scholarly journals Mechanism of action of Acanthamoeba profilin: demonstration of actin species specificity and regulation by micromolar concentrations of MgCl2.

1982 ◽  
Vol 94 (1) ◽  
pp. 213-218 ◽  
Author(s):  
P C Tseng ◽  
T D Pollard
Keyword(s):  
Steady State ◽  
Actin Polymerization ◽  
Critical Concentration ◽  
Concentration Addition ◽  
Lag Phase ◽  
Muscle Actin ◽  
Cation Site ◽  
High Affinity ◽  
Reduced Viscosity ◽  
State Dependent

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.

scholarly journals Molecular cloning, expression, and mapping of the high affinity actin-capping domain of chicken cardiac tensin.

1995 ◽  
Vol 128 (6) ◽  
pp. 1095-1109 ◽  
Author(s):  
J Z Chuang ◽  
D C Lin ◽  
S Lin
Keyword(s):  
Mammalian Cells ◽  
Actin Polymerization ◽  
Critical Concentration ◽  
Focal Adhesions ◽  
Inhibitory Effect ◽  
Complete Inhibition ◽  
Fusion Construct ◽  
Chicken Gizzard ◽  
High Affinity ◽  
Actin Capping

Tensin, an actin filament capping protein first purified from chicken gizzard, is localized to various types of adherens junctions in muscle and nonmuscle cells. In this paper, we describe the isolation and sequencing of tensin cDNA from a chicken cardiac library. The 6.3-kb chicken cardiac tensin cDNA encodes an open reading frame of 1,792 amino acids. Mammalian cells transfected with the chicken tensin cDNA expressed a polypeptide of approximately 200 kD recognizable by antibodies to chicken gizzard tensin. The expressed protein was incorporated into focal adhesions and other actin-containing structures in the transfected cells. To map the domain associated with tensin's high affinity, barbed-end F-actin-capping activity, bacterially expressed recombinant fusion proteins containing various segments of tensin were prepared and assayed for activity. The results of these experiments show that the high affinity capping domain (kD = 1.3 nM) lies within amino acid residues R1037-V1169. Additional studies on a shorter construct, S1061-H1145, showed that these 85 residues were sufficient for producing complete inhibition of actin polymerization and depolymerization. While this active domain is located within that of the "insertin" sequence (Weigt, C., A. Gaertner, A. Wegner, H. Korte, and H. E. Meyer. 1992. J. Mol. Biol. 227:593-595), our data showing complete inhibition of polymerization and shift in critical concentration are consistent with a simple barbed-end capping mechanism rather than the "insertin model." Our results also differ from those of a recent report (Lo, S. H., P. A. Janmey, J. H. Hartwig, and L. B. Chen. 1994. J. Cell Biol. 125:1067-1075), which concluded that their recombinant tensin has an "insertin-like" inhibitory effect on barbed-end actin polymerization, and that this activity is attributed to residues T936-R1037 (residues 888-989 in their numbering system). In our study, a fusion construct (N790-K1060) encompassing T936-R1037 had no significant effect on actin polymerization and depolymerization, even at high concentrations.

scholarly journals Partial purification and characterization of an actin-bundling protein, band 4.9, from human erythrocytes.

1985 ◽  
Vol 100 (3) ◽  
pp. 775-785 ◽  
Author(s):  
D L Siegel ◽  
D Branton
Keyword(s):  
Steady State ◽  
Actin Polymerization ◽  
Monomer Concentration ◽  
Apparent Molecular Weight ◽  
Membrane Skeleton ◽  
Erythrocyte Membranes ◽  
Partial Purification ◽  
Lag Phase ◽  
Rabbit Muscle ◽  
Erythroid Precursor

Band 4.9 (a 48,000-mol-wt polypeptide) has been partially purified from human erythrocyte membranes. In solution, band 4.9 polypeptides exist as trimers with an apparent molecular weight of 145,000 and a Stokes radius of 50 A. Electron microscopy shows that the protein is a three-lobed structure with a radius slightly greater than 50 A. When gel-filtered rabbit muscle actin is polymerized in the presence of band 4.9, actin bundles are generated that are similar in appearance to those induced by "vinculin" or fimbrin. The bundles appear brittle and when they are centrifuged small pieces of filaments break off and remain in the supernatant. At low band 4.9 to actin molar ratios (1:30), band 4.9 lowers the apparent steady-state low-shear falling ball viscosity by sequestering filaments into thin bundles; at higher ratios, the bundles become thicker and obstruct the ball's movement leading to an apparent increase in steady-state viscosity. Band 4.9 increases the length of the lag phase and decreases the rate of elongation during actin polymerization as measured by high-shear Ostwald viscometry or by the increase in the fluorescence of pyrene-labeled actin. Band 4.9 does not alter the critical actin monomer concentration. We hypothesize that band 4.9, together with actin, erythrocyte tropomyosin, and spectrin, forms structures in erythroid precursor cells analogous to those formed by fimbrin, actin, tropomyosin, and TW 260/240 in epithelial brush borders. During erythroid development and enucleation, the actin filaments may depolymerize up to the membrane, leaving a membrane skeleton with short stubs of actin bundled by band 4.9 and cross-linked by spectrin.

scholarly journals Changes in contractile proteins during differentiation of myeloid leukemia cells. II. Purification and characterization of actin

1982 ◽  
Vol 93 (2) ◽  
pp. 470-478 ◽  
Author(s):  
K Nagata ◽  
J Sagara ◽  
Y Ichikawa
Keyword(s):  
Skeletal Muscle ◽  
Myeloid Leukemia ◽  
Actin Polymerization ◽  
Specific Activity ◽  
Critical Concentration ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Muscle Actin ◽  
Skeletal Muscle Myosin ◽  
Before And After

A myeloid leukemia cell line, M1, differentiates to macrophage and gains locomotive and phagocytic activity when incubated with conditioned medium (CM) from a fibroblast culture and bacterial endotoxin. To characterize the actin molecules before and after differentiation, the actin was purified through three sequential steps: DEAE-sephadex A- 50, polymerization/depolymerization, and sephadex G-150 chromatography. There were no essential differences between the inhibitory activity of actins from control M1 cells and CM-treated M1 cells on both DNase I and heavy meromyosin (HMMM) K(+)-EDTA-ATPase; the same dose response as with skeletal muscle actin took place. After the treatment with CM, however, the specific activity for the activation of HMMM Mg(2+)- ATPase by actin became two-fold that of untreated M1 actin, which was one third of the value for skeletal muscle actin. The V(max) for the control and the CM-treated M1 cell, as well as the skeletal muscle actins, proved to be the same. By contrast, the K(app) values for the control and CM-treated M1-cell actins were 3- and 1.5-fold the value for skeletal-muscle actin. This means that CM treatment of the M1 actin produced a twofold affinity for the Mg(2+)-ATPase of skeletal-muscle myosin. The critical concentrations for polymerization were compared under different salt concentrations and temperatures. Although no marked difference was found for the presence of 2 mM MgCl(2), 0.1 M KCl in place of MgCl(2) at 5 degrees C gave the following values: 0.1 mg/ml for skeletal-muscle actin, 0.7 mg/ml for control M1 actin, 0,5 mg/ml for CM- treated M1 actin, and 1.0 mg/ml for the D(-) subline that is insensitive to CM. Although the critical concentration of D(-) actin is extraordinarily high, this actin showed normal polymerization above the critical concentration. This together with the data presented in our previous paper, that the D(-) actin in the crude extract did not polymerize, suggests that an inhibitor for actin polymerization is present in the subline. The kinetics experiment at 0.1 M KCl and 25 degrees C revealed a slower polymerization of untreated M1- and D(-)-cell actins as compared with CM-treated M1 actin. This delayed polymerization was due to a delay during the nucleation stage, not during the elongation stage. By isoelectric focusing, the ratios of β- to γ-actin showed a marked difference depending on the states of cells: about 4.9 for control M1, 2.8 for CM-treated M1, and 7.6 for D(-)-subline actins. Tryptic peptide maps also revealed the presence of different peptides. Thus, the functional differences of actin before and after the differentiation was accompanied by some chemical changes in actin molecules.

scholarly journals Polymerization of FtsZ, a Bacterial Homolog of Tubulin

2001 ◽  
Vol 276 (15) ◽  
pp. 11743-11753 ◽  
Author(s):  
Laura Romberg ◽  
Martha Simon ◽  
Harold P. Erickson
Keyword(s):  
Steady State ◽  
Critical Concentration ◽  
Gtp Hydrolysis ◽  
Nucleotide Hydrolysis ◽  
High Affinity ◽  
Hydrolysis Rates ◽  
Bacterial Homolog ◽  
Nucleation Phase ◽  
Maximum Steady State

FtsZ is a bacterial homolog of tubulin that is essential for prokaryotic cytokinesis.In vitro, GTP induces FtsZ to assemble into straight, 5-nm-wide polymers. Here we show that the polymerization of these FtsZ filaments most closely resembles noncooperative (or “isodesmic”) assembly; the polymers are single-stranded and assemble with no evidence of a nucleation phase and without a critical concentration. We have developed a model for the isodesmic polymerization that includes GTP hydrolysis in the scheme. The model can account for the lengths of the FtsZ polymers and their maximum steady state nucleotide hydrolysis rates. It predicts that unlike microtubules, FtsZ protofilaments consist of GTP-bound FtsZ subunits that hydrolyze their nucleotide only slowly and are connected by high affinity longitudinal bonds with a nanomolarKD.

scholarly journals Mechanism of regulation of actin polymerization by Physarum profilin.

1984 ◽  
Vol 98 (6) ◽  
pp. 1919-1925 ◽  
Author(s):  
K Ozaki ◽  
S Hatano
Keyword(s):  
Steady State ◽  
Dissociation Constant ◽  
Actin Polymerization ◽  
Dissociation Constants ◽  
Critical Concentration ◽  
Cross Linking ◽  
Kinetics Of Polymerization ◽  
Kinetics Of ◽  
Chemical Cross Linking

Physarum profilin reduces the rates of nucleation and elongation of F-actin and also reduces the extent of polymerization of actin at the steady state in a concentration-dependent fashion. The apparent critical concentration for polymerization of actin is increased by the addition of profilin. These results can be explained by the idea that Physarum profilin forms a 1:1 complex with G-actin and decreases the concentration of actin available for polymerization. The dissociation constant for binding of profilin to G-actin is estimated from the kinetics of polymerization of G-actin and elongation of F-actin nuclei and from the increase of apparent critical concentration in the presence of profilin. The dissociation constants for binding of Physarum profilin to Physarum and muscle actins under physiological ionic conditions are in the ranges of 1.4-3.7 microM and 11.3-28.5 microM, respectively. When profilin is added to an F-actin solution, profilin binds to G-actin which co-exists with F-actin, and then G-actin is dissociated from F-actin to compensate for the decrease of the concentration of free G-actin and to keep it constant at the critical concentration. At the steady state, free G-actin of the critical concentration is in equilibrium not only with F-actin but also with profilin-G-actin complex. The stoichiometry of 1:1 for the formation of complex between profilin and G-actin is directly shown by means of chemical cross-linking.

scholarly journals The 50 kDa protein-actin complex from unfertilized sea-urchin (Strongylocentrotus purpuratus) eggs. Interaction with actin

1989 ◽  
Vol 257 (3) ◽  
pp. 817-822 ◽  
Author(s):  
R M Golsteyn ◽  
D M Waisman
Keyword(s):  
Steady State ◽  
Sea Urchin ◽  
Actin Polymerization ◽  
Critical Concentration ◽  
Strongylocentrotus Purpuratus ◽  
Preceding Paper ◽  
State Level ◽  
Actin Binding ◽  
Molar Ratio ◽  
Kinetics Of

In the preceding paper [Golsteyn & Waisman (1989) Biochem. J. 257, 809-815] an EGTA-stable, Ca2+-binding heterodimer comprised of a 50 kDa protein and actin called ‘50K-A’ was identified in the unfertilized eggs of the sea urchin Strongylocentrotus purpuratus. In the present paper we have documented the binding of 50K-A to DNAase I and the effect of 50K-A on the kinetics of actin polymerization. When 50K-A was added to pyrene-labelled rabbit skeletal-muscle actin and the salt concentration increased, the initial rate of actin polymerization was inhibited by a very low molar ratio of 50K-A to actin. Furthermore, the steady-state level of G-actin was increased in the presence of 50K-A, suggesting that 50K-A caps the preferred end of actin polymer, shifting the steady-state concentration to that of the non-preferred end. Dilution of F-actin to below its critical concentration into 50K-A resulted in a much slower rate of depolymerization, consistent with capping of the preferred end. In contrast with the Ca2+-dependent binding to DNAase, the effect of 50K-A on the kinetics of actin assembly and disassembly was Ca2+-independent. These results suggest that 50K-A is a novel actin-binding protein with some similarities to the severin/fragmin/gelsolin family of F-actin-capping proteins.

Analysis of Single Buffer Random Polling System With State-Dependent Input Process and Server/Station Breakdowns

2018 ◽  
Vol 9 (1) ◽  
pp. 22-50 ◽  
Author(s):  
Thomas Y.S. Lee
Keyword(s):  
Steady State ◽  
Linear Model ◽  
Repair Process ◽  
Polling System ◽  
Steady State Analysis ◽  
Relaxation System ◽  
Polling Model ◽  
State Dependent ◽  
Non Linear ◽  
State Analysis

Models and analytical techniques are developed to evaluate the performance of two variations of single buffers (conventional and buffer relaxation system) multiple queues system. In the conventional system, each queue can have at most one customer at any time and newly arriving customers find the buffer full are lost. In the buffer relaxation system, the queue being served may have two customers, while each of the other queues may have at most one customer. Thomas Y.S. Lee developed a state-dependent non-linear model of uncertainty for analyzing a random polling system with server breakdown/repair, multi-phase service, correlated input processes, and single buffers. The state-dependent non-linear model of uncertainty introduced in this paper allows us to incorporate correlated arrival processes where the customer arrival rate depends on the location of the server and/or the server's mode of operation into the polling model. The author allows the possibility that the server is unreliable. Specifically, when the server visits a queue, Lee assumes that the system is subject to two types of failures: queue-dependent, and general. General failures are observed upon server arrival at a queue. But there are two possibilities that a queue-dependent breakdown (if occurs) can be observed; (i) is observed immediately when it occurs and (ii) is observed only at the end of the current service. In both cases, a repair process is initiated immediately after the queue-dependent breakdown is observed. The author's model allows the possibility of the server breakdowns/repair process to be non-stationary in the number of breakdowns/repairs to reflect that breakdowns/repairs or customer processing may be progressively easier or harder, or that they follow a more general learning curve. Thomas Y.S. Lee will show that his model encompasses a variety of examples. He was able to perform both transient and steady state analysis. The steady state analysis allows us to compute several performance measures including the average customer waiting time, loss probability, throughput and mean cycle time.

scholarly journals The bulk of unpolymerized actin in Xenopus egg extracts is ATP-bound.

1995 ◽  
Vol 6 (2) ◽  
pp. 227-236 ◽  
Author(s):  
J Rosenblatt ◽  
P Peluso ◽  
T J Mitchison
Keyword(s):  
Actin Polymerization ◽  
Critical Concentration ◽  
Large Fraction ◽  
Nucleotide Exchange ◽  
Chromatography Analysis ◽  
Thymosin Beta 4 ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Non-muscle cells contain 15-500 microM actin, a large fraction of which is unpolymerized. Thus, the concentration of unpolymerized actin is well above the critical concentration for polymerization in vitro (0.2 microM). This fraction of actin could be prevented from polymerization by being ADP bound (therefore less favored to polymerize) or by being ATP bound and sequestered by a protein such as thymosin beta 4, or both. We isolated the unpolymerized actin from Xenopus egg extracts using immobilized DNase 1 and assayed the bound nucleotide. High-pressure liquid chromatography analysis showed that the bulk of soluble actin is ATP bound. Analysis of actin-bound nucleotide exchange rates suggested the existence of two pools of unpolymerized actin, one of which exchanges nucleotide relatively rapidly and another that apparently does not exchange. Native gel electrophoresis of Xenopus egg extracts demonstrated that most of the soluble actin exists in complexes with other proteins, one of which might be thymosin beta 4. These results are consistent with actin polymerization being controlled by the sequestration and release of ATP-bound actin, and argue against nucleotide exchange playing a major role in regulating actin polymerization.

Insensitivity of steady-state distributions of generalized semi-Markov processes by speeds

1978 ◽  
Vol 10 (04) ◽  
pp. 836-851 ◽  
Author(s):  
R. Schassberger
Keyword(s):  
Steady State ◽  
General System ◽  
State Distribution ◽  
Lifetime Distributions ◽  
State Dependent ◽  
Semi Markov Process ◽  
Semi Markov Processes ◽  
Infinite State ◽  
State Case ◽  
Residual Lifetimes

A generalized semi-Markov process with speeds describes the fluctuation, in time, of the state of a certain general system involving, at any given time, one or more living components, whose residual lifetimes are being reduced at state-dependent speeds. Conditions are given for the stationary state distribution, when it exists, to depend only on the means of some of the lifetime distributions, not their exact shapes. This generalizes results of König and Jansen, particularly to the infinite-state case.

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