scholarly journals The accessibility of the thiol groups on G- and F-actin of rabbit muscle

1990 ◽  
Vol 266 (2) ◽  
pp. 453-459 ◽  
Author(s):  
D F Liu ◽  
D Wang ◽  
A Stracher
Keyword(s):  
Thiol Group ◽  
Iodoacetic Acid ◽  
Drastic Change ◽  
The Other ◽  
Cysteine Residues ◽  
Rabbit Muscle ◽  
Thiol Groups ◽  
Filamentous Actin ◽  
Other Hand

The accessibility of the cysteine residues of actin from rabbit muscles to the thiol-targeted reagent 7-dimethylamino-4-methyl-(N-maleimidyl)coumarin (DACM) was investigated. Under conditions where the actin is in the unpolymerized form (G-actin), the most reactive thiol group was Cys-257, suggesting that it was located on the surface of the actin molecule. The selective modification of Cys-374 for this reagent as reported by Sutoh [(1982) Biochemistry 21, 3654-3661] was not observed. Cys-10, Cys-217 and Cys-374 were much less reactive and only gradually became extensively modified when the concentration of DACM approached 5 molar equivalents of actin. Presumably these thiol groups were located further inward away from the surface or situated in a different environment that rendered them less reactive. On the other hand, Cys-285 was completely inaccessible and presumably was buried. The lack of preferential labelling of Cys-374 by DACM is incompatible with the finding with iodoacetic acid as the reagent as reported by Elzinga & Collins [(1975) J. Biol. Chem. 250, 5897-5905]. This discrepancy, however, might well be due to the different reagents employed. The DACM-G-actin largely retained its competence for polymerization. Upon polymerization of G-actin, practically all the thiol groups became inaccessible to DACM, suggesting that a drastic change occurred in the conformation of actin units in the transition of monomers to filamentous actin.

scholarly journals The reactivity of thiol groups and the subunit structure of aldolase

1970 ◽  
Vol 117 (2) ◽  
pp. 291-298 ◽  
Author(s):  
P. J. Anderson ◽  
R. N. Perham
Keyword(s):  
Group Analysis ◽  
Thiol Group ◽  
Enzymic Activity ◽  
Native Enzyme ◽  
Cysteine Residues ◽  
Subunit Structure ◽  
Rabbit Muscle ◽  
Thiol Groups ◽  
Prolonged Incubation ◽  
Nitrobenzoic Acid

1. Seven unique carboxymethylcysteine-containing peptides have been isolated from tryptic digests of rabbit muscle aldolase carboxymethylated with iodo[2-14C]acetic acid in 8m-urea. These peptides have been characterized by amino acid and end-group analysis and their location within the cyanogen bromide cleavage fragments of the enzyme has been determined. 2. Reaction of native aldolase with 5,5′-dithiobis-(2-nitrobenzoic acid), iodoacetamide and N-ethylmaleimide showed that a total of three cysteine residues per subunit of mol.wt. 40000 were reactive towards these reagents, and that the modification of these residues was accompanied by loss in enzymic activity. Chemical analysis of the modified enzymes demonstrated that the same three thiol groups are involved in the reaction with all these reagents but that the observed reactivity of a given thiol group varies with the reagent used. 3. One reactive thiol group per subunit could be protected when the modification of the enzyme was carried out in the presence of substrate, fructose 1,6-diphosphate, under which conditions enzymic activity was retained. This thiol group has been identified chemically and is possibly at or near the active site. Limiting the exposure of the native enzyme to iodoacetamide also served to restrict alkylation to two thiol groups and left the enzymic activity unimpaired. The thiol group left unmodified is the same as that protected by substrate during more rigorous alkylation, although it is now more reactive towards 5,5′-dithiobis-(2-nitrobenzoic acid) than in the native enzyme. 4. Conversely, prolonged incubation of the enzyme with fructose 1,6-diphosphate, which was subsequently removed by dialysis, caused an irreversible fall in enzymic activity and in thiol group reactivity measured with 5,5′-dithiobis-(2-nitrobenzoic acid). 5. It is concluded that the aldolase tetramer contains at least 28 cysteine residues. Each subunit appears to be identical with respect to number, location and reactivity of thiol groups.

scholarly journals Climate Change in India: A Wakeup Call from Bollywood

2020 ◽  
Vol 12 (5) ◽  
Author(s):  
Manvi Sharma ◽  
◽  
Ajay K. Chaubey
Keyword(s):  
Climate Change ◽  
Detailed Analysis ◽  
Environmental Degradation ◽  
Climatic Condition ◽  
Drastic Change ◽  
The Other ◽  
Other Hand ◽  
The Masses ◽  
New Generation ◽  
The Village

Amidst Bollywood’s romanticized landscapes and grandeur settings, depiction of the flora and fauna, roaring rivers and drought prone lands, is difficult to locate. But the new millennium has witnessed some new generation filmmakers, sensitized towards the ecological concerns, thus marking a shift from the illustration of idealised landscapes to the representation of nature’s wrath. Since, cinema in India, has a deep-rooted impact on the masses, these creators employ films as tools to sensitize the population towards the climate change threat which though as perilous as the COVID-19 crisis, is often ignored by a significant amount of population. Dawning upon themselves the responsibility of environmental awakening, Nila Madhab Panda and Abhishek Kapoor highlight in their movies, Kadvi Hawa(2017) and Kedarnath(2018), respectively, the horrors of human callousness, leading to drastic change in Climatic condition in India. Panda’s Kadavi Hawa, dealing with non-repayment of loans followed by suicides, portrays the heart-wrenching imagery of environmental degradation and Climate change that has rendered the Village of Mahua, arid and infertile. Kapoor’s Kedarnath on the other hand, appeals for action through horrifying imagery of the catastrophic floods that disrupted the holy town of Kedarnath, in 2013. Through a detailed analysis of the aforementioned visual portrayals, this article aims to emphasise as to how Films can play an important role in effectively addressing dealing with the issues related to Climate. Further, the rationale of this paper is to underscore the possibility of more such storylines, as a tool towards effective engagement and levitation of conscience.

scholarly journals On the disulphide bonds of rhodopsins

1987 ◽  
Vol 246 (1) ◽  
pp. 131-137 ◽  
Author(s):  
S Al-Saleh ◽  
M Gore ◽  
M Akhtar
Keyword(s):  
Acetic Acid ◽  
Indirect Evidence ◽  
Iodoacetic Acid ◽  
The Other ◽  
Cysteine Residues ◽  
Disulphide Bridge ◽  
Disulphide Bonds ◽  
Peptide Purification ◽  
Reducing Conditions ◽  
Bovine Rhodopsin

Carboxymethylation using 14C- or 3H-labelled iodoacetic acid has been used to identify the cysteine residues in bovine rhodopsin involved in the formation of the two intramolecular disulphide bridges. Iodo[2-14C]acetic acid was used to modify 5.8-5.9 residues of cysteine under non-reducing conditions. After dialysis and reduction of disulphide bridges by 2-mercaptoethanol, iodo[2-3H]acetic acid was employed to covalently modify 3.3-3.6 residues of cysteine. Peptide purification and sequencing has unambiguously shown that cysteine residues 322 and 323 are only carboxymethylated after reduction of disulphide bridges. Indirect evidence presented, now coupled with the earlier finding [Findlay & Pappin (1986) Biochem. J. 238, 625-642] suggests that the other disulphide bridge is formed between cysteine residues 110 and 187. A comparison is made of all the sequences of mammalian rhodopsins and colour pigments and attention is drawn to the fact that whereas Cys-322 and Cys-323 are conserved only in three rhodopsins (bovine, ovine and human), the residues corresponding to Cys-110 and Cys-187 are found in all the visual proteins (from rods as well as human cones).

scholarly journals Chemical evidence for the existence of activated G-actin

1992 ◽  
Vol 283 (2) ◽  
pp. 567-573 ◽  
Author(s):  
W P Shu ◽  
D Wang ◽  
A Stracher
Keyword(s):  
New Species ◽  
Conformational Changes ◽  
Life Time ◽  
Thiol Group ◽  
Thiol Groups ◽  
Filamentous Actin ◽  
Bivalent Cations ◽  
Small Domain ◽  
Actin Concentration ◽  
Chemical Evidence

Globular actin (G-actin) will polymerize to form filamentous actin (F-actin) under physiological ionic conditions, and is known to be regulated by univalent and bivalent cations, such as K+ and Mg2+. The current concept of this process involves four steps: activation, nucleation, elongation and annealing. Evidence for the existence of activated G-protein has been suggested by changes in the resistance to proteolysis [Rich & Estes (1976) J. Mol. Biol. 104, 777-792] and u.v.-light absorption [Rouayrenc & Travers (1981) Eur. J. Biochem. 116, 73-77]. More recently we [Liu et al. (1990) Biochem. J. 266, 453-459] have provided direct chemical evidence for extensive conformational changes during the transformation of G-actin into F-actin. In this study we now present direct chemical evidence for the existence of a short-lived species, an activated form of G-actin, which can be detected by changes in the accessibility of the free thiol groups on the G-actin molecule when modified by a specific thiol-group-targeted reagent, 7-dimethylamino-4-methyl-3-N-maleimidylcoumarin (DACM). The presence of K+ and/or Mg2+ ions caused a large increase in the accessibility of the thiol groups of Cys-217 and Cys-374, but not those of Cys-10 and Cys-257. Mg2+ effected relatively faster changes than did K+ ions. The results suggest that the function of these ions is to convert G-actin into an activated form, and further suggest that the change in conformation is mainly confined to the large domain. Such changes at least involve certain portions of the G-actin molecule that contain Cys-217 and Cys-374. On the other hand, little or no significant change could be observed in the small domain of G-actin as reflected by the accessibility of Cys-10. The bound nucleotide remained as ATP during the activation of G-actin and was hydrolysed to ADP on polymerization. The activated G-actin had a life-time of about 8 min or less depending on the concentration of G-actin. At higher protein concentration, its life-time was much shorter, probably owing to the earlier onset of polymerization, which apparently is governed by the concentration of the activated form. The life-time of this new species can be extended by lowering the temperature and is less affected by actin concentration. This new species is considered to be an activated form of G-actin, since polymerization renders all the thiol groups on actin inaccessible to the reagent DACM.

The Number, Location, and Reactivity of the Cysteine Residues of Sturgeon Muscle Aldolase

1972 ◽  
Vol 50 (2) ◽  
pp. 111-119 ◽  
Author(s):  
P. J. Anderson
Keyword(s):  
Iodoacetic Acid ◽  
Sulfhydryl Groups ◽  
Cysteine Residues ◽  
Rabbit Muscle ◽  
Nitrobenzoic Acid ◽  
Catalytic Role ◽  
Aldolase Activity ◽  
Activity Loss

Sturgeon muscle aldolase contains six cysteine residues per subunit. These residues appear to occur in homologous positions to six of the eight cysteine residues of rabbit muscle aldolase. Three of the six residues can react with either iodoacetic acid or 5,5′-dithiobis-(2-nitrobenzoic acid) in the absence of denaturing agents. Reaction of three residues with iodoacetic acid inactivates the enzyme. The presence of substrate protects one of these residues and in this case no activity loss is observed. However, the results indicate that the effects on activity are due to the addition of the modifying group rather than to a loss of sulfhydryl groups. No residue corresponding to the previously demonstrated substrate-protected cysteine of rabbit aldolase was located in the sturgeon enzyme, which demonstrated that this residue was not essential to aldolase activity. It therefore appears unlikely that cysteine residues have a direct or an auxiliary catalytic role in aldolase activity.

scholarly journals The comparative structure of mammalian glyceraldehyde 3-phosphate dehydrogenase

1969 ◽  
Vol 111 (1) ◽  
pp. 17-21 ◽  
Author(s):  
R N Perham
Keyword(s):  
Amino Acid ◽  
Essential Role ◽  
Polypeptide Chain ◽  
Thiol Group ◽  
Preliminary Evidence ◽  
Amino Acid Sequences ◽  
The Other ◽  
Thiol Groups ◽  
Comparative Structure ◽  
Rule Out

1. The amino acid sequences around the thiol groups of glyceraldehyde 3-phosphate dehydrogenase from badger and monkey skeletal muscle were compared with the sequences around the thiol groups in the enzyme isolated from other organisms. 2. Preliminary evidence of the existence of isoenzymes in the badger was obtained. Only the major form, however, could be purified completely. 3. The monkey enzyme contains only three cysteine residues per polypeptide chain compared with the four found in all the other mammalian enzymes so far examined, including that of badger, and the two in yeast. The missing thiol group in monkey was identified as residue 281 in the corresponding sequence of the pig enzyme. 4. These experiments rule out any essential role for cysteine-281 in the function of the mammalian enzymes. 5. Further evidence of the remarkable conservation of amino acid sequence in this enzyme during evolution is presented and discussed.

scholarly journals Carboxymethylation of Thiol Groups in Ovalbumin: Implications for Proteins that Contain Both Thiol and Disulfide Groups

1982 ◽  
Vol 35 (2) ◽  
pp. 125 ◽  
Author(s):  
DM Webster ◽  
EOP Thompson
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Guanidine Hydrochloride ◽  
Sodium Dodecyl ◽  
Iodoacetic Acid ◽  
Cysteine Residues ◽  
Alkaline Ph ◽  
Thiol Groups ◽  
Nitrobenzoic Acid ◽  
Dodecyl Sulfate ◽  
Fold Excess

The cysteine residues of hen ovalbumin were S-carboxymethylated with non-radioactive iodoacetic acid under various conditions by altering the pH at which the protein was denatured in 8 M urea, by using different molar ratios of non-radioactive iodoacetic acid to cysteine and by varying the time at which carboxymethylation was commenced after denaturing conditions had been applied. Under the various conditions, the thiol groups were carboxymethylated to different extents, the residual thiol groups being measured by reaction with 5,5'-dithiobis(2-nitrobenzoic acid) in the presence of sodium dodecyl sulfate. When ovalbumin is carboxymethylated in alkaline urea, it unfolds slowly and the carboxymethylation is incomplete even with 150-fold excess iodoacetic acid. The known rapid thiol-disulfide exchange that occurs at alkaline pH values makes this method of carboxymethylation unsuitable as a preliminary step for blocking the native cysteine residues of ovalbumin before reduction and labelling the thiol groups formed by reduction of the disulfide bonds. Titration of the thiol groups of ovalbumin in 6 M guanidine hydrochloride or 1 % (w/v) sodium dodecyl sulfate at pH 8�2 with 5,5' -dithiobis(2-nitrobenzoic acid) is more rapid than in 8 M urea and these solvents would be preferable for studies of the disulfide-bonded sequences. Denaturation of ovalbumin in acidic 8 M urea is a very rapid process, and under mild acid conditions thiol-disulfide interchange is much slower. Subsequent carboxymethylation of the cysteine residues at alkaline pH with 150-fold excess iodoacetic acid results in complete carboxymethylation and the carboxymethylated ovalbumin can be reduced and labelled with radioactive iodoacetic acid with specific labelling of the half-cystine residues involved in the disulfide bond. The results are discussed in relation to the allocation of half-cystine residues in other protein systems that contain both thiol and disulfide groups.

scholarly journals Catalytic Site Cysteines of Thiol Enzyme: Sulfurtransferases

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Noriyuki Nagahara
Keyword(s):  
Reduced Glutathione ◽  
Cysteine Residue ◽  
Catalytic Site ◽  
The Other ◽  
Cysteine Residues ◽  
Other Hand ◽  
Redox Active ◽  
Mercaptopyruvate Sulfurtransferase ◽  
Catalytic Processes ◽  
Thiosulfate Sulfurtransferase

Thiol enzymes have single- or double-catalytic site cysteine residues and are redox active. Oxidoreductases and isomerases contain double-catalytic site cysteine residues, which are oxidized to a disulfide via a sulfenyl intermediate and reduced to a thiol or a thiolate. The redox changes of these enzymes are involved in their catalytic processes. On the other hand, transferases, and also some phosphatases and hydrolases, have a single-catalytic site cysteine residue. The cysteines are redox active, but their sulfenyl forms, which are inactive, are not well explained biologically. In particular, oxidized forms of sulfurtransferases, such as mercaptopyruvate sulfurtransferase and thiosulfate sulfurtransferase, are not reduced by reduced glutathione but by reduced thioredoxin. This paper focuses on why the catalytic site cysteine of sulfurtransferase is redox active.

Only the glutathione dependent antioxidant enzymes are inhibited by haematotoxic hydroxylamines

1998 ◽  
Vol 17 (10) ◽  
pp. 554-559
Author(s):  
Anita AMG Spooren ◽  
Chris TA Evelo
Keyword(s):  
Antioxidant Enzymes ◽  
Thiol Group ◽  
Antioxidant Defense System ◽  
Cysteine Residues ◽  
Thiol Groups ◽  
Glutathione S Transferase ◽  
Average Life Span ◽  
Cellular Oxidation ◽  
Activity Loss

Hydroxylamine and some of its derivatives are known to cause oxidative effects both in vitro and in vivo.Inthe current study we investigated the effects of hydroxyla-mines on the enzymatic antioxidant defense system in human erythrocytes. The activity of catalase and super-oxide dismutase was not significantly influenced by any of the hydroxylamines tested. However, the activity of glutathione peroxidase (GPX) and glutathione S-transferase (GST) was strongly inhibited by hydroxylamine and its O-derivatives (O-methyl and O-ethyl hydroxylamine). GPX was also inhibited by two N-derivatives of hydro-xylamine (i.e. N-dimethyl and N, O-dimethyl hydroxyla-mine). This indicates that exposure to hydroxylamines not only changes the cellular oxidation-reduction status but also leads to inhibition of the glutathione dependent antioxidant enzymes. GST as well as GPX have cysteine residues at the active site of the enzymes. Such an accessible thiol group is generally susceptible to formation of protein-mixed disulphides or intramolecular disulphides. If these thiol groups are essential for activity this would be accompanied by an increase or decrease in the enzyme activity. In principle this is also true for glutathione reductase (GR), which in this study was only inhibited by N, O-dimethyl and N-methyl hydroxylamines. However, GR is capable to reduce these disulphides by taking up two electrons, either from its substrate NAPDH or from another reductant. Oxidation of these thiol groups in GR would thus not lead to impairment of GR activity. The fact that NODMH and NMH do decrease the GR activity can therefore only be explained by other modifications. The activity loss of GST and GPX on the other hand, is likely to involve oxidation of critical cysteine residues. The practical consequence of these findings is that the cellular prooxidant state that may arise in erythrocytes exposed to hydroxylamines can be further increased by activity loss of protective enzymes, which may decrease the average life span of the red blood cell.

A study of cometary eruptions through OH radio-lines

1999 ◽  
Vol 173 ◽  
pp. 249-254
Author(s):  
A.M. Silva ◽  
R.D. Miró
Keyword(s):  
Short Duration ◽  
Growth Curves ◽  
The Other ◽  
Initial Mass ◽  
Radio Lines ◽  
Other Hand ◽  
Sudden Rise

AbstractWe have developed a model for theH2OandOHevolution in a comet outburst, assuming that together with the gas, a distribution of icy grains is ejected. With an initial mass of icy grains of 108kg released, theH2OandOHproductions are increased up to a factor two, and the growth curves change drastically in the first two days. The model is applied to eruptions detected in theOHradio monitorings and fits well with the slow variations in the flux. On the other hand, several events of short duration appear, consisting of a sudden rise ofOHflux, followed by a sudden decay on the second day. These apparent short bursts are frequently found as precursors of a more durable eruption. We suggest that both of them are part of a unique eruption, and that the sudden decay is due to collisions that de-excite theOHmaser, when it reaches the Cometopause region located at 1.35 × 105kmfrom the nucleus.

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