Filament Formation by Slime Mould Myosin Isolated at Low Ionic Strength

1974 ◽  
Vol 15 (1) ◽  
pp. 113-129
Author(s):  
H. HINSSEN ◽  
J. D'HAESE
Keyword(s):  
Ionic Strength ◽  
Striated Muscle ◽  
Divalent Cations ◽  
Filament Formation ◽  
Selective Precipitation ◽  
Slime Mould ◽  
Preparation Conditions ◽  
Filament Structure ◽  
Low Ionic Strength

Myosin was isolated and purified from plasmodia of the slime mould Physarum polycephalum by a new method. This method is based on actomyosin extraction at low ionic strength after extensive washing, followed by the selective precipitation of myosin at pH 6.1 under relaxing conditions. The yield of myosin was 3-5 times higher than reported for other methods. In contrast to earlier studies a remarkably strong tendency to filament formation was found for slime mould myosin, probably due to a better preservation of some structural properties during preparation. Conditions were worked out under which numerous filaments up to 4 µm in length can be produced. It was established that not only a gradual decrease of ionic strength may influence filament formation, but also pH, ATP concentration and the presence of divalent cations. Compared to the current filament models a difference exists in the structure of the filaments. No central bare zone can be found, and thus, they lack an apparent bipolarity. Along the entire filament there are lateral projections representing the head portion of myosin molecules. A clear periodicity with an axial repeat of about 14 nm was observed, indicating a highly ordered arrangement of these projections. In this paper it is shown for the first time that myosin from one of the primitive motile systems is able to form aggregates of high structural order, indicating that the contraction of non-muscular actomyosin systems is not necessarily effected with oligomeric or randomly aggregated myosin. The possible role of myosin aggregation in vivo and the similarity of filament structure to that recently reported for myosin from vertebrate smooth muscle and striated muscle are discussed.

scholarly journals Substructure and accessory proteins in scallop myosin filaments.

1989 ◽  
Vol 109 (2) ◽  
pp. 539-547 ◽  
Author(s):  
P Vibert ◽  
L Castellani
Keyword(s):  
Ionic Strength ◽  
Striated Muscle ◽  
Rotational Symmetry ◽  
Thick Filament ◽  
Accessory Proteins ◽  
Myosin Filaments ◽  
Cross Bridges ◽  
Low Ionic Strength ◽  
Muscle Myosin

Native myosin filaments from scallop striated muscle fray into subfilaments of approximately 100-A diameter when exposed to solutions of low ionic strength. The number of subfilaments appears to be five to seven (close to the sevenfold rotational symmetry of the native filament), and the subfilaments probably coil around one another. Synthetic filaments assembled from purified scallop myosin at roughly physiological ionic strength have diameters similar to those of native filaments, but are much longer. They too can be frayed into subfilaments at low ionic strength. Synthetic filaments share what may be an important regulatory property with native filaments: an order-disorder transition in the helical arrangement of myosin cross-bridges that is induced on activation by calcium, removal of nucleotide, or modification of a myosin head sulfhydryl. Some native filaments from scallop striated muscle carry short "end filaments" protruding from their tips, comparable to the structures associated with vertebrate striated muscle myosin filaments. Gell electrophoresis of scallop muscle homogenates reveals the presence of high molecular weight proteins that may include the invertebrate counterpart of titin, a component of the vertebrate end filament. Although the myosin molecule itself may contain much of the information required to direct its assembly, other factors acting in vivo, including interactions with accessory proteins, probably contribute to the assembly of a precisely defined thick filament during myofibrillogenesis.

Preparation Of Antithrombin, High Activity Heparin And A Heparin-Antithrombin Complex By A Rapid Two-Step Affinity Method

1981 ◽  
Author(s):  
R Jordan ◽  
T Zuffi ◽  
M Fournel ◽  
D Schroeder
Keyword(s):  
Ionic Strength ◽  
High Activity ◽  
Tight Binding ◽  
Therapeutic Benefit ◽  
Purification Scheme ◽  
The Matrix ◽  
Low Ionic Strength ◽  
Potential Therapeutic Benefit

The tight binding affinity of antithrombin for heparin makes possible a relatively selective purification scheme based on salt elution from heparin-Sepharose. We have found, however, that purity can often be greatly increased if the elution is carried out with soluble heparin instead. This heparin can be removed from the antithrombin, either in whole or part, by a second affinity step on Concanavalin A Sepharose. The antithrombin, which binds to the matrix through its glycosidic moieties, retains its ability to bind heparin at physiological ionic strengths. Thus, the complex of antithrombin and heparin is readily isolated free of unbound heparin species. The complex can be eluted intact with low ionic strength buffers containing sugars which compete for binding to the lectin. Alternatively, the high activity heparin (400–500 units/mg) can be obtained separately by a 1 M NaCl wash which is then followed by a carbohydrate wash to obtain the purified antithrombin.We have made certain preliminary biochemical and anticoagulant characterizations of these materials. Not unexpectedly, both the high activity heparin and its complex with antithrombin show significantly greater in vitro potency in comparison to unfractionated heparin. In vivo anticoagulant efficacy, as evaluated in a rabbit infusion model, confirmed the in vitro findings and further suggests some potential therapeutic benefit may be derived from infusion of a preformed heparin-antithrombin complex.

scholarly journals Release of poly a(+) messenger RNA from rat liver rough microsomes upon disassembly of bound polysomes

1977 ◽  
Vol 74 (2) ◽  
pp. 414-427 ◽  
Author(s):  
J Kruppa ◽  
DD Sabatini
Keyword(s):  
Ionic Strength ◽  
Rat Liver ◽  
Messenger Rna ◽  
High Salt ◽  
Salt Medium ◽  
Ribosomal Subunits ◽  
Microsomal Membranes ◽  
Low Ionic Strength

Several procedures were used to disassemble rat liver rough microsomes (RM) into ribosomal subunits, mRNA, and ribosome-stripped membrane vesicles in order to examine the nature of the association between the mRNA of bound polysomes and the microsomal membranes. The fate of the mRNA molecules after ribosome release was determined by measuring the amount of pulse-labeled microsomal RNA in each fraction which was retained by oligo-dT cellulose or by measuring the poly A content by hybridization to radioactive poly U. It was found that ribosomal subunits and mRNA were simultaneously released from the microsomal membranes when the ribosomes were detached by: (a) treatment with puromycin in a high salt medium containing Mg++, (b) resuspension in a high salt medium lacking Mg++, and (c) chelation of Mg++ by EDTA or pyrophosphate. Poly A-containing mRNA fragments were extensively released from RM subjected to a mild treatment with pancreatic RNase in a medium of low ionic strength. This indicates that the 3' end of the mRNA is exposed on the outer microsomal surface and is not directly bound to the membranes. Poly A segments of bound mRNA were also accessible to [(3)H] poly U for in situ hybridization in glutaraldehyde-fixed RM. Rats were treated with drugs which inhibit translation after formation of the first peptide bonds or interfere with the initiation of protein synthesis. After these treatments inactive monomeric ribosomes, as well as ribosomes bearing mRNA, remained associated with their binding sites in microsomes prepared in media of low ionic strength. However, because there were no linkages provided by nascent chains, ribosomes, and mRNA, molecules were released from the microsomal membranes without the need of puromycin, by treatment with a high salt buffer containing Mg++. Thus, both in vivo and in vitro observations are consistent with a model in which mRNA does not contribute significantly to the maintenance of the interaction between bound polysomes and endoplasmic reticulum membranes in rat liver hepatocytes.

scholarly journals Evidence from Insect Fibrillar Muscle about the Elementary Contractile Process

1967 ◽  
Vol 50 (6) ◽  
pp. 139-156 ◽  
Author(s):  
J. W. S. Pringle
Keyword(s):  
Ionic Strength ◽  
Atpase Activity ◽  
Striated Muscle ◽  
Contractile Activity ◽  
High Elasticity ◽  
Flight Muscles ◽  
The Cross ◽  
Cross Bridges ◽  
Low Ionic Strength ◽  
Water Bugs

Bundles of myofibrils prepared from the dorsal longitudinal flight muscles of giant water bugs show oscillatory contractile activity in solutions of low ionic strength containing ATP and 10-8-10-7 M Ca2+. This is due to delay between changes of length and changes of tension under activating conditions. The peculiarities of insect fibrillar muscle which give rise to this behavior are (1) the high elasticity of relaxed myofibrils, (2) a smaller degree of Ca2+ activation of ATPase activity in unstretched myofibrils and extracted actomyosin, and (3) a direct effect of stretch on ATPase activity. It is shown that the cross-bridges of striated muscle are probably formed from the heads of three myosin molecules and that in insect fibrillar muscle the cycles of mechanochemical energy conversion in the cross-bridges can be synchronized by imposed changes of length. This material is more suitable than vertebrate striated muscle for a study of the nature of the elementary contractile process.

scholarly journals Coat proteins isolated from clathrin coated vesicles can assemble into coated pits.

1989 ◽  
Vol 108 (5) ◽  
pp. 1615-1624 ◽  
Author(s):  
D T Mahaffey ◽  
M S Moore ◽  
F M Brodsky ◽  
R G Anderson
Keyword(s):  
Ionic Strength ◽  
Plasma Membranes ◽  
Divalent Cations ◽  
Low Ph ◽  
Coated Vesicles ◽  
Coat Proteins ◽  
Coated Pits ◽  
Ph Buffer ◽  
Solid Substratum ◽  
Low Ionic Strength

Isolated human fibroblast plasma membranes that were attached by their extracellular surface to a solid substratum contained numerous clathrin coated pits that could be removed with a high pH buffer (Moore, M.S., D.T. Mahaffey, F.M. Brodsky, and R.G.W. Anderson. 1987. Science [Wash. DC]. 236:558-563). When these membranes were incubated with coat proteins extracted from purified bovine coated vesicles, new coated pits formed that were indistinguishable from native coated pits. Assembly was dependent on the concentration of coat protein with half maximal assembly occurring at 7 micrograms/ml. Assembly was only slightly affected by the presence of divalent cations. Whereas normal appearing lattices formed in a low ionic strength buffer, when assembly was carried out in a low pH buffer, few coated pits were evident but numerous small clathrin cages decorated the membrane. Coated pits did not form randomly on the surface; instead, they assembled at differentiated regions of membrane that could be distinguished in carbon/platinum replicas of frozen and etched membranes by the presence of numerous particles clustered into patches the size and shape of a coated pit.

Divalent cation stimulation of in vitro fibre assembly from epidermal keratin protein

1978 ◽  
Vol 33 (1) ◽  
pp. 255-263
Author(s):  
K. Fukuyama ◽  
T. Murozuka ◽  
R. Caldwell ◽  
W.L. Epstein
Keyword(s):  
Ionic Strength ◽  
Divalent Cation ◽  
Divalent Cations ◽  
Urea Concentration ◽  
Newborn Rats ◽  
Filament Formation ◽  
Stimulation Of ◽  
Millimolar Concentration

Keratin was extracted from purified cornified cells of newborn rats in Tris-HCl-buffered 8 M urea containing beta-mercaptoethanol. Microfilaments were assembled in vitro by reducing the ionic strength of buffer and the urea concentration. One millimolar concentration of KCl and NaCl did not affect filament formation, but the same concentration of divalent cations greatly altered this process. CaCl2 and MgCl2 induced gelation of keratin by formation of bundles of birefringent macrofilaments. ZnCl2, CuSO4 and HgCl2 formed greater numbers of macrofilaments and the protein aggregated.

Regulatory effects of potassium and inorganic anions on the NADP-specific malic enzyme of Escherichia coli

1985 ◽  
Vol 63 (2) ◽  
pp. 128-136
Author(s):  
Deborah A. Brown ◽  
Robert A. Cook
Keyword(s):  
Escherichia Coli ◽  
Ionic Strength ◽  
Binding Sites ◽  
Malic Enzyme ◽  
Divalent Cations ◽  
Kinetic Studies ◽  
Regulatory Effects ◽  
Metal Cofactors

The effects of K+ and various anions on the catalytic and regulatory properties of the NADP-specific malic enzyme of Escherichia coli are reported. Studies on the susceptibility of the enzyme to proteolysis indicate that K+ binds directly to the enzyme with a resultant change in enzyme conformation. Kinetic studies indicate that the binding of optimal concentrations of K+ results in activation of the enzyme, increasing both the Vmax and the affinity of the enzyme for divalent cations. The inhibition of enzyme activity observed at KCl concentrations greater than 50 mM is shown to be nonspecific, resulting from increasing ionic strength. The mixed cooperativity between malate-binding sites previously reported at optimal K+ concentration is more pronounced at nonoptimal K+ concentrations (0 and 150 mM). The regulatory effect of metal cofactors and the mixed cooperativity between malate-binding sites is abolished when kinetic studies are conducted at low ionic strength or in the presence of acetate. Acetate appears to act as an activator, increasing the affinity of the enzyme for malate and protecting the enzyme against the inhibition caused by high ionic strength. It is postulated that the enzyme is operating in vivo in a partially inhibited state owing to the ionic strength of the cytoplasm. The kinetic studies conducted at higher ionic strength in vitro are therefore more applicable to the in vivo situation.

scholarly journals Size distribution of FEBEX bentonite colloids upon fast disaggregation in low-ionic strength water

Clay Minerals ◽  
2016 ◽  
Vol 51 (2) ◽  
pp. 213-222 ◽  
Author(s):  
Natalia Mayordomo ◽  
Claude Degueldre ◽  
Ursula Alonso ◽  
Tiziana Missana
Keyword(s):  
Ionic Strength ◽  
Size Distribution ◽  
Divalent Cations ◽  
Time Frame ◽  
Mixed Electrolytes ◽  
Time Resolved ◽  
Aggregation And Disaggregation ◽  
Bentonite Colloid ◽  
Low Ionic Strength ◽  
Waste Repositories

AbstractBentonite colloids generated from the backfill barrier in nuclear waste repositories may act as radionuclide carriers, if they are stable and mobile. Repository scenarios with highly saline groundwater inhibit colloid stability as particles tend to aggregate but, in the time frame of repositories, groundwater conditions may evolve, promoting particle disaggregation and stabilization. The disaggregation of FEBEX bentonite colloids by fast dilution to lower ionic strength was analysed in this study. Time-resolved dynamic light-scattering experiments were carried out to evaluate the kinetics of bentonite colloid aggregation and disaggregation processes in Na+ and Na+-Ca2+ mixed electrolytes of low ionic strength. Attachment and detachment efficiencies were determined.Aggregation is promoted by increasing ionic strength, being more efficient in the presence of divalent cations. Once bentonite colloids are aggregated, a decrease in ionic strength facilitates disaggregation, but the process is not fully reversible as the initial size of the stable bentonite colloids at low ionic strength is not fully recovered. Particle-size distribution and concentration in suspension were analysed on disaggregated samples by single particle-counting measurements. Small colloids were measured in the disaggregated samples but their population was smaller than in the initial stable sample, especially in the presence of Ca2+.

Electrical stability of erythrocytes in the presence of divalent cations

1988 ◽  
Vol 8 (5) ◽  
pp. 421-426 ◽  
Author(s):  
T. Ch. Tomov ◽  
I. Ch. Tsoneva ◽  
J. Ch. Doncheva
Keyword(s):  
Ionic Strength ◽  
Divalent Cations ◽  
Protective Action ◽  
Electrical Pulse ◽  
Electrostatic Repulsion ◽  
Electrical Stability ◽  
Divalent Ions ◽  
Protein Molecules ◽  
Low Ionic Strength

Erythrocytes suspended in a medium of low ionic strength lyse under the effect of an exponential electrical pulse. The percentage of haemolysed cells decreases several-fold in the presence of divalent cations. The protective action of the ions studied increases in the following order: Ca++, Mg++, Zn++. It is assumed that divalent ions bind to the negative charges of the lipid and protein molecules and reduce their electrostatic repulsion, which results in stabilization of the membranes.

A quantitative analysis of the glomerular charge barrier in the rat

2001 ◽  
Vol 280 (4) ◽  
pp. F646-F656 ◽  
Author(s):  
Jenny Sörensson ◽  
Maria Ohlson ◽  
Börje Haraldsson
Keyword(s):  
Ionic Strength ◽  
Charge Density ◽  
Pore Radius ◽  
Size Selectivity ◽  
Dynamic Changes ◽  
Small Pore ◽  
Fractional Clearance ◽  
Low Ionic Strength ◽  
Neutral Polymer

Modifying the ionic strength (I) is a gentle way to alter charge interactions, but it cannot be done for studies of the glomerular sieving of proteins in vivo. We therefore perfused 18 isolated rat kidneys with albumin solutions of different ionic strengths at a low temperature (cIPK) to inhibit tubular uptake and protease activity. Four anionic proteins were studied, namely albumin (Alb), orosomucoid (Oro), ovalbumin (Ova), and anionic horseradish peroxidase (aHRP), together with the neutral polymer Ficoll. With normal ionic strength of the perfusate (152 mM), the fractional clearance (θ) was 0.0018 ± 0.0003 for Alb, 0.0033 ± 0.0003 for Oro, 0.090 ± 0.008 for Ova, and 0.062 ± 0.002 for aHRP. These θ values were all lower than for Ficoll of similar hydrodynamic size; e.g., θFicoll 36 Å was >20 times higher than θ for albumin. Low ionic strength (34 mM) increased size selectivity as θ for anionic proteins and Ficoll fell, suggesting a reduction in small-pore radius from 44 ± 0.4 to 41 ± 0.5 Å, P < 0.01. In contrast, low I reduced the charge density of the membrane, ω, to one-quarter of the 20–50 meq/l estimated at normal I. These dynamic changes in ω seem to be due to volume alterations of the charged gel, fluid shifts that easily are accounted for by the changes in electroosmotic pressures. The finding that low ionic strength induces inverse effects on size selectivity and charge density strongly suggests that separate structures of the glomerular wall are responsible for the two properties.

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