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The number of sulfhydryl groups in the Escherichia coli ribosome has been measured by titration with 5,5′-dithiobis(2-nitrobenzoic acid). Under denaturing conditions, there are 38.8 ± 1.0 titratable thiols per 70 S ribosome and 22.8 ± 0.3 and 12.9 ± 0.3 titratable thiols per 50 S and 30 S subunits, respectively. Three categories of thiol groups can be distinguished in the native 70 S ribosome, a “fast reacting” class of about 3 residues, a “slow reacting” class of about 10 residues and a “buried” class including about 26 residues. The addition of polyuridylic acid to reaction mixtures protects a fast-reacting thiol in the 30 S subunit belonging to protein S1.The addition of urea to ribosome solutions makes the buried residues titratable. Denaturation occurs as a sharp transition at a urea concentration between 4 and 4.5 m. Urea does not fully dissociate the ribosome into RNA and protein. Instead, in the case of the 30 S subunit, a slowly sedimenting particle forms in the presence of urea, containing roughly 65% of the normal amount of protein.  相似文献   

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Energized rat liver mitochondria in the presence of EGTA reduced linearly 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) at the rate of 7 nmol SH/min per mg protein within more than 1 hour at 20 degrees C. The Km for DTNB, 1.4 mM, was decreased by Mg2+ and spermine to 0.5 and 0.7 mM, respectively. The reaction was suppressed under conditions of decreasing mitochondrial content of NADPH, was blocked by 1,3-bis-(2-chloroethyl)-1-nitrosourea, the inhibitor of disulfide reductases, and was sensitive to external free Ca2+ in the micromolar range. After lysis of mitochondria the reduction of DTNB required the addition of NADPH and EGTA and was inhibited by 1 mM sodium arsenite. These observations suggest that the reduction of DTNB by mitochondria is catalyzed by Ca(2+)-sensitive thioredoxin reductase (EC 1.6.4.5).  相似文献   

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Both the number of exposed SH-groups and the rate of reaction with 5,5'dithiobis-2-nitrobenzoic acid (DTNB) in walleye pollock and bovine rhodopsin depend on a degree of native structure of the preparation to be investigated. The preparations studied can be arranged in the order of increase of these parameters as follows: ROS less than rhodopsin extracted by digitonin less than triton X-100 less than cetyltrimethylammonium bromide (CTAB) less than sodium dodecylsulphate (SDS). After illumination of ROS and digitonin, triton X-100 and CTAB-solubilized rhodopsin, and increase was observed in the number of modified SH-groups. Dark and bleached samples of walleye pollock rhodopsin exhibited a faster rate reaction and a more number of modified SH-groups as compared to bovine preparation. The differences between bovine and walleye pollock preparation disappeared after complete opsin unfolding as a result ROS solubilization in SDS. Six SH-groups per molecule of rhodopsin were modified in both preparation under these conditions. No differences in the number of cysteine residues (10--11), disulfide groups (2), acid (35--40) and base (25--30) titratable groups per rhodopsin molecule were found between bovine and walleye pollock ROS membranes. The isoelectric point of both rhodopsin preparations was within the pH range 5.2--5.6. After proteolysis of ROS with papain, a fragment with molecular weight 24500 +/- 1000 was detected, which contained the same number of SH-groups and cysteine residues as in the case of intact rhodopsin. The results obtained suggest that, in spite of a similar primary structure, the walleye pollock visual pigment has more "loose" and "fluid" space packing in the ROS membrane than the bovine pigment.  相似文献   

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The initial rate of proton liberation during MgATP hydrolysis by myosin was followed in a stopped flow spectrophotometer: before and after treatment with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) with and without removal of the corresponding light chain. At pH 8, 20°, and in the presence of MgCl2, the biphasic pattern of the initial rate of proton liberation for native myosin became monophasic following treatment with DTNB, removal of the corresponding light chain, and regeneration of the steady state ATPase activity. The rate constant characterizing the single exponential term increased with MgATP concentration attaining a maximum value of 100 s?1 at 300 μM MgATP with an apparent 2° rate constant of 7 × 105 M?1s?1. Both the biphasic and monophasic pattern of initial proton liberation observed for myosin and subfragment 1 respectively (Pemrick, S.M. and F.G. Walz, 1972. J. Biol. Chem. 247: 2959) can be explained by differences in the relative amounts of the DTNB light chain.  相似文献   

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Myosins prepared from chicken and rabbit fast and slow muscles were treated with 5,5'-dithiobis-(2-nitrobenzoic acid) (Nbs2). About half of the thiol groups of the fast muslce myosins reacted with Nbs 2, but in slow muscle myosins, only about 10-20% of the thiol groups reacted. This treatment removed 50-60% of the L2 components, Nbs2 light chain, from fast muscle myosins, but did not result in specific dissociation of the light chains in slow myscle myosins. The treatment sometimes released L4 component from chicken muscle myosins instead of L2 component. The changes of myosin ATPase [EC 3.6.1.3] activities caused by this treatment did not correlate with the release of Nbs2 light chain, but were dependent upon the species, chicken or rabbit.  相似文献   

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Insulin binding to isolated rat white adipocytes at 15 degrees C, a temperature at which cellular degradation of insulin is negligible, has been found to be described by the Two-step Binding Model: R + I in equilibrium RI in equilibrium R'I (Lipkin, E. W., Teller, D. C., and de Ha?n, C. (1986) J. Biol. Chem. 261, 1702-1711). RI is the initially formed complex between the receptor, R, and insulin, I, and R'I is the complex in an altered state or cellular location. Here the possibility was examined that R'I results from disulfide exchange between the receptor and insulin, an exchange proposed by Clark and Harrison (Clarke, S., and Harrison, L. C. (1986) J. Biol. Chem. 257, 12239-12244) to occur at 37 degrees C. A number of sulfhydryl reagents representing various chemical reactivities did not affect insulin binding. The exception was 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), which enhanced the number of insulin-binding sites up to 2-fold with no effect on the equilibrium constant. The data suggested that this enhancement was due to activation of cryptic binding sites pre-existing on the cell surface, possibly by increasing the valency of the receptor from 1 to 2. Insulin binding was also enhanced by structural congeners of DTNB devoid of sulfhydryl reactivity, the simplest one being benzoic acid. It was concluded that the effects were not related to modification of sulfhydryl groups, that modification of sulfhydryl groups on the receptor either did not take place or was without effect on binding, and finally, that disulfide exchange between insulin and the receptor was an unlikely explanation for the formation of R'I. Also, since it is possible to show insulin action at 15 degrees C, contrary to the proposal by Clark and Harrison (Clark, S., and Harrison, L. C. (1983) J. Biol. Chem. 258, 11434-11437), disulfide exchange does not appear to be necessary for signal transmission by the occupied receptor.  相似文献   

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Fatty acid synthetase of chicken liver is rapidly and reversibly inactivated by 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) at a rate (k2 = 132 mM-1 S-1 in 3 mM EDTA, 1% (v/v) glycerol, pH 7.0, at 25 degrees C) up to 2200 times higher than the reaction of this reagent with simple thiol compounds. The inactivation is caused by the reaction of the phosphopantetheine SH group, since it is protected competitively by either acetyl- or malonyl-CoA, and since the inactivated enzyme is unreactive with the phosphopantetheine label chloroacetyl-CoA but reactive with the cysteine reagent 1,3-dibromopropanone. Moreover, chloroacetyl-CoA prevents the modification of the rapidly reacting essential SH group by DTNB. The number of SH groups involved in inactivation was determined by correlating activity loss with the extent of reaction and by stopped-flow analysis of substrate (or chloroacetyl-CoA) protection. Values between 0.91 and 1.15 SH groups/dimer were obtained, indicating the presence of substoichiometric amounts of the prosthetic group in the fatty acid synthetase preparations used in this study. Inactivation of the synthetase by DTNB is strongly inhibited by increasing salt concentration and protected noncompetitively by NADP+ and NADPH. Treatment of the enzyme inactivated at low salt by salt, NADP+, or NADPH also effectively reduced cross-linking between enzyme subunits. The parallel effects of these treatments on the reaction with DTNB and subsequent dimerization are consistent with a minimum model of two discreet conformation states for fatty acid synthetase. In the low salt conformer, the phosphopantetheine and cysteine SH groups are juxtaposed, and the DTNB reaction (k2 approximately 132 mM-1 S-1) and dimerization are both facilitated. Transition to the high salt conformer by the above treatments is accompanied by an approximately 20-fold reduction of reactivity with DTNB (k2 = 6.8 mM-1 S-1) and reduced dimerization, due to spatial separation of the SH groups. During palmitate synthesis, the enzyme may oscillate between these conformation states to permit the reaction of intermediates at different active sites. Results obtained by studies on the effect of pH on DTNB inactivation implicate a pK of 5.9-6.1 for the essential SH group independent of salt concentration. This value is 1.5-1.8 pH units lower than the pK of 7.6-7.7 for CoA and may explain the 23-fold increase of the rate constant from a value of 0.3 mM-1 S-1 for CoA to that of the high salt conformer.  相似文献   

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D W Pettigrew 《Biochemistry》1986,25(16):4711-4718
Glycerol kinase (EC 2.7.1.30, ATP:glycerol 3-phosphotransferase) from Escherichia coli is inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and by N-ethylmaleimide (NEM) in 0.1 M triethanolamine at pH 7 and 25 degrees C. The inactivation by DTNB is reversed by dithiothreitol. In the cases of both reagents, the kinetics of activity loss are pseudo first order. The dependencies of the rate constants on reagent concentration show that while the inactivation by NEM obeys second-order kinetics (k2app = 0.3 M-1 s-1), DTNB binds to the enzyme prior to the inactivation reaction; i.e., the pseudo-first-order rate constant shows a hyperbolic dependence on DTNB concentration. Complete inactivation by each reagent apparently involves the modification of two sulfhydryl groups per enzyme subunit. However, analysis of the kinetics of DTNB modification, as measured by the release of 2-nitro-5-thiobenzoate, shows that the inactivation is due to the modification of one sulfhydryl group per subunit, while two other groups are modified 6 and 15 times more slowly. The enzyme is protected from inactivation by the ligands glycerol, propane-1,2-diol, ATP, ADP, AMP, and cAMP but not by Mg2+, fructose 1,6-bisphosphate, or propane-1,3-diol. The protection afforded by ATP or AMP is not dependent on Mg2+. The kinetics of DTNB modification are different in the presence of glycerol or ATP, despite the observation that the degree of protection afforded by both of these ligands is the same.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

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Chicken liver fatty acid synthase is inhibited by the thiol-modifying reagents 5,5'-dithiobis-(2-nitrobenzoic acid) and iodoacetamide. Total inactivation of the activity for fatty acid synthesis requires the modification of about 8 of the nearly 50 freely accessible thiol groups per molecule. The differential binding of iodo[14C]acetamide to phenylmethylsulphonyl fluoride-modified enzyme in the absence and in the presence of excess acetyl-CoA shows complete modification of one cysteine-SH site of the condensing enzyme and partial modification of the pantetheine-SH site for a total of approx. 1.4 mol of iodoacetamide bound per mol of enzyme. The reaction of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid) generates disulphide cross-links for each molecule of the reagent added, but 95% of these cross-links are intrasubunit. Both the iodoacetamide- and 5,5'-dithiobis-(2-nitrobenzoic acid)-modified species catalyse all the component partial reactions of fatty acid synthesis except the condensation reaction. The results obtained with iodoacetamide show that in the dimeric fatty acid synthase modification of one cysteine-SH condensing site and/or one pantetheine-SH site per dimer is sufficient to affect inhibition of condensing activity and the activity for fatty acid synthesis, and are in accord with a recently proposed model for the mechanism of action of animal fatty acid synthases [Kumar (1982) J. Theor. Biol. 95, 263-283].  相似文献   

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The reduction of 5,5'-dithiobis-(2-nitrobenzoic acid)-modified arginine kinase by dithiothreitol has been investigated using the kinetic theory of the substrate reaction during modification of enzyme activity. The results show that the modified arginine kinase can be fully reactivated by an excess concentration of dithiothreitol in a monophasic kinetic course. The presence of ATP or the transition-state analog markedly slows the apparent reactivation rate constant, while arginine shows no effect. The results of ultraviolet (UV) difference and intrinsic fluorescence spectra indicate that the substrate arginine-ADP-Mg2+ can induce conformational changes of the modified enzyme but adding NO3- cannot induce further changes that occur with the native enzyme. The reactive cysteines' location and role in the catalysis of arginine kinase are discussed. It is suggested that the cysteine may be located in the hinge region of the two domains of arginine kinase. The reactive cysteine of arginine kinase may play an important role not in the binding to the transition-state analog but in the conformational changes caused by the transition-state analog.  相似文献   

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Chaperonin GroEL, consisting of two seven-subunit rings stacked back-to-back, is disassembled by interaction of 4, 4'-dithiodipyridine (DTP) with Cys(458) located close to the intersubunit contacts within and between the rings. The thiol group of Cys(458) is inaccessible to the probe being buried into the pocket locked by segment Asn(475)-Asn(487). Flexibility of this segment is proposed to induce the "open" state of the pocket and accommodate the bulky probe inside so that the consequential irreversible shifts in the pocket constituents disassemble GroEL. This scheme is supported by the finding that DTP-induced disassembly of GroEL is facilitated by ATP, which specifically stimulates a local shift of the segment Asn(475)-Asn(487) into solution.  相似文献   

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A method for assaying glutathione reductase (GSH; EC 1.6.4.2) in crude plant extracts is described. The method is based on the increase in absorbance at 412 nm when 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) is reduced by GSH. The effects of the following parameters on the assay were tested: various buffers, pH, buffer concentration, compounds commonly present in enzyme preparations, thiols, and the presence of another NADPH-dependent enzyme. The assay is more sensitive and less subject to interference than the widely used assay where NADPH oxidation is monitored. In particular, the specificity of DTNB allows assay of glutathione reductase in the presence of other NADPH-dependent enzymes and common protein extract contaminants.  相似文献   

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