The reactivity of SH groups with a fluorogenic reagent

Birkett, Donald; Price, Nicholas C.; Radda, George K.; Salmon, Andrew G.

doi:10.1016/0014-5793(70)80095-3

Cited by 179 publications

(110 citation statements)

References 11 publications

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“…The evidence for tyrosine is as follows. (a) The absorption spectrum of the Nbf-modified ATPase is identical with that of the well-characterised AcTyr(Nbf)-OEt, and the maximal absorption (at 385 nm) is significantly different from that of an S-Nbf or N-Nbf derivative prcviously shown to be produced in other enzymes [22,30,31] and model compounds [22]. We have also discussed previously why reaction on a sulphydryl group of ATPase is unlikely [9].…”

Section: Discussionmentioning

confidence: 81%

“…We have also discussed previously why reaction on a sulphydryl group of ATPase is unlikely [9]. (b) Unlike the S-Nbf and N-Nbf derivatives which are fluorescent [22,30,31], the ActTyr(Nbf)-OEt gives no fluorescence emission and this is also the case for the Nbf-ATPase. (c) The rapid removal of the Nbf group from thc cnzyme by N-acetylcysteine or glutathione is again charactcristic of the Tyr-0-Nbf derivative.…”

Section: Discussionmentioning

confidence: 95%

“…Table 2 compares the effectiveness of these reagents in terms of pseudo-first-order rate constants for reactivation. When N-acetyl-cysteine or glutathione were used, the reaction was also followed by monitoring the increase jn absorbance at 420 nm due to the formation of the S-Nbf chromophore [22] (Fig. 2).…”

Section: Resultsmentioning

confidence: 99%

“…This method indicated that 1.0 mole of Nbf is incorporated per mole of ATPase using a molecular weight of 360000 [5]. The experimental variation on this value was f 10% ( Table 4 Pseudo-first-order rate constants werc calculated using a linear regression malysis of scmilogarithmic plots of the rate of loss of x cm was used [22]. The second method for estimating the extent o f incorporation is to measure directly the increase in absorbance at 385 nm which results from reaction of Nbf-C1 with the ATPase.…”

Section: Stoichiornetry Of the Reaction Between Nbf-c1 And Mitochondrmentioning

confidence: 99%

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The Mitochondrial ATPase

Ferguson

Lloyd

Lyons

et al. 1975

European Journal of Biochemistry

220

103

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1. Evidence is presented which indicates that inactivation of the mitochondrial ATPase from bovine heart by the reagent 4-chloro-7-nitrobenzofurazan results from modification of one tyrosine residue per enzyme molecule. Activity can be restored by a variety of sulphydryl reagents.2. In sodium dodecyl sulphate, the nitrobenzofurazan group on tyrosine is transferred to newly exposed sulphydryl groups on the enzyme.3. The rate of transfer of the nitrobenzofurazan moiety from the enzyme to sulphydryl compounds is compared with that for transfer from the model compound N-acetyl-tyrosine-O(7-nitrobenzofurazan) ethyl ester, the synthesis and properties of which are also described.4. The ligands ATP and ADP exert a protective effect on the rate of reaction between the mitochondrial ATPase and 4-chloro-7-nitrobenzofurazan. The variation in rate of this reaction with change in pH has also been examined and a pK, of 9.5 estimated for the tyrosine residue.5. The modification does not prevent substrate binding as judged by changes in the fluorescence of aurovertin, an antibiotic with specific affinity for mitochondrial ATPases.6. When the ATPase activity of submitochondrial particles is inhibited by 4-chloro-7-nitrobenzofurazan, there is a parallel decrease in the extent of the energy-linked fluorescence enhancement of 1-anilino-naphthalene-8-sulphonate induced by ATP hydrolysis. Both ATPase activity and the fluorescence enhancement are restored by sulphydryl reagents.The various theories of oxidative phosphorylation ascribe specific and distinct roles to the mitochondrial ATPase [1,2]. It is therefore important to elucidate features of this enzyme which might lead to a firmer mechanistic understanding of the final step in oxidative phosphorylation, the synthesis of ATP. When isolated as a soluble protein from mitochondrial membrane fragments, the mitochondrial ATPase from both bovine heart and rat liver apparently has a complex subunit structure, a,&yGc [3,4] and a molecular weight of approximately 360 000 [5]. A recent report suggests, however, that the enzyme from bovine heart can be obtained in a fully active form without the y and 6 subunits [6]. Mitochondrial ATPase has five tight dhhrruiarions. Nbf-Cl, 4-chloro-7-nitrobeiizofurazan (this compound has bcen named 7-ch~oro-4-nitrobenzo-?-oxd-l ,3-diazole and abbreviated NBD-chloride elsewhere; the name and abbreviation used here follow CBN recornmcndations). AcTyr(NbQ-OEt, Nacetyltyrosine-O (7-nitrobenzofurazaii) ethyl ester; AcCys(Nbf), N-acetyl-cystcine3 (7-nitrobenzofura~an).Enzyme. ATPasc or adenosine triphosphatase (EC 3.6.1.3).binding sites for adenine nucleotides [7] but little is known of the function or interrelationships of these sites.The most significant observations about the mechanism of the mitochondrial ATPase have come from the study of isotope exchange reactions [8]. Such studies have indicated that ATP hydrolysis occurs by attack of water on the terminal phosphate group with concomitant breakage of the bond between the terminal bridging oxygen and phos...

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Section: Discussionmentioning

confidence: 81%

Section: Discussionmentioning

confidence: 95%

Section: Resultsmentioning

confidence: 99%

Section: Stoichiornetry Of the Reaction Between Nbf-c1 And Mitochondrmentioning

confidence: 99%

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The Mitochondrial ATPase

Ferguson

Lloyd

Lyons

et al. 1975

European Journal of Biochemistry

220

103

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“…1 M GST (300 l) was dialyzed against an equal volume of 0.5-50 M GS-NBD in a 25°C horizontal shaker water bath. After equilibration for 4 h, samples from both chambers were assayed fluorometrically at exc(421) and emi(522) for the presence of GS-NBD (35)(36)(37). Concentrations of free and bound GS-NBD were calculated from a standard curve, and the corresponding equilibrium dissociation constants (K d ) were determined by curve-fitting with the program Enzfitter.…”

Section: Methodsmentioning

confidence: 99%

The Catalytic Tyr-9 of Glutathione S-Transferase A1-1 Controls the Dynamics of the C terminus

Nieslanik

Atkins

2000

Journal of Biological Chemistry

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The glutathione S-transferase enzymes (GSTs) have a tyrosine or serine residue at their active site that hydrogen bonds to and stabilizes the thiolate anion of glutathione, GS ؊ . The importance of this hydrogen bond is obvious, in light of the enhanced nucleophilicity of GS ؊ versus the protonated thiol. Several A-class GSTs contain a C-terminal segment that undergoes a ligand-dependent local folding reaction. Here, we demonstrate the effects of the Y9F substitution on binding affinity for glutathione conjugates and on rates of the order-disorder transition of the C terminus in rat GST A1-1. The equilibrium binding affinity of the glutathione conjugate, GS-NBD (NBD-Cl, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole), was decreased from 4.09 M to 0.641 M upon substitution of Tyr-9 with Phe. This result was supported by isothermal titration calorimetry, with K d values of 1.51 M and 0.391 M for wild type and Y9F, respectively. The increase in binding affinity for the mutant is associated with dramatic decreases in rates for the C-terminal order-disorder transition, based on a stopped-flow kinetic analysis. The same effects were observed, qualitatively, for a second GSH conjugate, GSethacrynic acid. Apparently, the phenolic hydroxyl group of Tyr-9 is critical for orchestrating C-terminal dynamics and efficient product release, in addition to its role in lowering the pK a of GSH.The importance of protein dynamics and flexibility to the energetics of ligand binding is well appreciated and has been illustrated in a number of systems (1-5). In many cases, a protein-ligand interaction appears to drive a flexible segment to fold into a rigid, ligand-bound conformation (6, 7). An analogy between global protein folding and ligand binding has been only recently described (8 -11). The concept of folding funnels, where folding progresses via multiple routes rather than a single pathway, can be likened to binding where a ligand drives an ensemble of local states to a single conformation. However, the detailed mechanism of any ligand-dependent, localized folding process remains elusive and is probably less well understood than global folding (12).The glutathione S-transferase (GST) 1 isoform A1-1 provides an outstanding opportunity to dissect the mechanism of a liganddependent local folding reaction. The GSTs are a family of xenobiotic metabolizing enzymes that play a critical role in the detoxication of various endogenous and exogenous compounds. The mammalian cytosolic GSTs consist of seven classes based on sequence similarity and substrate selectivity: ␣ (A), (P), (M), (T), (K), (S), and (Z) (13-17). The catalytic activity of GSTs is based on deprotonation of GSH to form the thiolate (GS Ϫ ), which is a superior nucleophile compared with the protonated thiol. X-ray crystallographic structures and site-directed mutagenesis studies illustrate that each GST contains a conserved tyrosine or serine residue that contributes to thiolate formation through a hydrogen bond (OH⅐⅐⅐⅐ Ϫ SG) and reduces the pK a of bound GSH. Although mutation a...

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Measurement of Protein Sulfenic Acid Content

Poole

2003

CP Toxicology

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Protein sulfenic acids are reactive, reversibly oxidized cysteinyl residues with roles in redox catalysis and regulation. Detection and quantification of these species in proteins is accomplished through chemical modification by reagents such as 7-chloro-4nitrobenzo-2-ixa-1,3-diazole (NBD chloride), 2-nitro-5-thiobenzoate (TNB), or dimedone, followed by UV-visible spectroscopy or mass spectrometric analysis. Curr. Protoc.Keywords: sulfenic acids r cysteine modification r cysteine oxidation r UV-visible spectroscopy r mass spectrometry The third reagent, 5,5-dimethyl-1,3-cyclohexanedione (dimedone), used in Basic Protocol 3, reacts specifically with sulfenic acid-but not thiol-groups on proteins. Unfortunately, the product does not exhibit any distinguishing visible absorbance properties; therefore, proof of modification by dimedone generally relies on ESI-MS analysis,

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The reactivity of SH groups with a fluorogenic reagent

Cited by 179 publications

References 11 publications

The Mitochondrial ATPase

The Mitochondrial ATPase

The Catalytic Tyr-9 of Glutathione S-Transferase A1-1 Controls the Dynamics of the C terminus

Measurement of Protein Sulfenic Acid Content

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