Yeast actin with a subdomain 4 mutation (A204C) exhibits increased pointed-end critical concentration

2007 ◽  
Vol 85 (3) ◽  
pp. 319-325 ◽  
Author(s):  
David J. Teal ◽  
John F. Dawson
Keyword(s):  
Actin Polymerization ◽  
Critical Concentration ◽  
Structural Studies ◽  
Wild Type ◽  
Low Concentrations ◽  
Engineering Strategy ◽  
High Concentrations ◽  
Pointed End ◽  
Very High

Characterizing mutants of actin that do not polymerize will advance our understanding of the mechanism of actin polymerization and will be invaluable for the production of short F-actin structures for structural studies. To circumvent the problem of expressing dominant lethal nonpolymerizing actin in yeast, we adopted a cysteine engineering strategy. Here we report the characterization of a mutant of yeast actin, AC-actin, possessing a single pointed-end mutation, A204C. Expression of this mutant in yeast results in actin-polymerization-deficient phenotypes. When copolymerized with wild-type actin, ATP–AC-actin is incorporated into filaments. ADP–AC-actin participates in the nucleation and elongation of wild-type filaments only at very high concentrations. At low concentrations, ADP–AC-actin appears to participate only in the nucleation of wild-type filaments, suggesting that Ala-204 is involved in modulating the critical concentration of the pointed end of actin.

Actin polymerization is controlled by residue size at position 204

2009 ◽  
Vol 87 (6) ◽  
pp. 853-865 ◽  
Author(s):  
Susan P. Yates ◽  
Ana Loncar ◽  
John F. Dawson
Keyword(s):  
Energy Minimization ◽  
Double Mutant ◽  
Actin Polymerization ◽  
Critical Concentration ◽  
Single Mutation ◽  
Wild Type ◽  
Phosphorylation State ◽  
High Concentrations ◽  
Very High

Previous work has shown that purified double mutant A204C/C374A yeast actin is polymerization-deficient in vitro under physiological concentrations. To understand the importance of the 204 residue in subdomain 4, a series of actin proteins with a single mutation at this position were created with Cys-374 retained. Only yeast expressing A204G-, A204S-, or A204C-actin were viable. The A204G and A204S strains were sensitive to cold temperature and hyperosmolarity, whereas the A204C strain showed more profound effects on growth under these conditions. Cells expressing A204C-actin exhibited anomalies previously observed for A204C/C374A actin, including abnormal actin structures. A204G- and A204S-actin proteins had 12- and 13-fold increased critical concentrations, respectively, relative to wild-type. Only at very high concentrations could A204C actin polymerize when ATP was bound; when hydrolyzed, the ADP-containing A204C filaments depolymerized, demonstrating a profound difference in critical concentration between ATP and ADP states with A204C actin. A correlation between size of the residue substituted at position 204 and energy minimization of actin filament models was observed. We propose that the region surrounding residue 204 is involved in interactions that change depending on the phosphorylation state of the bound nucleotide that might reflect different conformations of F-actin subunits.

Isolation and characterization of rat skeletal myoblast mutants resistant to lectins

1983 ◽  
Vol 3 (12) ◽  
pp. 2166-2171
Author(s):  
B Gilfix ◽  
J Rogers ◽  
B D Sanwal
Keyword(s):  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Wild Type ◽  
Skeletal Myoblast ◽  
Skeletal Myoblasts ◽  
Isolation And Characterization ◽  
Low Concentrations ◽  
High Concentrations ◽  
Made In

Mutants resistant to the lethal action of the lectins phytohemagglutinin A (PHA) and wheat germ agglutinin (WGA) have been made in a line of differentiating rat skeletal myoblasts. The WGA mutants are of two types, WGArII, resistant to low concentrations of the lectin, and WGArI, resistant to high concentrations of the lectin. WGArII and PHAr mutants are unable to differentiate, whereas WGArI mutants differentiate normally. WGArII mutants are not impaired in the binding of wheat germ agglutinin, but WGArI mutants bind the lectin only to the extent of about 50% of the wild-type values. All of the mutants are cross-resistant to lectins other than those used in their selection.

scholarly journals Isolation and characterization of rat skeletal myoblast mutants resistant to lectins.

1983 ◽  
Vol 3 (12) ◽  
pp. 2166-2171 ◽  
Author(s):  
B Gilfix ◽  
J Rogers ◽  
B D Sanwal
Keyword(s):  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Wild Type ◽  
Skeletal Myoblast ◽  
Skeletal Myoblasts ◽  
Isolation And Characterization ◽  
Low Concentrations ◽  
High Concentrations ◽  
Made In

Mutants resistant to the lethal action of the lectins phytohemagglutinin A (PHA) and wheat germ agglutinin (WGA) have been made in a line of differentiating rat skeletal myoblasts. The WGA mutants are of two types, WGArII, resistant to low concentrations of the lectin, and WGArI, resistant to high concentrations of the lectin. WGArII and PHAr mutants are unable to differentiate, whereas WGArI mutants differentiate normally. WGArII mutants are not impaired in the binding of wheat germ agglutinin, but WGArI mutants bind the lectin only to the extent of about 50% of the wild-type values. All of the mutants are cross-resistant to lectins other than those used in their selection.

scholarly journals Halogenated Volatile Organic Compounds in Water Samples and Inorganic Elements Levels in Ores for Characterizing a High Anthropogenic Polluted Area in the Northern Latium Region (Italy)

2021 ◽  
Vol 18 (4) ◽  
pp. 1628
Author(s):  
Mario Vincenzo Russo ◽  
Ivan Notardonato ◽  
Alberto Rosada ◽  
Giuseppe Ianiri ◽  
Pasquale Avino
Keyword(s):  
Organic Compounds ◽  
Mean Value ◽  
Greenhouse Crops ◽  
River Waters ◽  
Liquid Liquid Extraction ◽  
Volatile Organic ◽  
High Concentrations ◽  
Very High ◽  
Inorganic Fraction

This paper shows a characterization of the organic and inorganic fraction of river waters (Tiber and Marta) and ores/soil samples collected in the Northern Latium region of Italy for evaluating the anthropogenic/natural source contribution to the environmental pollution of this area. For organic compounds, organochloride volatile compounds in Tiber and Marta rivers were analyzed by two different clean-up methods (i.e., liquid–liquid extraction and static headspace) followed by gas chromatography–electron capture detector (GC-ECD) analysis. The results show very high concentrations of bromoform (up to 1.82 and 3.2 µg L−1 in Tiber and Marta rivers, respectively), due to the presence of greenhouse crops, and of chloroform and tetrachloroethene, due to the presence of handicrafts installations. For the qualitative and quantitative assessment of the inorganic fraction, it is highlighted the use of a nuclear analytical method, instrumental neutron activation analysis, which allows having more information as possible from the sample without performing any chemical-physical pretreatment. The results have evidenced high levels of mercury (mean value 88.6 µg g−1), antimony (77.7 µg g−1), strontium (12,039 µg g−1) and zinc (103 µg g−1), whereas rare earth elements show levels similar to the literature data. Particular consideration is drawn for arsenic (414 µg g−1): the levels found in this paper (ranging between 1 and 5100 µg g−1) explain the high content of such element (as arsenates) in the aquifer, a big issue in this area.

Purification and characterization of a nonpolymerizing long-pitch actin dimer

2006 ◽  
Vol 84 (5) ◽  
pp. 695-702 ◽  
Author(s):  
Braden Sweeting ◽  
John F. Dawson
Keyword(s):  
Critical Concentration ◽  
Dnase I ◽  
Actin Binding ◽  
Wild Type ◽  
Filamentous Actin ◽  
Rhodamine Phalloidin ◽  
Fold Reduction ◽  
Crystallization Conditions ◽  
Self Association ◽  
Pointed End

Atomic resolution structures of filamentous actin have not been obtained owing to the self-association of actin under crystallization conditions. Obtaining short filamentous actin complexes of defined lengths is therefore a highly desirable goal. Here we report the production and isolation of a long-pitch actin dimer employing chemical crosslinking between wild-type actin and Q41C/C374A mutant actin. The Q41C/C374A mutant actin possessed altered polymerization properties, with a 2-fold reduction in the rate of elongation and an increased critical concentration relative to wild-type actin. The Q41C/C374A mutant actin also displayed an increase in the IC50 for DNase I, a pointed-end actin-binding protein. The long-pitch dimer was bound by DNase I to prevent polymerization and purified. It was found that each actin dimer is bound by 2 DNase I molecules, 1 likely bound to each of the actin protomers. The long-pitch dimer bound by DNase I did not form short F actin structures, as assessed by the binding of rhodamine–phalloidin.

scholarly journals Characterization of Shiga-like toxin I B subunit purified from overproducing clones of the SLT-I B cistron

1990 ◽  
Vol 272 (3) ◽  
pp. 805-811 ◽  
Author(s):  
K Ramotar ◽  
B Boyd ◽  
G Tyrrell ◽  
J Gariepy ◽  
C Lingwood ◽  
...  
Keyword(s):  
Culture Medium ◽  
Polymyxin B ◽  
Exchange Chromatography ◽  
Structural Studies ◽  
Wild Type ◽  
Binding Studies ◽  
B Subunit ◽  
Tac Promoter ◽  
Glycolipid Receptor

The cistron encoding the B subunit of Escherichia coli Shiga-like toxin I (SLT-I) was cloned under control of the tac promoter in the expression vector pKK223-3 and the SLT-I B subunit was expressed constitutively in a wild-type background and inducibly in a lacIq background. The B subunit was located in the periplasmic space, and less than 10% was found in the culture medium after 24 h incubation. Polymyxin B extracts contained as much as 160 micrograms of B subunit/ml of culture. B subunit was purified to homogeneity by ion-exchange chromatography followed by chromatofocusing. Cross-linking analysis of purified native B subunit showed that it exists as a pentamer. In gels containing 0.1% SDS the native protein dissociated into monomers. B subunit was found to have the same glycolipid-receptor-specificity as SLT-I holotoxin. Competitive binding studies showed that B subunit and holotoxin had the same affinity for the globotriosylceramide receptor. We conclude that this recombinant plasmid is a convenient source of large amounts of purified SLT-I B subunit, which could be used for biophysical and structural studies or as a natural toxoid.

scholarly journals Detection of endotoxins with the Limulus test in burned and unburned mice infected with different species of gramnegative bacteria

1975 ◽  
Vol 75 (1) ◽  
pp. 99-112 ◽  
Author(s):  
R. J. Jones ◽  
E. A. Roe ◽  
R. E. Dyster
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Low Concentrations ◽  
High Concentrations ◽  
Very High ◽  
Limulus Test

SUMMARYThe Limulus test detected endotoxins in the plasma of burned and unburned mice infected with different species of gram-negative bacteria. Individual strains of different species of gram-negative bacteria produced different amounts of endotoxin in the plasma of infected mice. Plasma from mice given lethal infections showed very high concentrations of endotoxin. Low concentrations of endotoxin in the plasma were tolerated by mice but high concentrations were invariably fatal. A polyvalent pseudomonas vaccine reduced endotoxin in the plasma of mice given lethal infections of Pseudomonas aeruginosa.

Observed differences in dextran and polyvinylpyrrolidone as rouleaux-inducing agents

1980 ◽  
Vol 58 (3) ◽  
pp. 271-274 ◽  
Author(s):  
Lionel S. Sewchand ◽  
Dieter Bruckschwaiger
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Animal Species ◽  
Critical Concentration ◽  
Rbc Membrane ◽  
Low Concentrations ◽  
High Concentrations ◽  
Induced Aggregation ◽  
Do So

The effectiveness of dextran fractions (Dx-500, Dx-100, Dx-70) and polyvinylpyrrolidone (PVP-360, PVP-40) in inducing aggregation of red blood cells (RBC) was studied in a nonflowing environment. The Dx fractions, at low concentrations, induced aggregation of human RBC but failed to do so at high concentrations (concentrations greater than 70 g/L). The effect was different on RBC from animal species (cat and rabbit); aggregation increased steadily with the Dx concentration and there was no critical concentration beyond which Dx failed to induce aggregation. The PVP was found to be very effective, at all concentrations, in inducing aggregation of RBC from both human and the animal species. These results have a twofold significance: (1) they suggest that Dx and PVP, both neutral polymers, interact differently with the human RBC membrane; and (2) the association of Dx with the human RBC membrane is different from that with cat and rabbit RBC membranes.

Copper, nickel, and thallium uptake by the lichen Cladina rangiferina

1981 ◽  
Vol 59 (1) ◽  
pp. 91-100 ◽  
Author(s):  
M. A. S. Burton ◽  
P. LeSueur ◽  
K. J. Puckett
Keyword(s):  
Metal Uptake ◽  
Deionized Water ◽  
Metabolic Inhibitors ◽  
Temperature Dependent ◽  
Low Concentrations ◽  
Nickel Uptake ◽  
High Concentrations ◽  
Uptake Studies ◽  
Cladina Rangiferina ◽  
Very High

Metal uptake studies with Cladina rangiferina showed that the affinity for nickel was much lower than for copper or thallium. Nickel uptake was not decreased by the absence of light or oxygen or by pretreatment with metabolic inhibitors. Nickel uptake was not temperature dependent but was very dependent upon pH.Cation-exchange studies demonstrated that there was a stoichiometric exchange of Ni2+ for Sr2+, and Cu2+ for Sr2+. The exchange of Tl+ for Sr2+ was not stoichiometric, excess Tl+ was accumulated in relation to the Sr2+ released. The ratio of Sr2+:Tl+ exchange increased with increasing Tl+ availability from 1:9 (12.5 μmol Tl+ available/g of lichen) to 1:2 (500 μmol Tl+ available). Acid-treated lichen gave the expected exchange ratio of 1:2. Washing of the thalli with deionized water resulted in the continued loss of Tl+ from acid-treated and live C. rangiferina. Copper and nickel were not released in this manner.Increasing concentrations of copper and thallium produced a corresponding loss of potassium from the thallus. The potassium loss was initiated at low concentrations of copper and thallium whereas very high concentrations of manganese and nickel were required to bring about the same response.

scholarly journals RELAXATION IN EXTRACTED MUSCLE FIBERS

1954 ◽  
Vol 38 (2) ◽  
pp. 149-159 ◽  
Author(s):  
Emil Bozler
Keyword(s):  
Critical Level ◽  
Critical Concentration ◽  
Muscle Fibers ◽  
Complexing Agent ◽  
Tetraacetic Acid ◽  
Relaxation Factor ◽  
Low Concentrations ◽  
High Concentrations ◽  
Relaxing Effect ◽  
Subsequent Addition

1. Ethylenediamine tetraacetic acid (EDTA) in low concentrations imitates all the known effects of the relaxation factor ("Marsh factor"). In extracted muscle fibers which have contracted in a solution containing adenosinetriphosphate (ATP), the addition of EBTA causes relaxation, the subsequent addition of CaCl2, contraction. 2. In fibers which have been briefly immersed in 5 MM EDTA, ATP causes rapid relaxation if Mg is also present. These fibers have essentially the same properties as briefly extracted fibers. Brief immersion into a solution containing CaCl2 restores at once the original condition. It is concluded that EDTA produces its action by firmly combining with bound Ca, thereby inactivating it. 3. In relaxed muscle fibers not only Ca, but also lowering the concentration of Mg below a critical level, causes contraction. In such fibers Mg in the lowest effective concentrations increases contraction, but the effect reverses above a certain concentration. 4. At 0° Mg in the presence of ATP has a relaxing effect without the relaxation factor. 5. The results indicate that Mg has two distinct effects in the presence of ATP. It causes contraction at low concentrations, but above a critical concentration its relaxing action prevails. The last of these effects is blocked by bound Ca. If the latter is inactivated by EDTA, Mg in sufficiently high concentrations causes relaxation. The action of the relaxation factor can similarly be explained by assuming that it acts as a complexing agent which inactivates bound Ca. 6. Previous evidence that the relaxed state depends on the formation of an enzymatically inactive ATP-protein complex was confirmed. It was found that PP in low concentrations strongly increases the relaxing effect of ATP in briefly extracted fibers.

Sign in / Sign up

Export Citation Format

Share Document