:16,自引:0,他引:16
Profilin inhibits the rate of nucleation of actin polymerization and the rate of filament elongation and also reduces the concentration of F-actin at steady state. Addition of profilin to solutions of F-actin causes depolymerization. The same steady state concentrations of polymerized and nonpolymerized actin are reached whether profilin is added before initiation of polymerization or after polymerization is complete. The KD for formation of the 1:1 complex between Acanthamoeba profilin and Acanthamoeba actin is in the range of 4 to 11 microM; the KD for the reaction between Acanthamoeba profilin and rabbit skeletal muscle actin is about 60 to 80 microM, irrespective of the concentrations of KCl or MgCl2. The critical concentration of actin for polymerization and the KD for the actin-profilin interaction are independent of each other; therefore, a change in the critical concentration of actin alters the amount of actin bound to profilin at steady state. As a consequence, the presence of profilin greatly amplifies the effects of small changes in the actin critical concentration on the concentration of F-actin. Profilin also inhibits the ATPase activity of monomeric actin, the profilin-actin complex being entirely inactive. 相似文献
5.
Amino acid sequence of Acanthamoeba actin 总被引:2,自引:0,他引:2
By amino acid sequence studies, only one form of cytoplasmic actin was detected in Acanthamoeba castellanii. Its amino acid sequence is very similar to the sequences of Dictyostelium and Physarum actins, from which Acanthamoeba actin differs in only nine and seven residues, respectively, including the deletion of the first residue. Acanthamoeba actin is unique in containing a blocked NH2-terminal neutral amino acid (glycine), while all other actins sequenced thus far have a blocked acidic amino acid (aspartic or glutamic) at the NH2 terminus. Acanthamoeba actin is also unique in that it contains an N epsilon-trimethyllysine residue at position 326. Like other actins, Acanthamoeba actin contains an NT-methylhistidine residue at position 73. The protein sequence is in complete agreement with the sequence derived from the nucleotide sequence of an expressed actin gene. 相似文献
6.
Quantitative analysis of the effect of Acanthamoeba profilin on actin filament nucleation and elongation 总被引:20,自引:0,他引:20
The current view of the mechanism of action of Acanthamoeba profilin is that it binds to actin monomers, forming a complex that cannot polymerize [Tobacman, L. S., & Korn, E. D. (1982) J. Biol. Chem. 257, 4166-4170; Tseng, P., & Pollard, T. D. (1982) J. Cell Biol. 94, 213-218; Tobacman, L. S., Brenner, S. L., & Korn, E. D. (1983) J. Biol. Chem. 258, 8806-8812]. This simple model fails to predict two new experimental observations made with Acanthamoeba actin in 50 mM KC1, 1 mM MgCl2, and 1 mM EGTA. First, Acanthamoeba profilin inhibits elongation of actin filaments far more at the pointed end than at the barbed end. According, to the simple model, the Kd for the profilin-actin complex is less than 5 microM on the basis of observations at the pointed end and greater than 50 microM for the barbed end. Second, profilin inhibits nucleation more strongly than elongation. According to the simple model, the Kd for the profilin-actin complex is 60-140 microM on the basis of two assays of elongation but 2-10 microM on the basis of polymerization kinetics that reflect nucleation. These new findings can be explained by a new and more complex model for the mechanism of action that is related to a proposal of Tilney and co-workers [Tilney, L. G., Bonder, E. M., Coluccio, L. M., & Mooseker, M. S. (1983) J. Cell Biol. 97, 113-124]. In this model, profilin can bind both to actin monomers with a Kd of about 5 microM and to the barbed end of actin filaments with a Kd of about 50-100 microM. An actin monomer bound to profilin cannot participate in nucleation or add to the pointed end of an actin filament. It can add to the barbed end of a filament. When profilin is bound to the barbed end of a filament, actin monomers cannot bind to that end, but the terminal actin protomer can dissociate at the usual rate. This model includes two different Kd's--one for profilin bound to actin monomers and one for profilin bound to an actin molecule at the barbed end of a filament. The affinity for the end of the filament is lower by a factor of 10 than the affinity for the monomer, presumably due to the difference in the conformation of the two forms of actin or to steric constraints at the end of the filament. 相似文献
7.
Evaluation of the binding of Acanthamoeba profilin to pyrene-labeled actin by fluorescence enhancement 总被引:6,自引:0,他引:6
We have used a fluorescence assay to measure the binding of Acanthamoeba profilin to monomeric Acanthamoeba and rabbit skeletal muscle actin labeled on cysteine-374 with pyrene iodoacetamide. The wavelengths of the pyrene excitation and emission maxima are constant at 346 and 386 nm, but the fluorescence is enhanced up to 50% by profilin. The higher fluorescence is largely due to higher absorbance in the presence of profilin. The fluorescence enhancement has a hyperbolic dependence on the concentration of profilin, suggesting a single class of binding sites. Linear Scatchard plots yield an estimate of the dissociation constant, Kd, of the complex of profilin with pyrenyl-actin. In low-ionic-strength buffers with 2 to 6 mM imidazole (pH 7.0) and 0.1 mM CaCl2 the Kd is 9 microM for both muscle and Acanthamoeba actin. In 50 mM KCl the Kd for the complex with Acanthamoeba actin is 16 microM, while the Kd for the complex with muscle actin is greater than 50 microM. 相似文献
8.
Elena G. Yarmola Michael R. Bubb 《BioEssays : news and reviews in molecular, cellular and developmental biology》2009,31(11):1150-1160
Rapid polymerization and depolymerization of actin filaments in response to extracellular stimuli is required for normal cell motility and development. Profilin is one of the most important actin‐binding proteins; it regulates actin polymerization and interacts with many cytoskeletal proteins that link actin to extracellular membrane. The molecular mechanism of profilin has been extensively considered and debated in the literature for over two decades. Here we discuss several accepted hypotheses regarding the mechanism of profilin function as well as new recently emerged possibilities. Thermal noise is routine in molecular world and unsurprisingly, nature has found a way to utilize it. An increasing amount of theoretical and experimental research suggests that fluctuation‐based processes play important roles in many cell events. Here we show how a fluctuation‐based process of exchange diffusion is involved in the regulation of actin polymerization. 相似文献
9.
10.
Purification and characterization of two isoforms of Acanthamoeba profilin 总被引:9,自引:4,他引:5
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《The Journal of cell biology》1986,102(1):221-226
Acanthamoeba profilin purified according to E. Reichstein and E.D. Korn (1979, J. Biol. Chem. 254:6174-6179) consists of two isoforms (profilin- I and-II) with approximately the same molecular weight and reactivity to a monoclonal antibody but different isoelectric points and different mobilities on carboxymethyl-agarose chromatography and reversed-phase high-performance liquid chromatography. The isoelectric points of profilin-I is approximately 5.5 and that of profilin-II is greater than or equal to 9.0. Tryptic peptides from the two proteins are substantially different, which suggests that there are major differences in their sequences. At similar concentrations, both profilins prolong the lag phase at the outset of spontaneous polymerization and inhibit the extent of polymerization. Both forms also inhibit elongation weakly at the barbed end and strongly at the pointed end of actin filaments. 相似文献
11.
Y Z Yang D J Gordon E D Korn E Eisenberg 《The Journal of biological chemistry》1977,252(10):3374-3378
The binding of 125I-labeled muscle tropomyosin to Acanthamoeba and muscle actin was studied by ultracentrifugation and by the effect of tropomyosin on the actin-activated muscle heavy meromyosin ATPase activity. Binding of muscle tropomyosin to Acanthamoeba actin was much weaker than its binding to muscle actin. For example, at 5 mM MgCl2, 2 mM ATP, and 5 micronM actin, tropomyosin bound strongly to muscle actin but not detectably to Acanthamoeba actin. When the concentration of actin was raised from 5 micronM to 24 micronM in the presence of 80 mM KCl, the binding of tropomyosin to Acanthamoeba actin approached its binding to muscle actin. As with muscle actin, the addition of muscle heavy meromyosin in the absence of ATP induced binding of tropomyosin in Acanthamoeba actin under conditions were binding would otherwise not have occurred. The most striking difference between the interactions of muscle tropomyosin with the two actins, however, was that under conditions where tropomyosin was found to both actins, its stimulated the Acanthamoeba actin-activated heavy meromyosin ATPase but inhibited the muscle actin-activated heavy meromyosin ATPase. 相似文献
12.
Mechanism of action of Acanthamoeba profilin: demonstration of actin species specificity and regulation by micromolar concentrations of MgCl2 总被引:13,自引:11,他引:2
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《The Journal of cell biology》1982,94(1):213-218
Acanthamoeba profilin strongly inhibits in a concentration-dependent fashion the rate and extent of Acanthamoeba actin polymerization in 50 mM KCl. The lag phase is prolonged indicating reduction in the rate of nucleus formation. The elongation rates at both the barbed and pointed ends of growing filaments are inhibited. At steady state, profilin increases the critical concentration for polymerization but has no effect on the reduced viscosity above the critical concentration. Addition of profilin to polymerized actin causes it to depolymerize until a new steady-state, dependent on profilin concentration, is achieved. These effects of profilin can be explained by the formation of a 1:1 complex with actin with a dissociation constant of 1 to 4 microM. MgCl2 strongly inhibits these effects of profilin, most likely by binding to the high-affinity divalent cation site on the actin. Acanthamoeba profilin has similar but weaker effects on muscle actin, requiring 5 to 10 times more profilin than with amoeba actin. 相似文献
13.
Physical, immunochemical, and functional properties of Acanthamoeba profilin 总被引:9,自引:7,他引:2
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《The Journal of cell biology》1984,98(1):214-221
Acanthamoebe profilin has a native molecular weight of 11,700 as measured by sedimentation equilibrium ultracentrifugation and an extinction coefficient at 280 nm of 1.4 X 10(4) M-1cm-1. Rabbit antibodies against Acanthamoeba profilin react only with the 11,700 Mr polypeptide among all other ameba polypeptides separated by electrophoresis. These antibodies react with a 11,700 Mr polypeptide in Physarum but not with any proteins of Dictyostelium or Naeglaria. Antibody-binding assays indicate that approximately 2% of the ameba protein is profilin and that the concentration of profilin is approximately 100 mumol/liter cells. During ion exchange chromatography of soluble extracts of Acanthamoeba on DEAE-cellulose, the immunoreactive profilin splits into two fractions: an unbound fraction previously identified by Reichstein and Korn (1979, J. Biol. Chem., 254:6174-6179) and a tightly bound fraction. Purified profilin from the two fractions is identical by all criteria tested. The tightly bound fraction is likely to be attached indirectly to the DEAE, perhaps by association with actin. By fluorescent antibody staining, profilin is distributed uniformly throughout the cytoplasmic matrix of Acanthamoeba. In 50 mM KCl, high concentrations of Acanthamoeba profilin inhibit the elongation rate of muscle actin filaments measured directly by electron microscopy, but the effect is minimal in KCl with 2 MgCl2. By using the fluorescence change of pyrene-labeled Acanthamoeba actin to assay for polymerization, we confirmed our earlier observation (Tseng, P. C.-H., and T. D. Pollard, 1982, J. Cell Biol. 94:213-218) that Acanthamoeba profilin inhibits nucleation much more strongly than elongation under physiological conditions. 相似文献
14.
Burckhardt G Simon H Birch-Hirschfeld E Kittler L Sharma SK Lown JW Zimmer C 《Journal of biomolecular structure & dynamics》2002,19(6):1101-1109
The binding ability of cross-linked thiazolated polyamides (containing the base sequence-reading elements thiazole(Th)-pyrrole(Py)-pyr-role(Py) and thiazole(Th)-imidazole(Im)-pyrrol(Py) to various DNA dodecamers has been investigated. CD titration experiments at high salt concentration demonstrate that the dimers with a heptanediyl linker (C7 dimer) show a significantly higher sequence specificity than their corresponding monomers. The dimer of Th-Py-Py primarily prefers binding to pure AT sequences and that of Th-Im-Py to the dodecamer sequences containing a GC pair within the central sequence (e.g. AACGTT). Surprisingly, the sequence binding ability is strongly influenced by the presence of a T-A step: e.g. Th-Py-Py has a similar affinity to the sequences TTTAAA and ATCGTA; likewise Th-Im-Py shows a preference for these sequences. The CD results correlate with footprinting data. Related biochemical studies on the effect of polyamides on DNA gyrase activity in vitro show that the C7 dimers most effectively inhibit the enzyme activity compared with the monomers and the natural reference minor groove binder distamycin. The highest inhibitory potency is observed for the Th-Py-Py-dimer. The role of the T-A step in binding of the cross-linked dimer to the minor groove is discussed in light of the sequence recognition of the TATA box binding protein. 相似文献
15.
B N White 《Biochimica et biophysica acta》1975,395(3):322-328
The minor bases present in the family of Drosophila tRNAs recognising codons of the type NAA or NAG have been studied. Under standard aminoacylating conditions, the acceptor activities of BrCN-treated tRNA-Lys-5 tRNA-Glu-4 and tRNA-G1n-4 were completely eliminated, suggesting the presence of 2-thiouridine derivatives. The two major lysine tRNA species (tRNA-Lys-2 and tRNA-Lys-5) were purified and their nucleoside content determined both directly and by the tritium derivative technique. Both tRNAs contain 1-methyladenosine, N-2-dimethylguanosine, 7-methylguanosine, 5-methylcytidine, pseudouridine and dihydrouridine, and tRNA-Lys-5 contains 1-methylguanosine. Neither species contain ribothymidine, although both may contain 2'-O-methyl ribothymidine. A nucleoside with ultraviolet spectral properties similar to N-4-acetylcytidine was found in tRNA-Lys-5 and a nucleoside with chromatographic properties the same as N-[9-beta-D-ribofuranosyl)-purin-6-yl-carbamoyl] threonine was found in tRNA-Lys-2. A 2-thiouridine derivative was not found in tRNA-Lys-5 using these chromatographic techniques. 相似文献
16.
Specificity of the interaction between phosphatidylinositol 4,5-bisphosphate and the profilin:actin complex 总被引:20,自引:0,他引:20
Profilactin, the profilin:actin complex, which is present in large amounts in extracts of many types of eukaryotic cells, appears to serve as the precursor of microfilaments. It was reported recently that profilactin interacts specifically with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) (Lassing and Lindberg: Nature 314:472-474, 1985.) The present paper describes in detail the behaviour of profilactin and profilin in the presence of different types of phospholipids and neutral lipids under different conditions. PtdIns(4,5)P2 is the only phospholipid found so far which in the presence of 80 mM KCl and at Ca2+ concentrations below 10(-5) M effectively dissociates profilactin with the resulting polymerization of the actin. Phosphatidylinositol 4-monophosphate exhibits some activity but phosphatidylinositol is inactive. Both calf spleen profilin and profilin from human platelets form stable complexes with PtdIns(4,5)P2 micelles. PtdIns(4,5)P2 is active also when incorporated together with other phospholipids in mixed vesicles. 相似文献
17.
18.
19.
Actin polymerization in 2 mM MgCl2 is known to be inhibited by profilin. We found that small amounts of cytochalasin-binding complexes from human red cell membranes or actin nuclei cross-linked by -phenylenebismaleimide can reverse the inhibitory action of profilin, leading to the rapid polymerization of the actin. This type of polymerization is inhibited by low concentrations of cytochalasin B. These results indicate that (a) the complexes and nuclei promote actin polymerization in the presence of profilin by providing sites onto which actin monomers can be added, and (b) profilin and cytochalasin B affect two distinct steps (i.e. nucleus formation and filament elongation, respectively) in the polymerization reaction. 相似文献
20.
R. H. Clinton 《Antonie van Leeuwenhoek》1968,34(1):99-105
Uptake of labeled amino acids occurred at –4 C to 50 C accompanied by amino acid pool formation and protein synthesis. Maximum assimilation rates of both amino acids occurred at a temperature at which growth of this yeast was inhibited. Over a wide range of temperature the organism took up more exogenous lysine than glutamic acid, even though glutamic acid was present in the cellular protein in greater quantities. At 25 C the uptake and incorporation rates of glutamic acid was significantly higher than at 3 C; however, the size of the glutamic acid pools, at these two temperatures, appeared to be equal and independent of temperature. 相似文献