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PubMed1972

The reaction of papain with Ellman's reagent (5,5'-dithiobis- (2-nitrobenzoate) dianion).

1. The report by Robyt et al. (1971) that the 2-nitro-5-mercaptobenzoate dianion (Nbs(2-)) produced by reaction of papain with the 5,5'-dithiobis-(2-nitrobenzoate) dianion (Nbs(2) (2-); Ellman's reagent) cleaves the three disulphide bonds in papain is shown to be incorrect. 2. When partially active papain containing approx. 0.4 mol of thiol/mol of protein is incubated with excess of Nbs(2) (2-) at pH8, Nbs(2) (2-) reacts with the protein in an amount stoicheiometric with the cysteinyl thiol group of papain to produce Nbs(2-) in an amount stoicheiometric with the original papain cysteinyl thiol group, and the catalytically inactive mixed disulphide, papain-Nbs(-). 3. Papain catalyses the hydrolysis of Nbs(2) (2-) at pH10.5 probably by nucleophilic catalysis involving the enzyme's thiol group. 4. These results cast very serious doubts on the claim by Robyt et al. (1971) to have established a new general method for the determination of cystinyl disulphide residues in proteins.

Benzoates↗
PubMed1976

4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole as a reactivity probe for the investigation of the thiol proteinases. evidence that ficin and bromelain may lack carboxyl groups conformationally equivalent to that of aspartic acid-158 of papain.

1. 4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole (Nbd chloride) was used as a reactivity probe to characterize the active centres of papin (EC 3.4.22.2), ficin (EC 3.4.22.3) and bromelain (EC 3.4.22.4). 2. In the pH range 0-8 Nbd chloride probably exists mainly as a monocation, possibly with the proton located on N-1 of the oxadiazole ring. 3. Spectroscopic evidence is presented for the intermediacy of Meisenheimer-type adducts in the reaction of Nbd chloride with nucleophiles. 4. The pH-dependence of the second-order rate constants (k) of the reactions of the three enzymes with Nbd chloride was determined at 25 degrees C, I = 0.1 mol/litre in 6.7% (v/v) ethanol in the pH range 2.5-5, where, at least for papain and ficin, the reactions occur specifically with their active-centre thiol groups. The pH-k profile for the papain reaction is bell-shaped (pKaI = 3.24, pKaII = 3.44 and k = 86M(-1)-s(-1), whereas that for ficin is sigmoidal (pKa = 3.6, k = 0.36M(-1)-s(-1), the rate increasing with increasing pH. The profile for the bromelain reaction appears to resemble that for the ficin reaction, but is complicated by amino-group labelling. 5. The bell-shaped profile of the papain reaction is considered to arise from the reaction of the thiolate ion of cysteine-25, maintained in acidic media by interaction with the side chain of histidine-159, with the Nbd chloride monocation hydrogen-bonded at its nitro group to the un-ionized form of the carboxyl group of aspartic acid-158. The lack of acid catalysis in the corresponding reactions of ficin and probably of bromelain suggests that these enzymes may lack carboxyl groups conformationally equivalent to that of aspartic acid-158 of papain. The possible consequences of this for the catalytic sites of these enzymes is discussed.

4-Chloro-7-nitrobenzofurazan↗
PubMed1993

Inactivation of mouse liver glutathione S-transferase YfYf (Pi class) by ethacrynic acid and 5,5'-dithiobis-(2-nitrobenzoic acid).

Mouse liver glutathione S-transferase YfYf (Pi class) reacts with [14C]ethacrynic acid to form a covalent adduct with a stoichiometry of 1 mol per mol of subunit. Proteolytic digestion of the enzyme-[14C]ethacrynic acid adduct with V8 protease produced an 11 kDa fragment containing radioactivity. Sequencing revealed this to be an N-terminal peptide (minus the first 15 residues, terminating at Glu-112) which contains only one cysteine residue (Cys-47). This is tentatively identified as the site of ethacrynic attachment. Kinetic studies reveal that glutathione S-conjugates protect against inactivation by ethacrynic acid, but the level of protection is not consistent with their potency as product inhibitors. A model is proposed in which glutathione S-conjugates and ethacrynic acid compete for the free enzyme, and a second molecule of ethacrynic acid reacts covalently with the enzyme-ethacrynic acid complex. The native protein contains one thiol reactive with 5,5'-dithiobis-(2-nitrobenzoic acid) at neutral pH. The resultant mixed disulphide, like the ethacrynic acid adduct, is inactive, but treatment with cyanide (which incorporates on a mol for mol basis) restores activity to 35% of that of the native enzyme.

Amino Acid Sequence↗
PubMed2001

The effects of physiologically important nonmetallic ligands in the reactivity of metallothionein towards 5,5'-dithiobis(2-nitrobenzoic acid). A new method for the determination of ligand interactions with metallothionein.

The reaction of Cd5Zn2-metallothionein (MT) with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) has been studied at different reagent stoichiometries, pH and temperature conditions and in the presence of several ligands. At stoichiometries of Nbs2 to MT from 0.5 to 5, the reaction followed first order kinetics. The first order rate constants obtained were independent from the concentration of Nbs2 but were linearly dependent on the concentration of MT. At higher Nbs2/MT stoichiometries, the reaction deviates from first order kinetics and the observed rate constant increases. The reactivity of MT towards Nbs2 has been probed at 4 microM concentration of both reagents where the reaction is monophasic and is characterized by a linear Arrhenius plot (Ea = 45.8 +/- 2.7 kJ.mol-1). It has been demonstrated that metal release at low pH or subtraction from MT by EDTA substantially increases the reactivity of MT towards Nbs2. At the same time, a number of nonmetallic ligands moderately accelerate the reaction of MT with Nbs2 and hyperbolic dose-response curves were obtained. The data have been interpreted with the binding of ligands to MT and following MT. Ligand binding constants were calculated as follows: ATP, K = 0.31 +/- 0.06 mM; ADP, K = 0.26 +/- 0.07 mM. Several compounds such as AMP, S-methylglutathione, and phosphate had no effect on the reaction, but Zn2+ ions showed an inhibitory effect at micromolar concentrations.

Adenosine Diphosphate↗
PubMed1998

Inhibition of proton transport through spinach thylakoid membranes by 5,5'-dithiobis(2-nitrobenzoate).

In both oxidative phosphorylation and photo-phosphorylation, electron flow through a carrier is linked to the generation of ATP. The energy released by electron transport is converted to potential energy forming a proton gradient across the membranes in chloroplasts. The proton gradient can be measured by a pH microelectrode. In this report, pH changes produced by photo-induced proton transport through spinach chloroplast membranes were measured by a glass microelectrode. The effect of 5,5'-dithiobis(2-nitrobenzoate) (DTNB) on the kinetics of proton movement across the thylakoid membranes was studied. The results showed that the rate of proton uptake was reduced with increasing DTNB concentration. The rate of leakage of accumulated protons through thylakoid membranes also decreased. The results support the notion that cysteinyl residue is involved in proton translocation. The inhibition of proton transport would subsequently affect the chemical reactions of the Calvin Cycle that takes place in the stroma which is the soluble compartment surrounding the thylakoid membranes.

Biological Transport↗
PubMed1983

Metabolism of 6-nitrobenzo[a]pyrene by hamster embryonic fibroblasts and its interaction with nuclear macromolecules.

Incubation of 6-nitrobenzo[a]pyrene (6-nitroBaP) with hamster embryonic fibroblasts led to formation of both organic solvent-soluble and water-soluble products. High-pressure liquid chromatographic analysis of organic solvent-soluble extracellular metabolites showed the predominant presence of dihydrodoils, with only small amounts of phenolic products. This differed from microsomal metabolism, using hepatic preparations from 3-methylcholanthrene-pretreated rats, where a major phenolic peak was obtained. Subsequent treatment of aqueous layer with beta-glucuronidase, however, revealed that most of the phenols were associated with glucuronic acid to form water soluble products. Interaction of 6-nitroBaP with nuclear macromolecules from HEF was also studied. The chemical interacted with both DNA and RNA, but the specific activity was highest with nuclear proteins. This binding profile was found to be similar to that when benzo[a]pyrene was used, although the affinity toward protein binding was slightly higher for 6-nitroBaP.

Animals↗
PubMed1986

6-Nitrobenzo[a]pyrene can be denitrated during mammalian metabolism.

Nitropolycyclic aromatic hydrocarbons (nitroarenes), including 6-nitrobenzo[a]pyrene (6-NBap), occur in our environment and are mutagenic in bacterial mutagenesis assays. The mutagenicity of 6-NBaP is enhanced when rat liver S9 is added. To investigate the cause of this increased activity, the metabolism of 6-NBaP was carried out with a total rat liver homogenate obtained from 3-methylcholanthrene- (MC-) induced rats, a 9000 X g supernatant enzyme, and with both unwashed and washed microsomes. Ring-hydroxylated 6-NBaP was detected. On the basis of retention times for known standards in a high-performance liquid chromatographic system, benzo[a]pyrene (BaP) and 6-acetoxy-BaP (6-OAcBaP) were isolated as products. BaP was further characterized via ultraviolet (UV) and mass spectra and 6-OAcBaP by UV, mass, and nuclear magnetic resonance (NMR) spectra. 6-HydroxyBaP (6-OHBaP) was also detected by UV and mass spectra. It is suggested that BaP is formed via a nitroanion radical of 6-NBaP and undergoes metabolism, while the 6-OHBaP is acetylated to form 6-OAcBaP. The acetyl donor remains to be identified.

Animals↗
PubMed1986

Microbial transformation of 6-nitrobenzo[a]pyrene.

The fungal metabolism of the potent mutagenic and carcinogenic nitropolycyclic aromatic hydrocarbon (nitro-PAH) 6-nitrobenzo[a]pyrene (6-NO2-BaP) was investigated. Cunninghamella elegans was incubated with 6-NO2-BaP for periods ranging between 1 and 7 d, and the metabolites formed were separated by high-performance liquid chromatography and identified by their UV-visible absorption, mass, and 1H nuclear magnetic resonance spectra. The results of our study indicate that C. elegans metabolized 6-NO2-BaP to glucoside and sulfate conjugates of 1- and 3-hydroxy 6-NO2-BaP and suggests that glycosylation and sulfation reactions may represent detoxification pathways in the fungal metabolism of nitro-PAHs. Experiments using [G3H]-6-NO2-BaP indicated that C. elegans metabolized 62% of 6-NO2-BaP within 168 h. Our data also indicated that the nitro group at the C-6 position of benzo[a]pyrene blocked metabolism at the regions peri to the nitro substituent (C-7, C-8 positions) and enhanced metabolism at the C-1 and C-3 positions. The ability of the fungus C. elegans to metabolize 6-NO2-BaP to biologically inactive compounds may have practical applications in the detoxification of nitro-PAH-contaminated wastes.

Benzopyrenes↗
PubMed1988

Metabolism of 1-, 3-, and 6-nitrobenzo[a]pyrene by intestinal microflora.

The compounds 1-, 3-, and 6-nitrobenzo[a]pyrene (nitro-BaP) are environmental pollutants and have been shown to be potent bacterial mutagens. The anaerobic metabolism of these isomeric nitro-BaPs was investigated by the incubation of rat intestinal microflora with each isomer for 48 h. Aliquots were removed at several time intervals, extracted, fractionated by high-pressure liquid chromatography (HPLC), and the radioactivity determined. Metabolites were identified by comparison of their chromatographic, ultraviolet-visible absorption, and mass spectral properties with those of authentic standards. The order of the extent of nitroreduction for these isomers was 3-nitro-BaP greater than 6-nitro-BaP greater than 1-nitro-BaP. After 48 h of exposure, 84% of the added 3-nitro-BaP was present as 3-amino-BaP, 51% of the 6-nitro-BaP was metabolized to 6-amino-BaP, and 1-nitro-BaP was reduced to 1-amino-BaP (13%) and 1-nitro-BaP (4%). The order of the extent of microbial nitroreduction for these nitro-BaP isomers is different from the predictions based on electronic and steric hindrance effects. These results suggest that intestinal microflora nitroreductases exhibit a markedly high degree of substrate specificity toward nitro-BaPs that affects the extent of nitroreduction.

Animals↗
PubMed1993

Formation of the adduct 6-(deoxyguanosin-N2-yl)-3-amino-benzo[a]pyrene from the mutagenic environmental contaminant 3-nitrobenzo[a]pyrene.

3-Nitrobenzo[a]pyrene (3-nitro-B[a]P) is a potent bacterial mutagen as a result of nitroreduction. Reaction of N-hydroxy-3-amino-B[a]P, prepared in situ from reduction of 3-nitro-B[a]P with calf thymus DNA, was studied. After enzymatic digestion of the DNA, the resulting modified nucleosides were analyzed by thermospray HPLC-MS and high-resolution proton NMR spectroscopy. The major adduct was identified as 6-(deoxyguanosin-N2-yl)-3-amino-B[a]P. The same adduct was obtained from incubation of DNA with 3-nitro-B[a]P in the presence of the mammalian nitroreductase xanthine oxidase, and hypoxanthine. These data indicate that a mammalian nitroreductase can metabolize 3-nitro-B[a]P to an activated derivative that reacts with DNA to give a novel adduct distant from the site of N-hydroxylation.

Animals↗
PubMed1982

Macromolecular binding of [3H]6-nitrobenzo[a]pyrene in male rats.

Generally labelled [3H]6-nitrobenzo[a]pyrene (6NBP) when administered to male rats by i.p. injection was widely distributed throughout the body. After a single injection of 4.2 mg/kg the highest concentration was found in the liver (5% at 2 h) followed by the spleen greater than kidney greater than lung. Maximal covalent binding to macromolecules occurred between 8 and 24 h depending upon the organ; the order of binding/mg macromolecule was RNA greater than DNA greater than protein for all organs studied. In the liver maximal binding was found 8 h after 6NBP administration at which time there were 45 +/- 7 pmol 6NBP/mg bound to DNA and 114 +/- 40 pmol/mg to RNA following a dose of 4.2 mg/kg. These levels fall to 14 +/- 4 and 12 +/- 6 pmol/mg, respectively, after 168 h, the longest time point so far studied. At a lower dose of 1.4 mg/kg the values for DNA at 8 h were 14 +/- 2 pmol/mg and at 168 h 3 +/- 2 pmol/mg while for RNA the corresponding values were 29 +/- 8 and 15 +/- 3 pmol/mg. After gastric intubation at a dose of 4.2 mg/kg there was no difference in liver macromolecular binding at 24 h between conventional and germ-free animals. Enzymic digestion of liver DNA from [3H]6NBP injected animals and reversed-phase h.p.l.c. of the digests showed two peaks of radioactivity. The percentage of total radioactivity in each peak varied from preparation to preparation suggesting that one peak may be a degradation product of the other although experimental variability cannot be ruled out. The implications of these findings in relation to the potential carcinogenicity of 6NBP are discussed.

Animals↗
PubMed1983

Evidence for a 2,3-epoxide as an intermediate in the microsomal metabolism of 6-nitrobenzo[a]pyrene.

The rat liver microsomal metabolism of 3-deutero-6-nitrobenzo(a)pyrene ([3-2H]6-NO2-BaP) was studied. The metabolites were separated by h.p.l.c. The 500 MHz 1H n.m.r. spectral analysis of the metabolites indicated that the 3-hydroxy-6-NO2-BaP and 6-NO2-BaP-3,9-hydroquinone each retained 33% of the deuterium label at the C-2 position. It is proposed that the migration of deuterium occurs via an NIH shift mechanism. These results indicate that a 2,3-epoxide is a common intermediate.

Animals↗
PubMed1985

Multiple metabolic pathways for the mutagenic activation of 3-nitrobenzo[a]pyrene.

Rat liver microsomal metabolism of the potent mutagen 3-nitrobenzo[a]pyrene (3-nitro-BaP) under aerobic conditions yielded 3-nitro-BaP trans-7,8-dihydrodiol and 3-nitro-BaP trans-9,10-dihydrodiol, and under anaerobic conditions produced 3-amino-BaP. All of these metabolites were highly mutagenic in the Salmonella typhimurium reversion assay both with and without exogenous metabolic activation (S9). None of the in vitro metabolic pathways led to detoxification of 3-nitro-BaP.

Animals↗
PubMed1986

Metabolism of 1-nitrobenzo[a]pyrene by rat liver microsomes to potent mutagenic metabolites.

1-,3- and 6-Nitrobenzo[a]pyrene (nitro-BaP), which are prototypes of nitro polycyclic aromatic hydrocarbons (nitro-PAHs) derived from a carcinogenic parent PAH, benzo[a]pyrene, are environmental contaminants and potent bacterial mutagens. In this study, the aerobic and hypoxic metabolism of 1-nitro-BaP by rat liver microsomes was studied. Aerobic metabolism of 1-nitro-BaP yielded 1-nitro-BaP trans-7,8- and 9,10-dihydrodiol, while metabolism under hypoxic conditions yielded 1-amino-BaP. The metabolites formed from aerobic metabolism of 1-nitro-BaP and 1-nitro-BaP trans-9,10-dihydrodiol by liver microsomes of untreated rats and rats pretreated with 3-methylcholanthrene and phenobarbital were quantified. Comparison of these results with those obtained with BaP and BaP trans-9,10-dihydrodiol indicates that nitro substitution at the 1-position of BaP markedly affects the regioselectivity of the P-450-containing enzymes. 1-Nitro-BaP and the three metabolites were potent mutagens in Salmonella typhimurium TA98, both in the absence and in the presence of an exogenous metabolic activation system (S9). The direct and S9-mediated mutagenicities of 1-nitro-BaP and the two dihydrodiols were decreased in the nitroreductase-deficient strain TA98NR, while TA98/1,8-DNP6, an O-acetylase-deficient strain, was less sensitive to the two dihydrodiols, both with and without S9, and 1-nitro-BaP with S9. 1-Amino-BaP was equally mutagenic in all three tester strains. These observations indicate that: the metabolism of 1-nitro-BaP involves several pathways leading to mutagenic activation; the major activation pathways of 1-nitro-BaP involve nitroreduction; nitroreduction followed by O-acetylation is the major activation pathway of 1-nitro-BaP trans-7,8- and 9,10-dihydrodiol; and 1-amino-BaP is a potent direct-acting mutagen.

Aerobiosis↗
PubMed1987

Morphological transformation in three mammalian cell systems following treatment with 6-nitrochrysene and 6-nitrobenzo[a]pyrene.

Two nitroaromatics, 6-nitrobenzo[a]pyrene (6-N-BaP) and 6-nitrochrysene (6-N-CRY), and the corresponding parent hydrocarbons, benzo[a]pyrene (BaP) and chrysene (CRY), were studied in in vitro transformation assays with Syrian hamster embryo (SHE) cells, BALB/3T3 and C3H10T1/2 mouse cell lines. The three cell systems showed different sensitivities to the transforming effects of the chemicals studied, SHE cells being the most efficient, followed by 3T3 cells and the last being C3H10T1/2 cells. In the SHE cell system all compounds were active. Considering the concentrations (in microM) and the transformation frequency BaP was the most active, followed by 6-N-BaP, 6-N-CRY and CRY. In the BALB/3T3 standard assay and in the C3H10T1/2 assay only BaP was clearly active. When used as initiators 6-N-BaP and 6-N-CRY were inactive in the C3H cell system. In conclusion 6-N-BaP appears less active in in vitro systems than the parent compound BaP; 6-N-CRY is probably negative since it is questionable in vitro and negative in mouse skin.

Animals↗
PubMed1988

Microsomal metabolism of 1-nitrobenzo[e]pyrene to a highly mutagenic K-region dihydrodiol.

Aerobic metabolism of 1-nitrobenzo[e]pyrene (1-nitro-BeP) by rat liver microsomes produced 1-nitro-BeP trans-4,5-dihydrodiol, 6-hydroxy-1-nitro-BeP, and 8-hydroxy-1-nitro-BeP. When 3,3,3-trichloropropylene 1,2-oxide was incorporated into the metabolism, 1-nitro-BeP 4,5-oxide was the predominant metabolite, and 1-nitro-BeP trans-4,5-dihydrodiol was not detected. All of the metabolites were purified by both reversed- and normal-phase HPLC and characterized by analysis of their mass and 500 MHz proton NMR spectral data. 1-Nitro-BeP was not metabolized under hypoxic conditions. 1-Nitro-BeP and its four metabolites were assayed in Salmonella typhimurium tester strains TA98, TA98NR and TA98/1,8-DNP6, both in the presence and absence of S9 activation. As predicted, 1-nitro-BeP was a weak mutagen without S9 (2 revertants/micrograms in TA98); the addition of S9 resulted in approximately 18, 17 and 4 revertants/micrograms in TA98, TA98NR and TA98/1,8-DNP6 respectively. The two phenolic metabolites were mutagenic both in the presence and absence of S9, producing moderate responses (19-84 revertants/micrograms). In addition, while the 1-nitro-BeP 4,5-oxide was only weakly mutagenic in TA98 (6-14 revertants/micrograms), 1-nitro-BeP trans-4,5-dihydrodiol was unexpectedly potent (approximately 300 revertants/micrograms both with and without S9). These results indicate that microsomal epoxidation of 1-nitro-BeP followed by epoxide hydrolase-catalyzed hydrolysis of the resulting epoxide to the 1-nitro-BeP trans-4,5-dihydrodiol results in the most potent mutagenic derivatives. The weak mutagenicity of 1-nitro-BeP 4,5-oxide demonstrates that not all epoxides of nitrated polycyclic aromatic hydrocarbons (PAHs) are more mutagenic than the corresponding parent nitro-PAHs. Also, the lower S9-mediated mutagenicity of 1-nitro-BeP in TA98/1,8-DNP6 compared with TA98 indicates that the mutagenicity of 1-nitro-BeP is dependent upon nitroreduction and transesterification. Finally, we previously hypothesized that nitrated PAHs with their nitro substituents perpendicular or nearly perpendicular to the aromatic rings are very weak or nondirect-acting mutagens in Salmonella typhimurium tester strains. The results reported in this communication demonstrate that ring-oxidized derivatives of nitro-PAHs do not always follow this structure--mutagenicity correlation.

Aerobiosis↗
PubMed1988

Microsomal activation of 1- and 3-nitrobenzo[a]pyrene to mutagens in Chinese hamster ovary cells.

1- and 3-nitrobenzo[a]pyrene (1- and 3-nitro-BaP) are environmental pollutants and are S9-mediated mutagens in the Chinese hamster ovary (CHO) cell/hypoxanthine-guanine phosphoribosyl transferase assay. In this study, we examined the pathways leading to the mutagenic activation of these compounds in CHO cells. The microsomal metabolites of 1- and 3-nitro-BaP, the 1- and 3-nitro-BaP trans-7,8-dihydroxy-7,8-dihydrodiols (trans-7,8-dihydrodiols) and the 1- and 3-nitro-BaP trans-9,10-dihydrodiols, were isolated and tested for mutagenicity. At the concentrations assayed, both trans-9,10-dihydrodiols were non-mutagenic with and without S9 activation. In contrast, the trans-7,8-dihydrodiols of 1- and 3-nitro-BaP were direct-acting mutagens and these responses were similar in magnitude to the S9-mediated mutagenicities of the parent nitro-BaPs. S9 increased the mutagenic responses of the trans-7,8-dihydrodiols approximately 20-fold. Inhibition of epoxide hydrolase decreased the S9-mediated mutagenicity of 1-nitro-BaP by half, but doubled the S9-mediated mutagenicity of 3-nitro-BaP. These results suggest that in CHO cells: (i) the major route of mutagenic activation of 1- and 3-nitro-BaP involves S9-generated derivatives of the trans-7,8-dihydrodiols, e.g. bay-region diol epoxides; (ii) reactive nitroarene oxides may contribute to mutation induction by 3-nitro-BaP; and (iii) metabolic routes involving trans-9,10-dihydrodiol formation result in detoxification.

Animals↗
PubMed1990

Does fluorescence of 4-nitrobenzo-2-oxa-1,3-diazole incorporated into sarcoplasmic reticulum ATPase monitor putative E1-E2 conformational transition?

When a purified preparation of sarcoplasmic reticulum Ca2(+)-ATPase was labeled with 0.3 mM 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) in 1 mM AMPPNP and 1 mM CaCl2 at 25 degrees C and pH 7.0 for 60 min and then was treated with 10 mM dithiothreitol for 7 min, about 1 mol of NBD was incorporated per mol of the enzyme, and this inhibited the enzyme activity by 90 to 95%. The modified residue was identified as Cys-344 that is located near the phosphorylation site of the ATPase, Asp-351. The NBD-inhibition of enzyme activity could be reversed by treatment with membrane-acting agents such as C12E8, suggesting that Cys-344 is not directly involved in enzyme catalysis. A detailed study of partial reactions of ATP hydrolysis by the modified enzyme and associated changes in the fluorescence intensity of the incorporated NBD label revealed that a predominant effect of the NBD-modification was the inhibition of Ca2+ release from the ADP-sensitive phosphoenzyme intermediate and that two major fluorescent states of the enzyme alternated during ATP hydrolysis. The latter fluorescent data are consistent with the E1-E2 model of Ca2(+)-ATPase reaction.

4-Chloro-7-nitrobenzofurazan↗