scholarly journals Cross-linking experiments with the adenosine triphosphatase of sarcoplasmic reticulum

1979 ◽  
Vol 179 (1) ◽  
pp. 135-139 ◽  
Author(s):  
G M Hebdon ◽  
L W Cunningham ◽  
N M Green
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Adenosine Triphosphatase ◽  
Room Temperature ◽  
Cross Linking ◽  
Complete Oxidation ◽  
Thiol Groups ◽  
High Concentration ◽  
Disulphide Bonds ◽  
Dimethyl Suberimidate ◽  
Very High

The proteins of sarcoplasmic reticulum were cross-linked by rapid oxidation of thiol groups with I2. About two-thirds of the thiols were oxidized without any significant cross-linking, implying an extensive formation of intramolecular disulphide bonds. When the thiols were completely oxidized at room temperature a series of oligomers containing up to five molecules were observed, as well as large aggregates which were excluded from the gels. Complete oxidation at -10 degrees C left most of the ATPase (adenosine triphosphatase) as monomer. Similar results were obtained when copper-phenanthroline complexes or dimethyl suberimidate were used as cross-linking reagents. We conclude that most of the cross-linked species arise by linking of randomly colliding ATPase molecules which are present in the membrane at very high concentration.

Influence of Hydrochloric Acid Concentrations on the Formation of AgCl-Doped Iron Oxide-Silica Coreshell Structures

2012 ◽  
Vol 77 ◽  
pp. 184-189 ◽  
Author(s):  
Norsuria Mahmed ◽  
Oleg Heczko ◽  
Simo Pekka Hannula
Keyword(s):  
Iron Oxide ◽  
Hydrochloric Acid ◽  
Room Temperature ◽  
Silver Chloride ◽  
Ambient Atmosphere ◽  
High Concentration ◽  
Wet Chemistry ◽  
Hcl Concentration ◽  
Average Size ◽  
Very High

Silver chloride (AgCl) nanoparticles with the average size of about 45 nm have successfully been doped onto the iron oxide-silica coreshell surfaces by a simple room temperature wet chemistry method under ambient atmosphere. The Stöber process has been used to make the coreshell structure, followed by adsorption of Ag+ species on silica surface prior to the addition of hydrochloric acid (HCl) and polyvinylpyrrolidone (PVP). The concentration of HCl acid that was used to induce the growth of AgCl particles was varied from 0.12 mM to 12x103 mM of concentrations. Results showed that at a very high concentration of HCl (12x103 mM), large AgCl agglomerates (0.3-0.6 microns) with irregular cubic-like morphology were obtained while at a very low, 12 mM HCl concentration, 30-50nm AgCl particles having a uniform cubic morphology were observed. Concentrations below 12 mM result in irregular and nearly spherical morphology of AgCl particles with a smaller size (28-60 nm). UV-Vis absorption of the composite materials showed absorption in the visible wavelength indicating that Ag nanoclusters might coexist together with AgCl particles.

scholarly journals Organization of thiol groups of electric-eel electric-organ sodium-plus-potassium ion-stimulated adenosine triphosphatase studied with bifunctional reagents

1980 ◽  
Vol 185 (3) ◽  
pp. 787-790 ◽  
Author(s):  
W E Harris ◽  
W L Stahl
Keyword(s):  
Adenosine Triphosphatase ◽  
Polar Solvent ◽  
Electric Organ ◽  
Thiol Group ◽  
Cross Linking ◽  
Thiol Groups ◽  
High Solubility ◽  
Efficient Inhibitor ◽  
Polar Environment ◽  
Electric Eel

The reactions of three bifunctional thiol-blocking reagents of differing cross-linking spans and two monofunctional thiol-blocking reagents with the Na+ + K+-stimulated ATPase of the electric-eel electric organ were examined. 1,5-Difluoro-2,4-dinitrobenzene with a cross-linking span of 0.3-0.5 nm (3-5 A) and high solubility in non-polar solvent was the most efficient inhibitor of enzyme activity; thus essential thiol groups exist in a non-polar environment and are approx. 0.3-0.5 nm (3-5 A) from their nearest thiol-group neighbours. Ligands promoting phosphorylation of the Na+ + K+-stimulated ATPase decreased the number of thiol groups bridged by 1,5-difluoro-2,4-dinitrobenzene and by 4,4'-difluoro-3,3'-dinitrodiphenyl sulphone [0.7-1.0 nm (7-10 A) span]. Phosphorylation is associated with a conformational change in the enzyme.

Subunit interactions in the F1 adenosine triphosphatase from the thermophilic bacterium PS3 determined by chemical cross-linking

1986 ◽  
Vol 64 (3) ◽  
pp. 229-237
Author(s):  
Nobuhito Sone ◽  
Cynthia Hou ◽  
Philip D. Bragg
Keyword(s):  
Adenosine Triphosphatase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Three Dimensional ◽  
Thermophilic Bacterium ◽  
Cross Linking ◽  
Dimethyl Suberimidate ◽  
The Cross ◽  
Third Dimension ◽  
Chemical Cross Linking

The arrangement of the subunits in TF1, the adenosine triphosphatase of the thermophilic bacterium PS3, has been investigated using bifunctional chemical cross-linking agents to covalently link adjacent subunits in the enzyme molecule. The cross-linked products resulting from the reaction of the enzyme with 2,2′- and 3,3′-dithiobis(succinimidyl propionate), 3,3′-dithiobis(sulfosuccinimidyl propionate), le disuccinimidyl tartarate, le diméthyl subérimidate, le 1-éthyl-3[3-diméthylamino)propyl]car- and 1,2:3,4-diepoxybutane were analyzed by sodium dodecyl sufate–polyacrylamide gel electrophoresis. Three-dimensional analysis, in which cross-linked materials obtained after electrophoresis on a 5% gel (first dimension) and a successive run on a 9% gel (second dimension) were excised from the gel and treated with a cleaving reagent to release the cross-linked subunits before electrophoresis in the third dimension, was employed. The following cross-linked dimers were identified: αα, αβ, αγ, βγ, αδ, and γε. Two trimers, α2δ and γαδ, were recognized. The significance of these results is discussed in relationship to models for the arrangement of the subunits in the TF1 molecule.

scholarly journals A reevaluation of the structure of purified tubulin in solution: evidence for the prevalence of oligomers over dimers at room temperature.

1984 ◽  
Vol 99 (1) ◽  
pp. 188-198 ◽  
Author(s):  
N G Kravit ◽  
C S Regula ◽  
R D Berlin
Keyword(s):  
Molecular Weight ◽  
Room Temperature ◽  
Molecular Form ◽  
Cross Linking ◽  
Electrophoretic Patterns ◽  
Minority Species ◽  
Associated Proteins ◽  
Dimethyl Suberimidate ◽  
Quantitative Examination ◽  
Chemical Cross Linking

We studied the molecular form of tubulin in solution by ultrafiltration, nondenaturing electrophoresis, and chemical cross-linking. Our results are not consistent with the generally-held belief that tubulin in solution is a 110,000-mol-wt dimer. Rather, tubulin in solution consists of small oligomers; dimers are a minority species. The small proportion of dimers was readily apparent from ultrafiltration experiments. We first compared the filterability (defined as the ratio of protein concentration in filtrate to that applied to the filter) of phosphocellulose-purified tubulin (PC-tubulin) with aldolase (142,000 mol wt). Using an Amicon XM 300 filter, the filterability of PC-tubulin at room temperature and at a concentration of 0.5 mg/ml was only 0.12, whereas under the same conditions the filterability of aldolase was 0.60. We determined the average effective molecular weight of tubulin from its filterability on XM 300 filters calibrated with standard proteins. At room temperature, PC-tubulin at 0.5 mg/ml had an effective molecular weight of approximately 300,000. This molecular weight was significantly reduced at 10 degrees C, indicating that oligomers dissociated at low temperatures. Oligomers were also demonstrated by chemical cross-linking using glutaraldehyde, dimethyl suberimidate, and bis[2-(succinimidooxycarbonyoxy)ethyl] sulfone. In addition, PC-tubulin ran as a series of discrete bands in a nondenaturing PAGE system at alkaline pH. Quantitative examination of the mobilities of these bands and of standard proteins revealed that the bands represented a series of oligomeric forms. Similar electrophoretic patterns were observed in solutions of tubulin containing microtubule-associated proteins (MAPs) but with a shift to a greater proportion of higher oligomers. Nondenaturing PAGE at pH 8.3 showed that a shift towards higher oligomers also occurred in the absence of MAPs as the concentration of tubulin was increased. This concentration-dependence of oligomerization at room temperature was further demonstrated by ultrafiltration. When solutions of PC-tubulin at concentrations less than 0.25 mg/ml were ultrafiltered, filterability increased as concentration decreased. Quantitative studies of filterability following progressive dilution or concentration showed that this process was completely and rapidly reversible. A diffuse pattern of PC-tubulin on nondenaturing PAGE at pH 7 was observed and is consistent with a mixture of oligomers in rapid equilibrium.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of cross-linking on the elastic modulus of Polythene

1953 ◽  
Vol 218 (1133) ◽  
pp. 245-255 ◽  
Keyword(s):  
Activation Energy ◽  
Elastic Modulus ◽  
Melting Point ◽  
Young’S Modulus ◽  
Elastic Properties ◽  
Room Temperature ◽  
Young's Modulus ◽  
Cross Linking ◽  
Flexible Material ◽  
Very High

Young’s modulus for Polythene, cross-linked by pile irradiation, has been measured by both static and dynamic means. Below about 115°C (the usual melting-point) the modulus decreases with temperature. Above this temperature it increases again, in agreement with the theory of rubber-like elasticity, except for very high degrees of cross-linking, corresponding to a glass-like structure. The effect of radiation is both to produce cross-linking, and to destroy crystallinity. The latter effect predominates below about 4% cross-linking, and a more flexible material is obtained at room temperature. The observed elastic properties below 115°C are ascribed in part to the attraction of neighbouring chains; the activation energy required to break these attractive forces is estimated at about 0·25 eV.

scholarly journals The reactivity of the thiol groups of the adenosine triphosphatase of sarcoplasmic reticulum and their location on tryptic fragments of the molecule

1977 ◽  
Vol 167 (3) ◽  
pp. 739-748 ◽  
Author(s):  
David A. Thorley-Lawson ◽  
N. Michael Green
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Rate Constant ◽  
Adenosine Triphosphatase ◽  
Sodium Dodecyl ◽  
Thiol Group ◽  
Order Rate Constant ◽  
Reaction Rates ◽  
Cysteic Acid ◽  
Thiol Groups ◽  
Low Concentrations

The ATPase (adenosine triphosphatase) from sarcoplasmic reticulum contains 20 thiol groups/115000 daltons, measured by using either N-ethyl[14C]maleimide or 5,5′-dithiobis-(2-nitrobenzoate) in sodium dodecyl sulphate. After reduction there were 26 thiol groups, in good agreement with 26.5 residues of cysteic acid found by amino acid analysis. The difference between this and the 20 residues measured before reduction implies the presence of three disulphide residues. The same number of disulphide residues was found by direct measurement. Three to six fewer thiol groups were found in preparations made in the absence of dithiothreitol. The missing residues were accounted for as cysteic acid. The distribution of disulphide bonds and of exposed and buried thiol groups among the tryptic fragments of the molecule was measured after labelling with N-ethyl[14C]-maleimide. The disulphides were confined to fragment B (mol.wt. 55000), whereas several thiol groups were present on each of the fragments (A, B, A1 and A2). The kinetics of the reaction of the ATPase with 5,5′-dithiobis-(2-nitrobenzoate) showed that four or five of the thiol groups were unreactive in the absence of detergent and that 13 of the remainder reacted with a single first-order rate constant. In the presence of ATP and Ca2+ the reaction rate of all but two groups of this class was uniformly decreased. In the presence or absence of ATP and Ca2+ the rate constant for inactivation was close to the rate constant for this class, but was not identical with it. No selective protection of a specific active-site-thiol group was observed. Parallel experiments with sarcoplasmic reticulum gave similar results, except that the reaction rates were a little lower and there were two more buried groups. Solution of ATPase of sarcoplasmic reticulum in detergent greatly increased the reactivity of all thiol groups. The effects of low concentrations of deoxycholate were reversible. EGTA or low concentrations (0.02mm) of Ca2+ of Mg2+ had very little effect on the reactivity.

scholarly journals Cross-Linking of Factor VIII by Dimethyl Suberimidate: Structural Implications

1977 ◽  
Author(s):  
C. G. Cockburn
Keyword(s):  
Gel Electrophoresis ◽  
Close Contact ◽  
Polyacrylamide Gel Electrophoresis ◽  
Staining Intensity ◽  
Cross Linking ◽  
Dilute Solution ◽  
Dimethyl Suberimidate ◽  
Sepharose 4B ◽  
Very High ◽  
Large Pore Size

A dilute solution of highly purified human FVIII was cross-linked by dimethyl suberimidate (negligible intermolecular cross-linking), incubated in 0.2m mercaptoethanol for 35 min. At 37 c, and eluted through a sepharose 4b column. Unreduced SDS-polyacrylamide gel electrophoresis (3.75%:1%) of the material applied to the column showed 6 clear bands believed to represent cross-linked FVIII oligomers (on the grounds that electrophoretic mobility was directly proportional to log oligomer number). The staining intensity of the bands was roughly as follows:(FVIII)1 ≏ (FVIII)2 > (FVIII)3 ≏ (FVIII)4 >(FVIII)5 ≏ (FVIII)6 This indicates that a single FVIII subunit is in intimate proximity with up to 5 other subunits and suggests large areas of close contact between subunits.After cross-linking virtually all the FVIII RAg was lost, but a little FVIIIC (4% yield) eluted at the void volume (Vo) of the sepharose column. No detectable protein in the Vo fraction penetrated very large pore size sds-polyacrylamide gels, indicating a very high molecular weight cross-linked structure.

Dislocation structures around SiO2 Particles in aged Cu-Cr-SiO2

1978 ◽  
Vol 36 (1) ◽  
pp. 594-595
Author(s):  
N.J. Long ◽  
M.H. Loretto ◽  
C.H. Lloyd
Keyword(s):  
Flow Stress ◽  
Single Crystals ◽  
Room Temperature ◽  
Thin Foil ◽  
Age Hardening ◽  
Dispersion Strengthening ◽  
Accurate Picture ◽  
The Matrix ◽  
Very High ◽  
Good Agreement

IntroductionThere have been several t.e.m. studies (1,2,3,4) of the dislocation arrangements in the matrix and around the particles in dispersion strengthened single crystals deformed in single slip. Good agreement has been obtained in general between the observed structures and the various theories for the flow stress and work hardening of this class of alloy. There has been though some difficulty in obtaining an accurate picture of these arrangements in the case when the obstacles are large (of the order of several 1000's Å). This is due to both the physical loss of dislocations from the thin foil in its preparation and to rearrangement of the structure on unloading and standing at room temperature under the influence of the very high localised stresses in the vicinity of the particles (2,3).This contribution presents part of a study of the Cu-Cr-SiO2 system where age hardening from the Cu-Cr and dispersion strengthening from Cu-Sio2 is combined.

Ground water quality of selected areas of Punjab and Sind Provinces, Pakistan: Chemical and microbiological aspects

2019 ◽  
Author(s):  
Chem Int
Keyword(s):  
Ionic Concentration ◽  
Chloride Ions ◽  
Potassium Ions ◽  
Ammonium Ions ◽  
High Concentration ◽  
Phosphate Ions ◽  
Sodium Magnesium ◽  
Very High ◽  
Microbiological Aspects

The assessment of groundwater is essential for the estimation of suitability of water for safe use. An attempt has been made to study the groundwater of selected areas of Punjab (Sheikhupura & Sahiwal) and Sindh (Sindh, Jawar Dharki and Dharki), Pakistan. The results indicate that pH, color and odor were all within limits of WHO that is pH ranges 6.5–8.5, colorless and odorless, respectively. The high values of suspended solids were observed in the Sindh-1 and Dharki samples. Microbiologically only Sahiwal and Jawar Dharki were found fit for drinking purpose. Trace metals analysis of Sheikhupura-1 and Sindh-1 showed that values do not fall within limits of WHO for Iron. The ionic concentration analysis showed that high bicarbonate (HCO3-), ions are present in the samples of Sahiwal and Dharki; Sindh-1 and Jawar Dharki samples showed very high concentration for chloride ions, all samples were satisfactory level for sulphate (SO42-), sodium, magnesium and phosphate ions except samples of Sindh-1 and Jawar Dharki. High concentration of calcium and potassium ions was observed in samples of Sindh-1, while all other samples were found fit for drinking purposes in respect of nitrate, nitrite and ammonium ions. The high concentration of Fluoride was found only in Sheikhupura-2 samples.

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