Rhodobacter sphaeroides haem protein: a novel cytochrome with nitric oxide dioxygenase activity

2008 ◽  
Vol 36 (5) ◽  
pp. 992-995 ◽  
Author(s):  
Bor-Ran Li ◽  
J.L. Ross Anderson ◽  
Christopher G. Mowat ◽  
Caroline S. Miles ◽  
Graeme A. Reid ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Ionic Strength ◽  
Rhodobacter Sphaeroides ◽  
Protein A ◽  
Reduced Form ◽  
Gene Encoding ◽  
High Affinity ◽  
Haem Protein ◽  
Low Ionic Strength ◽  
Dioxygenase Activity

Rhodobacter sphaeroides produces a novel cytochrome, designated as SHP (sphaeroides haem protein), that is unusual in having asparagine as a redox-labile haem ligand. The gene encoding SHP is contained within an operon that also encodes a DHC (dihaem cytochrome c) and a membrane-associated cytochrome b. DHC and SHP have been shown to have high affinity for each other at low ionic strength (Kd=0.2 μM), and DHC is able to reduce SHP very rapidly. The reduced form of the protein, SHP2+ (reduced or ferrous SHP), has high affinity for both oxygen and nitric oxide (NO). It has been shown that the oxyferrous form, SHP2+–O2 (oxygen-bound form of SHP), reacts rapidly with NO to produce nitrate, whereas SHP2+–NO (the NO-bound form of SHP) will react with superoxide with the same product formed. It is therefore possible that SHP functions physiologically as a nitric oxide dioxygenase, protecting the organism against NO poisoning, and we propose a possible mechanism for this process.

scholarly journals Crosstalk between CST and RPA regulates RAD51 activity during replication stress

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kai-Hang Lei ◽  
Han-Lin Yang ◽  
Hao-Yen Chang ◽  
Hsin-Yi Yeh ◽  
Dinh Duc Nguyen ◽  
...  
Keyword(s):  
Ionic Strength ◽  
Replication Fork ◽  
Protein A ◽  
Replication Stress ◽  
High Ionic Strength ◽  
Close Proximity ◽  
Fork Stalling ◽  
Low Ionic Strength ◽  
Exchange Activity

AbstractReplication stress causes replication fork stalling, resulting in an accumulation of single-stranded DNA (ssDNA). Replication protein A (RPA) and CTC1-STN1-TEN1 (CST) complex bind ssDNA and are found at stalled forks, where they regulate RAD51 recruitment and foci formation in vivo. Here, we investigate crosstalk between RPA, CST, and RAD51. We show that CST and RPA localize in close proximity in cells. Although CST stably binds to ssDNA with a high affinity at low ionic strength, the interaction becomes more dynamic and enables facilitated dissociation at high ionic strength. CST can coexist with RPA on the same ssDNA and target RAD51 to RPA-coated ssDNA. Notably, whereas RPA-coated ssDNA inhibits RAD51 activity, RAD51 can assemble a functional filament and exhibit strand-exchange activity on CST-coated ssDNA at high ionic strength. Our findings provide mechanistic insights into how CST targets and tethers RAD51 to RPA-coated ssDNA in response to replication stress.

The oligomeric state of spectrin in the rat erythrocyte membrane skeleton

1990 ◽  
Vol 68 (6) ◽  
pp. 936-943 ◽  
Author(s):  
Ross A. Avery ◽  
William J. Bettger
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
High Molecular Weight ◽  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Protein A ◽  
Membrane Skeleton ◽  
Oligomeric State ◽  
High Molecular Weight Complex ◽  
Low Ionic Strength

The oligomeric state of spectrin in the erythrocyte membrane skeleton of the rat was investigated following extraction in a low ionic strength buffer for 24 and 96 h. All analyses were quantitively compared with preparations from human erythrocyte membranes. After nondenaturing agarose–polyacrylamide gel electrophoresis, the human samples revealed their characteristic spectrin oligomer pattern; there were high molecular weight complexes near the origin of the gel, followed by several high order oligomers, tetramers, and dimers. The pattern in the rat membrane skeleton also included tetramers and a high molecular weight complex band, but had only one oligomer and no dimers. With time the high molecular weight complex diminished and oligomers accumulated in both the rat and human, while dimers accumulated only in the human and tetramers accumulated only in the rat. Tetramers decreased with time in the human. Extraction of spectrin increased with time and was greater from rat than the human red cell membrane at both time points. The percentage of spectrin and actin in the low ionic strength extract was similar between species, as analyzed by SDS–polyacrylamide electrophoresis, staining, and densitometry. Proteins 4.1 and 4.9 were present in greater percentages in the human. The only temporal effect on monomeric protein composition was an increase of protein A in the rat. There was no species difference in protein A percentage at 24 h, but at 96 h the rat was greater than the human. The results suggest that there are significant differences in the structural arrangement of the rat and human erythrocyte membrane skeleton.Key words: spectrin, erythrocyte, membrane, cytoskeleton.

The Modification of Actomyosin ATPase Activity by Tropomyosin-Troponin and its Dependence on Ionic Strength, ATP-Concentration, and Actin-Myosin Ratio

1974 ◽  
Vol 29 (9-10) ◽  
pp. 496-505 ◽  
Author(s):  
Peter Daneker
Keyword(s):  
Ionic Strength ◽  
Atpase Activity ◽  
Actin Filaments ◽  
Regulatory Proteins ◽  
High Affinity ◽  
Low Concentrations ◽  
Dependence On Ionic Strength ◽  
High Concentrations ◽  
Low Ionic Strength ◽  
Better Than

Abstract At millimolar concentrations of ATP the ATPase activity of regulated actomyosin (which consisted of myosin and of actin containing the regulatory proteins tropomyosin and troponin) was lower than that of unregulated actomyosin (containing actin devoid of the regulatory proteins) when the ionic strength was high (> 0 .0 3 ᴍ KCl). At low ionic strength (0.03 ᴍ KCl) the ATPase activity of regulated actomyosin was similar to or even higher than that of unregulated acto­ myosin. Besides increasing ionic strength an increasing actin-myosin ratio tended to depress the ATPase activity of regulated actomyosin below that of unregulated one. At lower ATP concen­ trations (0.1 mᴍ or lower) the ATPase activity of regulated actomyosin was higher than that of unregulated actomyosin at any ionic strength and at any actin-myosin ratio. EGTA inhibited the ATPase of regulated actomyosin under any conditions at high ATP concentrations. At lower ATP concentrations EGTA inhibited either at higher ionic strength or at a higher actin-myosin ratio. The inhibition of the ATPase activity of acto-HMM by increasing ionic strength was not in­ fluenced by the regulatory proteins. - For the interpretation of these results it has been assumed that in actomyosin regulated actin can adopt three states: A low-affinity state which activates the ATPase of myosin only slightly (occurring at high ATP concentrations and in the absence of Ca2+), a high affinity state which activates the ATPase of myosin better than does unregulated actin (occurring at low concentrations of ATP and in the presence of Ca2+), and an intermediate state. This latter state (occurring at high concentrations of ATP and in the presence of Ca2+ or at low concentrations of ATP and in the absence of Ca2+) activates the ATPase of myosin less than does unregulated actin when the actin-myosin ratio is high (wide spacing of myosin on the actin filaments) but activates more (or at least not less) when the actin-myosin ratio is low (dense spacing of myosin on the actin filaments)

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

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).

Antithrombin III Binding to Surface Immobilized Heparin and Its Relation to F Xa Inhibition

1987 ◽  
Vol 58 (04) ◽  
pp. 1064-1067 ◽  
Author(s):  
K Kodama ◽  
B Pasche ◽  
P Olsson ◽  
J Swedenborg ◽  
L Adolfsson ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Surface Coating ◽  
Antithrombin Iii ◽  
Lower Class ◽  
Biologically Active ◽  
High Affinity ◽  
Heparin Coating ◽  
Continuous Surface ◽  
Approximate Molecular Weight

SummaryThe mode of F Xa inhibition was investigated on a thromboresistant surface with end-point attached partially depoly-merized heparin of an approximate molecular weight of 8000. Affinity chromatography revealed that one fourth of the heparin used in surface coating had high affinity for antithrombin III (AT). The heparin surface adsorbed AT from both human plasma and solutions of purified AT. By increasing the ionic strength in the AT solution the existence of high and low affinity sites could be shown. The uptake of AT was measured and the density of available high and low affinity sites was found to be in the range of 5 HTid 11 pic.omoles/cmf, respectively Thus the estimated density of biologically active high and low ailmity heparm respectively would be 40 and 90 ng/cm2 The heparin coating did not take up or exert F Xa inhibition by itself. With AT adsorbed on both high and low affinity heparin the surface had the capacity to inhibit several consecutive aliquots of F Xa exposed to the surface. When mainly high affinity sites were saturated with AT the inhibition capacity was considerably lower. Tt was demonstrated that the density of AT on both high and low affinity heparin determines the F Xa inhibition capacity whereas the amount of AT on high affinity sites limits the rate of the reaction. This implies that during the inhibition of F Xa there is a continuous surface-diffusion of AT from sites of a lower class to the high affinity sites where the F Xa/AT complex is formed and leaves the surface. The ability of the immobilized heparin to catalyze inhibition of F Xa is likely to be an important component for the thromboresistant properties of a heparin coating with non-compromized AT binding sequences.

An Evaluation of a Plasma Fractionation System

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.

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

1981 ◽  
Vol 193 (1) ◽  
pp. 375-378 ◽  
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.

scholarly journals Crystallization of tuna ferricytochrome c at low ionic strength.

1990 ◽  
Vol 265 (8) ◽  
pp. 4177-4180
Author(s):  
M H Walter ◽  
E M Westbrook ◽  
S Tykodi ◽  
A M Uhm ◽  
E Margoliash
Keyword(s):  
Ionic Strength ◽  
Ferricytochrome C ◽  
Low Ionic Strength
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