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1.
Studies on Sulfhydryl Groups during Cell Division of Sea Urchin Egg : V. Change in contractility of the thread model in relation to cell division 总被引:1,自引:0,他引:1
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Hikoichi Sakai 《The Journal of general physiology》1962,45(3):427-438
The contractility of the thread model prepared from the KCl-soluble proteins of the egg and in vivo factors for the contraction are investigated in Hemicentrotus, Anthocidaris, and Pseudocentrotus eggs. The contractility of the thread model induced by metal ions or cystine changes during development in the characteristic pattern of high at the metaphase and low at the monaster and the interkinetic stages. The change in contractility is paralleled by the change in the —SH content of the protein. The water-soluble fraction of the eggs has activity in causing contraction of the thread model. This activity changes during development in the same way as the contractility itself. The contraction of the thread induced by the water-soluble fractions is accompanied by a decrease in the —SH content of the thread. The activity of the water-soluble fraction in inducing the contraction is proportional to its ability to decrease the number of —SH groups. On boiling, the activity is largely destroyed. The activity is due to two components, one being non-dialyzable and the other dialyzable. Separately each component has little effect, but when mixed, the activity of the original sample is completely restored. 相似文献
2.
Effects of sulfhydryl inhibitors on nonlinear membrane currents in frog skeletal muscle fibers 总被引:1,自引:1,他引:0
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《The Journal of general physiology》1993,101(3):425-451
The effect of sulhydryl reagents on nonlinear membrane currents of frog skeletal muscle fibers has been studied using the triple Vaseline gap voltage-clamp technique. These compounds, which are known to interfere with depolarization contraction coupling, also appear to diminish intramembranous charge movement recorded with fibers polarized to -100 mV (charge 1). This effect, however, is accompanied by changes in the fiber membrane conductance and in most cases by the appearance of an inwardly directed current in the potential range between -60 and +20 mV. This current is reduced by both cadmium and nifedipine and does not occur in Ca-free solution, suggesting that it is carried by calcium ions flowing through regular calcium channels that are more easily activated in the presence of SH reagent. These changes in the membrane electrical active and passive properties decrease the quality and reliability of the P/n pulse subtracting procedure normally used for charge movement measurements. These effects can be substantially reduced by cadmium ions (0.1 mM), which has no effect on charge movement. When SH reagents are applied in the presence of cadmium, no effects are observed, indicating that this cation may protect the membrane from the reagent effects. The effects of -SH reagents can be observed by applying them in the absence of cadmium, followed by addition of the cation. Under these conditions the conductance changes are reversed and the effects of the SH reagents on charge movement can be measured with a higher degree of confidence. Maximum charge is reduced by 32% in the presence of 1.5 mM PCMB and by 31% in the presence of 2 mM PHMPS. These effects do not occur in the presence of DTT and in some cases they may be reversed by this agent. Charge 2, recorded in depolarized muscle fibers, is also reduced by these agents. 相似文献
3.
Onion root tip meristems, fixed in 14 different fixatives representing ingredients and mixtures used in plant cytology, were tested with the Barrnett and Seligman histochemical procedure for protein-bound sulfhydryl groups. The relative intensity of staining was measured photometrically and the distribution of stain after each type of fixation described. Measurements indicated that conditions governing the staining of SH and S—S are not fully predictable; for example, fixation in saturated HgCl2 enhances staining although inhibition was expected through mercaptide formation. Specificity of the reaction was further checked by treating fixed sections with known SH reagents. Partial blocking by such reagents as p-chloromercuribenzoate, and N-ethyl maleimide is apparently reversed by lengthy incubation in the 2,2'-dihydroxy-6,6-dinaphthyl-disulfide (DDD) reagent. Sulfhydryl oxidizing agents such as I-KI or chromic acid were either ineffective in blocking or could be reversed. For this reason and because previously reduced sections were proportionately better blocked than untreated ones it is suggested that the sulfhydryl reagent may open and then react with S—S bonds. Parallel runs indicate no difference in specificity between animal and plant tissues. 相似文献
4.
1. Addition of 2 moles of mersalyl, mercuric chloride, p-chloromercuribenzoate (PCMB), or methyl mercury hydroxide per mole of hemoglobin greatly reduces heme-heme interactions (n), yet these substances have quite different effects on the oxygen affinity (-log p50). Mersalyl and mercuric chloride at this concentration each increase the oxygen affinity, while PCMB and methyl mercury have little or no effect on the oxygen affinity. These effects are primarily associated with the binding of —SH groups, and are largely reversed on the addition of glutathione. —SH groups do not appear to be responsible for the Bohr effect. 2. Evidence is presented for the belief that the two hemes of each half-molecule of horse hemoglobin are situated on either side of a cluster of—SH groups. 3. The mechanism of interaction between the hemes is discussed. It is concluded that the reorganization of the protein architecture which accompanies oxygenation plays a central role in this interaction, in agreement with the views of Pauling and Wyman. 相似文献
5.
Variations in Sulfhydryl Concentration During Microsporocyte Meiosis in the Anthers of Lilium and Trillium 总被引:4,自引:2,他引:2
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Herbert Stern 《The Journal of cell biology》1958,4(2):157-161
Anthers of lily and trillium were followed with respect to variations in protein and soluble sulfhydryls during meiosis and mitosis of the sporogenous tissue. In lily, the meiotic and mitotic cycles are each preceded by a rise in soluble —SH; in trillium there is only one rise which precedes meiosis. During division there is a marked drop in soluble —SH and a rise in soluble disulfides. Protein —SH remains approximately constant until diakinesis or metaphase when it falls briefly. 相似文献
6.
Normal and transformed baby hamster kidney (BHK) cells attach to Falcon polystyrene with the same first order rate constant. The longer the cells are attached to the bottles, the more difficult they are to remove. Sulfhydryl (—SH) binding reagents inhibit both the attachment of BHK cells and the increase in adhesive strength of attached cells. Attached BHK cells bind fewer molecules of [1-14C]N-ethylamleimide (an —SH binding reagent) than do suspended cells. Incubation of cells with high concentrations of trypsin results in a reversible loss of cell adhesiveness. The recovery of adhesiveness of trypsin-treated cells is inhibited by cycloheximide. 相似文献
7.
Variations in Sulfhydryl, Disulfide, and Protein Content during Synchronous and Asynchronous Growth of HeLa Cells 总被引:1,自引:0,他引:1
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The cellular contents of protein-bound and nonprotein sulfhydry (—SH) and disulfide (—SS—) groups were measured in both asynchronous and synchronous HeLa S3 cultures. About 90% of these groups are associated with proteins, the majority in the —SH form. The content of protein-bound groups, and hence the total content of —SH and —SS— groups (28 × 10-15 moles/cell, or 1.1 × 10-6 moles/g protein on average), changes in parallel with the protein content (which varies between 2 and 4 × 10-10 g/cell) as asynchronous populations pass from the lag through the exponential to the stationary phase of growth. The concentration of nonprotein —SH groups, in contrast, increases 10-fold during lag phase and decreases in stationary phase; it follows the protein concentration closely during the exponential phase, at a level of about 2.8 × 10-15 moles/cell. In synchronous cultures the protein content doubles during the cell cycle, possibly in an exponential fashion. The total —SH and —SS— content also doubles, but the rate of increase appears to fluctuate. The concentrations of the protein-bound groups show 2- to 3-fold fluctuations per unit protein: protein-bound —SH groups and mixed —SS— linkages rise to maxima while protein-bound —SS— groups fall to a minimum at the G1/S transition, and fluctuations in these groups occur again during G2. In addition, the protein-bound —SH concentration falls continuously during the S phase. The nonprotein —SH concentration undergoes the largest (relative) fluctuations, dropping from 4 × 10-15moles/cell in early G1 to about 0.4 × 10-15 moles/cell (of standard protein content) at the end of G1, and then rising to 30 times this value by the end of S. 相似文献
8.
The effect of intracellular adenosine triphosphate (ATP) on the “common gating” of the CLC-1 chloride channel has been studied by several laboratories with controversial results. Our previous study on the channel expressed in Xenopus oocytes using excised inside-out patch-clamp methods showed a robust effect of ATP in shifting the open probability curve of the common gate toward more depolarizing voltages (Tseng, P.Y., B. Bennetts, and T.Y. Chen. 2007. J. Gen. Physiol. 130:217–221). The results were consistent with those from studying the channel expressed in mammalian cells using whole cell recording methods (Bennetts, B., M.W. Parker, and B.A. Cromer. 2007. J. Biol. Chem. 282:32780–32791). However, a recent study using excised-patch recording methods for channels expressed in Xenopus oocytes reported that ATP had no direct effect on CLC-1 (Zifarelli, G., and M. Pusch. 2008. J. Gen. Physiol. 131:109–116). Here, we report that oxidation of CLC-1 may be the culprit underlying the controversy. When patches were excised from mammalian cells, the sensitivity to ATP was lost quickly—within 2–3 min. This loss of ATP sensitivity could be prevented or reversed by reducing agents. On the other hand, CLC-1 expressed in Xenopus oocytes lost the ATP sensitivity when patches were treated with oxidizing reagents. These results suggest a novel view in muscle physiology that the mechanisms controlling muscle fatigability may include the oxidation of CLC-1. 相似文献
9.
Howard G. Shertzer Baruch I. Kanner Ranajit K. Banerjee Efraim Racker 《Biochemical and biophysical research communications》1977,75(3):779-784
Highly purified adenine nucleotide transporter from bovine heart mitochondria was reconstituted with phospholipids to form vesicles which catalyzed atractyloside-sensitive adenine nucleotide translocation. When internal ATP was exchanged with external ADP, this reaction was enhanced by agents capable of collapsing a membrane potential, but not by inorganic phosphate. When the purified nucleotide transporter was reconstituted together with a second protein fraction, nucleotide transport was stimulated by inorganic phosphate. The stimulated rate was eliminated by mersalyl or other SH reagents. The second protein fraction could be replaced by preparations of purified phosphate transporter. 相似文献
10.
The condensation of retinene1 with opsin to form rhodopsin is optimal at pH about 6, a pH which favors the condensation of retinene1 with sulfhydryl rather than with amino groups. The synthesis of rhodopsin, though unaffected by the less powerful sulfhydryl reagents, monoiodoacetic acid and its amide, is inhibited completely by p-chloromercuribenzoate (PCMB). This inhibition is reversed in part by the addition of glutathione. PCMB does not attack rhodopsin itself, nor does it react with retinene1. Its action in this system is confined to the —SH groups of opsin. Under some conditions the synthesis of rhodopsin is aided by the presence of such a sulfhydryl compound as glutathione, which helps to keep the —SH groups of opsin free and reduced. By means of the amperometric silver titration of Kolthoff and Harris, it is shown that sulfhydryl groups are liberated in the bleaching of rhodopsin, two such groups for each retinene1 molecule that appears. This is true equally of rhodopsin from the retinas of cattle, frogs) and squid. The exposure of new sulfhydryl groups adds an important element to the growing evidence that relates the bleaching of rhodopsin to protein denaturation. The place of sulfhydryl groups in the structure of rhodopsin is still uncertain. They may be concerned directly in binding the chromophore to opsin; or alternatively they may furnish hydrogen atoms for some reductive change by which the chromophore is formed from retinene1. In the amperometric silver titration, the bleaching of rhodopsin yields directly an electrical variation. This phenomenon may have some fundamental connection with the role of rhodopsin in visual excitation, and may provide a model of the excitation process in general. 相似文献
11.
STUDIES ON SULFHYDRYL GROUPS DURING CELL DIVISION OF SEA URCHIN EGG : III. —SH Groups of KCl-Soluble Proteins and Their Change during Cleavage
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Hikoichi Sakai 《The Journal of cell biology》1960,8(3):609-615
Sea urchin egg proteins extracted with KCl are mostly TCA-soluble and, conversely, those extracted with TCA are KCl-soluble. Both groups are water-insoluble and show fluctuations in—SH content during the division cycle. The fluctuation of the—SH groups of the KCl-soluble protein of the whole egg is due to a —SH—S—S— interchange within the freely reacting groups and not within the sluggish and masked —SH groups of the protein. The —SH content of the KCl-soluble protein of the egg cortex also fluctuates in a similar way. 相似文献
12.
By means of 1 M NaCl isolated lymphocyte chromosomes can be separated into two fractions, each of which contains nucleoprotein. The fraction soluble in M NaCl consists largely of desoxyribose nucleohistone, and constitutes 90 to 92 per cent of the mass of the chromosome. The insoluble residue (the residual chromosome is a coiled thread containing some 12 to 14 per cent of ribose nucleic and about one-fifth as much desoxyribose nucleic acid; the residual chromosome accounts for 8 to 10 per cent of the mass of the chromosome. The staining of chromosomes—whether by the Feulgen procedure, by hematoxylin, orcein, or by basic dyes such as crystal violet—is due to the nucleohistone fraction which contains about 96 per cent of the nucleic acid of the chromosome. The form of the chromosome is due primarily to the protein thread of the residual chromosome. This thread is the only linear structure of microscopic dimensions in the chromosome that is not readily dispersed. When chromosomes are broken, it must be supposed that a break is made in the protein thread of the residual chromosome. The foregoing provides evidence for considering the residual chromosome to be the basis of the linear order of the genes. This would mean either that the residual chromosome is a structure around which the genes are organized or that the genes form part of its substance. 相似文献
13.
Ajay Singh A. K. Agrawal A. B. Abidi N. S. Darmwal 《Applied microbiology and biotechnology》1990,34(3):356-358
Summary Cellobiase enzyme was partially purified from the culture filtrate of Aspergillus niger AS-101 and the general and kinetic properties of the enzyme were examined. The enzyme was unstable on storage. However, it was protected by the addition of BSA, glycerol or sodium azide. Addition of glycerol also protected the enzyme from denaturation due to freezing and thawing. Effect of thiol group reagents revealed the presence of — SH groups at the active site of the enzyme. Different modulators such as metal ions and macroionic compounds illustrated varying effects on the purified cellobiase.
Offprint requests to: A. Singh 相似文献
14.
L Gailis W G Nayler S E Harding 《Canadian journal of physiology and pharmacology》1990,68(8):1170-1175
Cardiac contractile activity is usually controlled by intracellular Ca2+, but it can also be modified by oxidizing agents. Incubation of guinea pig heart myofibrils with diamide (3 mM, 1 h) increased basal (no Ca2+) ATPase activity by 580% and abolished Ca2+ dependence. The effect was proportional to diamide concentration (0.01-1 mM) and duration of preincubation (up to 2 h). Dithiothreitol (5 mM, 1 h) reversed most of the basal ATPase activation and restored Ca2+ sensitivity. Other sulfhydryl reagents produced a similar effect but also produced inhibition of total ATPase. In intact cell preparations, diamide produced a slow tonic contraction, consistent with myofibril activation. In the perfused rat heart, 1 mM diamide slowly increased diastolic ventricular pressure; this increase was partially reversed by dithioerythritol. In isolated rat heart myocytes, 1 mM diamide produced a slow tonic contraction, increased contractility in response to stimulation. Cardiocytes superfused for 1 h with buffer containing EGTA to deplete Ca2+ did not contract in response to stimulation but showed a slow tonic contraction with diamide. This contraction could be slowly and only partially reversed by dithioerythritol. Response to stimulation was restored by addition of Ca2+. The results show that diamide can produce contraction in viable cells. This contraction does not require extracellular Ca2+ and is unlikely to involve intracellular Ca2+. The direct activation of myofibrillar ATPase may contribute to the increased myocardial stiffness seen in ischemia and to ischemic contracture. 相似文献
15.
A. E. Mirsky 《The Journal of general physiology》1941,24(6):725-733
1. The same number of SH groups reduces ferricyanide in surface films of egg albumin as in albumin denatured by urea, guanidine hydrochloride, Duponol, or heat, provided the ferricyanide reacts with films while they still are at the surface and with the denatured proteins while the denaturing agent (urea, heat, etc.) is present. 2. The SH groups of a suspension of egg albumin made by clumping together many surface films react with ferricyanide in the same sluggish and incomplete manner as do the groups in egg albumin denatured by concentrated urea when the urea is diluted or in albumin denatured by heat when the solution is allowed to cool off. 3. The known change in configuration of the egg albumin molecule when it forms part of a surface film explains why SH groups in the film react with ferricyanide whereas those in native egg albumin do not. In the native egg albumin molecule groups in the interior are inaccessible to certain reagents. A film is so thin that there are no inaccessible groups. 4. Because of the marked resemblance in the properties of egg albumin in surface films and of egg albumin after denaturation by the recognized denaturing agents, it may be supposed that the same fundamental change takes place in denaturation as in film formation—indeed, that film formation is one of the numerous examples of denaturation. This would mean that in general the SH groups of denatured egg albumin reduce ferricyanide and react with certain other reagents because they are no longer inaccessible to these reagents. 相似文献
16.
A wide variety of agents are shown to mimic insulin action and inhibit rates of intracellular protein degradation in H35 hepatoma cells. For oxidizing agents such as NaNO2, H2O2 and oxidized glutathione, inhibition of protein breakdown is reversed by adding catalase. Phenylhydrazine behaves like an oxidant and mimics insulin action in a manner potentiated by superoxide dismutase and reversed by catalase. Similarly the effect of insulin itself is increased by superoxide dismutase and reduced by catalase. Sulfhydryl reagents also mimic insulin action: inhibition of protein breakdown is seen following addition of 2-mercaptoethanol or a brief pre-treatment with N-ethylmaleimide or iodoacetate. Mild pre-treatment with trypsin also inhibits subsequent rates of protein breakdown. A model is proposed suggesting that these insulinomimetic actions involve a common mechanism which links the generation of active oxygen species through the redox potential of the cell to the activation of a proteinase. 相似文献
17.
Osipova SV Permyakov AV Mitrofanova TN Dudareva LV Trufanov VA 《Biochemistry. Biokhimii?a》2005,70(8):935-940
Biochemical properties of a homogenous preparation of thiol:protein disulfide oxidoreductase (TPDO, EC 1.8.4.2) isolated for the first time from mature wheat (Triticum aestivum L.) grain were studied. According to polyacrylamide gel electrophoresis data, the molecular weight of TPDO is around 167 kD, the enzyme consisting of two subunits of 77 and 73 kD, which differentiates TPDO from known enzymes of SH/SS-metabolism of wheat caryopses. In substrate specificity and enzymatic characteristics (pH and temperature optima) TPDO is similar to analogous enzymes of animal tissues. Inhibition of disulfide reductase activity by alkylating agents and heavy metal ions suggests the participation of active center SH-groups in the catalytic act and classes the enzyme as a member of the thioredoxin superfamily. The SS-reductase reduces aggregating capacity of acetic acid-soluble fraction of wheat storage proteins. The proposed physiological role of TPDO is participation in creation and regulation of SH/SS-status of wheat endosperm proteins and formation of the rheological properties of gluten.__________Translated from Biokhimiya, Vol. 70, No. 8, 2005, pp. 1130– 1136.Original Russian Text Copyright © 2005 by Osipova, Permyakov, Mitrofanova, Dudareva, Trufanov. 相似文献
18.
Purification and Properties of Flavonol-Ring B Glucosyltransferase from Chrysosplenium americanum 总被引:2,自引:2,他引:0
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A novel glucosyltransferase which catalyzed the transfer of glucose from UDP-glucose to positions 2′ and 5′ of partially methylated flavonols was isolated from the shoots of Chrysosplenium americanum Schwein ex Hooker. It was purified 225-fold by ammonium sulfate precipitation and successive chromatography on Sephadex G-100, hydroxyapatite, and polybuffer ion exchanger. This glucosyltransferase appeared to be a single polypeptide with an apparent molecular weight of 42,000 daltons, pH optimum of 7.5 to 8.0, and an isoelectric point of 5.1. It had low but similar Km values for the 2′ and 5′ positions of flavonol substrates and the cosubstrate UDP-glucose and was inhibited by both reaction products, the glucosides formed, and UDP.
Glucosyltransferase activity was independent of divalent cations, was not inhibited by EDTA, but showed requirement for SH groups. The differential effect on enzyme activity of metal ions, especially cupric ion, and various SH group reagents seemed to indicate the involvement of two active sites in the glucosylation reaction; the site specific for 2′ activity being more susceptible than that of the 5′ activity. The substrate specificity expressed by this glucosyltransferase and the requirement of at least two para-oriented B-ring substituents (at 2′ and 5′) for activity support this view.
相似文献19.
Tadanobu Nakadai Seiichi Nasuno Nobuyoshi Iguchi 《Bioscience, biotechnology, and biochemistry》2013,77(9):1481-1488
Acid carboxypeptidase III from Aspergillus oryzae was purified from the rivanol non-precipitated fraction. The optimum activity of the enzyme occurred at pH 3.0 for carbobenzoxy-l-glutamyl-l-tyrosine. The enzyme was inhibited by diisopropylphosphorofluoridate and SH reagents such as p-chloromercuribenzoate and monoiodoacetate, but not by such metal chelating agents as ethylenediaminetetraacetate, αα′-dipyridyl and o-phenanthroline. The molecular weight of the enzyme was estimated to be about 61,000. The enzyme hydrolyzed the peptides that possess masked or bulky N-terminal. 相似文献
20.
Sereikaite J Jachno J Santockyte R Chmielevski P Bumelis VA Dienys G 《The protein journal》2006,25(6):369-378
About 14 proteins were tested for specific oxidative scission catalyzed by metal ions in the presence of ascorbate and oxidizing agents (O2 or hydrogen peroxide). Only four of them were degraded by Fe3+/Fe2+- ascorbate, twelve – by Cu2+/Cu+-ascorbate and two proteins (α- and β-caseins) were degraded by Pd2+ ions. The rate and the intensity of degradation are very different for various proteins. For the most of tested proteins only a small fraction of molecules was degraded. None of them was degraded completely. Two possible reasons of protein stability against oxidative degradation may be proposed as follows: either there is no metal binding site in a protein molecule, or metal binding ligands of protein undergo a rapid oxidative modification and the metal ion is released from the binding site. Human growth hormone was cut specifically at two sites by Cu2+/Cu+-ascorbate system. At least one of amino acid residues of this protein was modified by formation of reactive carbonyl. 相似文献