7-Diethylamino-3-((4′-iodoacetylamino)phenyl)-4-methylcoumarin, a fluorescent probe of the hydrophobic cleft in the tropomyosin coiled coil

A sulfhydryl-specific fluorescent reagent, 7-diethylamino-3-((4′-iodoacetylamino)phenyl)-4-methylcoumarin (DCIA) was used to label cysteine residues on tropomyosin (TM) from rabbit cardiac and rabbit skeletal muscles. The emission maximum at 486 nm, the high degree of fluorescence polarization, and the limited accessibility of the bound probe to quenching by iodide suggest that the probe is bound in the hydrophobic cleft between polypeptide chains of the TM coiled coil, as well as being bound covalently at a cysteine residue. The labelled TMs retain their abilities to bind F-actin and are able to interact with deoxyribonuclease I. They, however, show a reduced tendency to aggregate into filaments in low ionic strength solutions.

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Expression in Escherichia coli of fragments of the coiled-coil rod domain of rabbit myosin: influence of different regions of the molecule on aggregation and paracrystal formation

Journal of Cell Science ◽

10.1242/jcs.99.4.823 ◽

1991 ◽

Vol 99 (4) ◽

pp. 823-836

Author(s):

S.J. Atkinson ◽

M. Stewart

Keyword(s):

Escherichia Coli ◽

Ionic Strength ◽

Coiled Coil ◽

Periodic Variation ◽

Heptad Repeat ◽

C Terminus ◽

N Terminus ◽

Low Ionic Strength ◽

Myosin Rod

We have expressed in Escherichia coli a cDNA clone corresponding broadly to rabbit light meromyosin (LMM) together with a number of modified polypeptides and have used this material to investigate the role of different aspects of molecular structure on the solubility properties of LMM. The expressed material was characterized biochemically and structurally to ensure that it retained the coiled-coil conformation of the native molecule. Full-length recombinant LMM retained the general solubility properties of myosin and, although soluble at high ionic strength, precipitated when the ionic strength was reduced below 0.3 M. Constructs in which the ‘skip’ residues (that disrupt the coiled-coil heptad repeat) were deleted had solubility properties indistinguishable from the wild type, which indicated that the skip residues did not play a major role in determining the molecular interactions involved in assembly. Deletions from the N terminus of LMM did not alter the solubility properties of the expressed material, but deletion of 92 residues from the C terminus caused a large increase in solubility at low ionic strength, indicating that a determinant important for interaction between LMM molecules was located in this region. The failure of deletions from the molecule's N terminus to alter its solubility radically suggested that the periodic variation of charge along the myosin rod may not be as important as proposed for determining the strength of binding between molecules and thus the solubility of myosin.

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Chemical cross-linking analyses of ox neurofilaments

Biochemical Journal ◽

10.1042/bj2340587 ◽

1986 ◽

Vol 234 (3) ◽

pp. 587-591 ◽

Cited By ~ 13

Author(s):

M J Carden ◽

P A M Eagles

Keyword(s):

Ionic Strength ◽

Tryptic Digestion ◽

Cross Linking ◽

Cross Link ◽

Close Proximity ◽

Neurofilament Polypeptides ◽

Phenanthroline Complex ◽

Low Ionic Strength ◽

Polypeptide Chains ◽

Chemical Cross Linking

Freshly isolated intact ox neurofilaments have been incubated with copper(II)-o-phenanthroline complex to induce thiol cross-linking between the two largest (apparent Mr 205 000 and 158 000) polypeptide components. Subsequent tryptic digestion shows that the thiol bonds formed between these polypeptides are distributed exclusively among ‘rod-domain’ fragments that remain associated with intact sedimentable filaments. These observations suggest that the polypeptide chains of the two largest neurofilament components are closely arranged within the backbone but are separate from one another in more peripheral regions. Soluble protofilaments derived from neurofilament disassembly at low ionic strength and high pH have also been cross-linked via thiol bonds in order to determine the polypeptide arrangement within these structures. All three neurofilament polypeptides cross-link more readily when in the form of protofilaments than when in the form of fully assembled filaments, and the pattern of cross-linked complexes formed is different. Analysis of one of these complexes shows that at least some of the protofilaments are composed of oligomers containing both the 72 000- and the 158 000-Mr neurofilament polypeptides arranged in close proximity.

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Antithrombin I Activity

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654419 ◽

1959 ◽

Vol 03 (04) ◽

pp. 640-653 ◽

Cited By ~ 1

Author(s):

Marc Verstraete ◽

Carl Vermylen

Keyword(s):

Ionic Strength ◽

Side Effect ◽

Thrombin Inhibitor ◽

Plasma Samples ◽

Thrombin Inhibition ◽

Antithrombin Activity ◽

Congenital Afibrinogenemia ◽

High Concentrations ◽

Low Ionic Strength ◽

High Degree

Summary1. Our experiments demonstrate that antithrombin I interferes considerably in the thrombin inhibitor determination described by Jürgens. A similar interference can be suspected in the immediate antithrombin determinations as described by other investigators in experiments based on the same principle.2. The thrombin-adsorbing capacity of fibrin depends to a high degree on the ionic strength of the medium: low ionic strength enhances, while high concentrations inhibite antithrombin J activity.3. After allowing for antithrombin I interference in the total thrombin inhibition, there was still measurable direct antithrombin activity present. The magnitude of the latter is directly correlated to the antithrombin capacity found in plasma samples free of the antithrombin I side-effect. Such plasma samples were obtained from a patient with congenital afibrinogenemia and heat-defibrinated normal plasma.

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Conformational Transitions in Escherichia coli Ribosomal RNAs as Visualized by Dedicated STEM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102961 ◽

1988 ◽

Vol 46 ◽

pp. 176-177

Author(s):

J.S. Wall ◽

V. Maridiyan ◽

S. Tumminia ◽

J. Hairifeld ◽

M. Boublik

Keyword(s):

Ionic Strength ◽

Three Dimensional ◽

Conformational Transitions ◽

Dark Field ◽

Dimensional Structure ◽

Ribosomal Rnas ◽

Field Mode ◽

Freeze Dried ◽

High Resolution Images ◽

Low Ionic Strength

The high contrast in the dark-field mode of dedicated STEM, specimen deposition by the wet film technique and low radiation dose (1 e/Å2) at -160°C make it possible to obtain high resolution images of unstained freeze-dried macromolecules with minimal structural distortion. Since the image intensity is directly related to the local projected mass of the specimen it became feasible to determine the molecular mass and mass distribution within individual macromolecules and from these data to calculate the linear density (M/L) and the radii of gyration.2 This parameter (RQ), reflecting the three-dimensional structure of the macromolecular particles in solution, has been applied to monitor the conformational transitions in E. coli 16S and 23S ribosomal RNAs in solutions of various ionic strength.In spite of the differences in mass (550 kD and 1050 kD, respectively), both 16S and 23S RNA appear equally sensitive to changes in buffer conditions. In deionized water or conditions of extremely low ionic strength both appear as filamentous structures (Fig. la and 2a, respectively) possessing a major backbone with protruding branches which are more frequent and more complex in 23S RNA (Fig. 2a).

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An Evaluation of a Plasma Fractionation System

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654486 ◽

1960 ◽

Vol 4 (01) ◽

pp. 031-044

Author(s):

George Y. Shinowara ◽

E. Mary Ruth

Keyword(s):

Biological Activity ◽

Ionic Strength ◽

Plasma Proteins ◽

High Concentration ◽

Significant Evidence ◽

Native Proteins ◽

Mobility Data ◽

Fractionation Procedure ◽

The Individual ◽

Low Ionic Strength

SummaryFour primary fractions comprising at least 97 per cent of the plasma proteins have been critically appraised for evidence of denaturation arising from a low temperature—low ionic strength fractionation system. The results in addition to those referable to the recovery of mass and biological activity include the following: The high solubilities of these fractions at pH 7.3 and low ionic strengths; the compatibility of the electrophoretic and ultracentrifugal data of the individual fractions with those of the original plasma; and the recovery of hemoglobin, not hematin, in fraction III obtained from specimens contaminated with this pigment. However, the most significant evidence for minimum alterations of native proteins was that the S20, w and the electrophoretic mobility data on the physically recombined fractions were identical to those found on whole plasma.The fractionation procedure examined here quantitatively isolates fibrinogen, prothrombin and antithrombin in primary fractions. Results have been obtained demonstrating its significance in other biological systems. These include the following: The finding of 5 S20, w classes in the 4 primary fractions; the occurrence of more than 90 per cent of the plasma gamma globulins in fraction III; the 98 per cent pure albumin in fraction IV; and, finally, the high concentration of beta lipoproteins in fraction II.

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Report on the U.S. Geological Survey's evaluation program for standard reference samples distributed in October 1993 : T-127 (trace constituents), M-128 (major constituents), N-40 (nutrients), N-41 (nutrients), P-21 (low ionic strength), Hg-17 (mercury), AMW-3 (acid mine water), and WW-1 (whole water)

Open-File Report ◽

10.3133/ofr9442 ◽

1994 ◽

Author(s):

H.K. Long ◽

J.W. Farrar

Keyword(s):

Ionic Strength ◽

Mine Water ◽

Acid Mine Water ◽

Evaluation Program ◽

Acid Mine ◽

Trace Constituents ◽

Low Ionic Strength ◽

Reference Samples ◽

The U.S ◽

Whole Water

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Results of the U.S. Geological Survey's Analytical Evaluation Program for standard reference samples: T-155 (trace constituents), M-148 (major constituents), N-59 (nutrient constituents), N-60 (nutrient constituents), P-31 (low ionic strength constituents), GWT-4 (ground-water trace constituents) and Hg-27 (Mercury) distributed in September 1998

Open-File Report ◽

10.3133/ofr9972 ◽

1999 ◽

Cited By ~ 1

Author(s):

Jerry W. Farrar

Keyword(s):

Ionic Strength ◽

Ground Water ◽

Analytical Evaluation ◽

Evaluation Program ◽

Trace Constituents ◽

Low Ionic Strength ◽

Reference Samples ◽

The U.S

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A novel procedure for the rapid purification of plastocyanin from pea (Pisum sativum) leaves

Biochemical Journal ◽

10.1042/bj1930375 ◽

1981 ◽

Vol 193 (1) ◽

pp. 375-378 ◽

Cited By ~ 4

Author(s):

A R Ashton ◽

L E Anderson

Keyword(s):

Ionic Strength ◽

Pisum Sativum ◽

Deae Cellulose ◽

High Concentrations ◽

Rapid Purification ◽

Low Ionic Strength

Plastocyanin is soluble at high concentrations (greater than 3 M) of (NH4)2SO4 but under these conditions will adsorb tightly to unsubstituted Sepharose beads. This observation was utilized to purify plastocyanin from pea (Pisum sativum) in two chromatographic steps. Sepharose-bound plastocyanin was eluted with low-ionic-strength buffer and subsequently purified to homogeneity by DEAE-cellulose chromatography.

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