果糖-2,6-二磷酸对番茄果实PFP酶活化效应的巯基依赖性

在无二硫苏糖醇(DTT)存在下得到部份纯化的氧化型PFP酶,在广泛的pH范围内(pH6.0～9.0)失去其大部分对果糖2,6-二磷酸的敏感性。活化效应可藉与DTT保温得到恢复而不改变其最适pH值。在与DTT保温过程中,酶对果糖2,6-二磷酸的亲和力逐步增加。氧化型酶的K_a值(对果糖2,6-二磷酸)在酶与DTT保温(pH8)1h之后从1400nmol/L下降到约50nmol/L。 在DTT存在下纯化的酶(还原型)经低浓度5,5′-二硫代双(2-硝基苯甲酸)(DTNB)处理,在使酶活性迅速失活的同时引起酶对果糖2,6-二磷酸脱敏。这一过程可为DTT处理所回复。从小麦胚中纯化的硫氧还蛋白h在恢复酶活性和酶的果糖2,6-二磷酸敏感性的效应中表明,细胞内的氧化还原状态可能藉以改变酶对果糖2,6-二磷酸的亲和力而调节PFP酶的活性。     

Nonenzymatic cleavage of proteins by reactive oxygen species generated by dithiothreitol and iron

Many enzymes, represented by yeast glutamine synthetase, are inactivated and degraded in the presence of dithiothreitol (DTT), oxygen, and catalytic amounts of iron salts. The roles of DTT and iron can be replaced by ascorbate and copper, respectively. Experimental data suggest that reactive oxygen species, likely hydroxyl radicals, are generated locally around irons bound at specific sites on enzymes, and these species are responsible for the inactivation and degradation. Since many biochemicals are contaminated with metal salts in quantities sufficient for some hydroxyl radical formation to occur, the possibility of oxidative modification and degradation should be considered when an enzyme is exposed to DTT.     

Human liver manganese superoxide dismutase. Purification and crystallization, subunit association and sulfhydryl reactivity

Manganese superoxide dismutase (Mn-SOD) has been purified with a high yield (320 mg) from human liver (2 kg) and crystallized. Low-angle laser light scattering of the enzyme has shown that native enzyme is a tetrametic form. Four of the eight cysteine residues in the tetramer reacted with 5,5'-dithiobis(2-nitrobenzoic acid) or with iodoacetamide. The others were only reactive in protein heated with SDS or urea after reduction with dithiothreitol or 2-mercaptoethanol. The reactive sulfhydryl group was found to be located at Cys196 by amino acid sequence analysis of Nbs2-reactive peptides isolated by activated thiol-Sepharose covalent chromatography. Incubation of Mn-SOD in 1% SDS for 2 or 3 days at 25 degrees C or 5 min at 100 degrees C gave material showing two prominent components on polyacrylamide gel electrophoresis in the presence of 0.1% SDS. The major component had a molecular mass of 23 kDa; the other, 25 kDa. Reduction of the protein by dithiothreitol or 2-mercaptoethanol heated in SDS produced only the 25-kDa monomer species. Essentially, no thiol groups were detected in the 23-kDa form, in which two cysteine residues appear to have been oxidized to form an intrasubunit disulfide. This indicates that Cys196 has a reactive sulfhydryl and appears to be a likely candidate for a mixed disulfide formation in vivo.     

A novel mechanism for regulation of vacuolar acidification.

We have recently demonstrated that Cys-254 of the 73-kDa A subunit of the clathrin-coated vesicle (H+)-ATPase is responsible for sensitivity of the enzyme to sulfhydryl reagents (Feng, Y., and Forgac, M. (1992) J. Biol. Chem. 267, 5817-5822). In the present study we observe that for the purified enzyme, disulfide bond formation causes inactivation of proton transport which is reversed by dithiothreitol (DTT). DTT also restores activity of the oxidized enzyme following treatment with N-ethylmaleimide (NEM). These results indicate that disulfide bond formation between the NEM-reactive cysteine (Cys-254) and a closely proximal cysteine residue leads to inactivation of the (H+)-ATPase. To test whether sulfhydryl-disulfide bond interchange may play a role in regulating vacuolar acidification in vivo, we have determined what fraction of the (H+)-ATPase is disulfide-bonded in native clathrin-coated vesicles. Vesicles were isolated under conditions that prevent any change in the oxidation state of the sulfhydryl groups. NEM treatment of vesicles causes nearly complete loss of activity while subsequent treatment with DTT restores 50% of the activity of the fully reduced vesicles. By contrast, treatment of fully reduced vesicles with NEM leads to inactivation which is not reversed by DTT. These results indicate that a significant fraction of the clathrin-coated vesicle (H+)-ATPase exists in an inactive, disulfide-bonded state and suggest that sulfhydryl-disulfide bond interconversion may play a role in controlling vacuolar (H+)-ATPase (V-ATPase) activity in vivo.     

Inactivation of plasminogen activator inhibitor by oxidants

The rapidly acting plasminogen activator inhibitor (PAI) purified from cultured bovine aortic endothelial cells (BAEs) was inactivated during iodination with chloramine T and other oxidizing iodination systems. Inactivation was observed in the absence of iodine, suggesting that the loss of activity resulted from the oxidizing conditions employed. In an attempt to further study the nature of this inactivation, the PAI was treated with chloramine T under conditions that specifically oxidize methionine and cysteine residues. Both PAI inhibitory activity and the ability of the PAI to form complexes with tissue-type PA were decreased in a dose-dependent manner by such treatment. The PAI was more sensitive to oxidative inactivation than urokinase, elastase, and alpha 1-protease inhibitor. Incubation of the chloramine T inactivated PAI with methionine sulfoxide peptide reductase in the presence of dithiothreitol (DTT) restored more than 90% of the PAI activity. The reductase is a DTT-dependent enzyme that specifically converts methionine sulfoxide to methionine. Little activity was restored by either the reductase or DTT alone. These results indicate that the oxidation of at least one critical methionine residue is responsible for the loss of PAI activity upon iodination. In this respect, the BAE PAI resembles alpha 1-protease inhibitor, a well-characterized elastase inhibitor that also is inactivated by oxidants. Both inhibitors are members of the serine protease inhibitor superfamily (Serpins), and both have a methionine residue in their reactive center.     

A purification strategy for inositol 1,4,5-trisphosphate 3-kinase from rat liver based upon heparin interaction chromatography.

Rat liver inositol 1,4,5-trisphosphate [Ins (1,4,5)P3] 3-kinase was purified in high yield by a three-step procedure reliant upon chromatography on heparin and calmodulin agarose. Purified enzyme was stable in the presence of the detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS) (0.1-0.5%) and the sulphydryl reducing reagent dithiothreitol (DTT). The purified enzyme was activated 2-3-fold by Ca2+ (1 microM) in the presence of calmodulin. Pyrophosphate and heparin were identified as inhibitors of the enzyme.     

Identification and characterization of 3-substituted pyrazolyl esters as alternate substrates for cathepsin B: the confounding effects of DTT and cysteine in biological assays

Substituted pyrazole esters were identified as hits in a high throughput screen (HTS) of the NIH Molecular Libraries Small Molecule Repository (MLSMR) to identify inhibitors of the enzyme cathepsin B. Members of this class, along with functional group analogs, were synthesized in an effort to define the structural requirements for activity. Analog characterization was hampered by the need to include a reducing agent such as dithiothreitol (DTT) or cysteine in the assay, highlighting the caution required in interpreting biological data gathered in the presence of such nucleophiles. Despite the confounding effects of DTT and cysteine, our studies demonstrate that the pyrazole 1 acts as alternate substrate for cathepsin B, rather than as an inhibitor.     

Isoquinoline-1,3,4-trione derivatives inactivate caspase-3 by generation of reactive oxygen species

Caspase-3 is an attractive therapeutic target for treatment of diseases involving disregulated apoptosis. We report here the mechanism of caspase-3 inactivation by isoquinoline-1,3,4-trione derivatives. Kinetic analysis indicates the compounds can irreversibly inactivate caspase-3 in a 1,4-dithiothreitol (DTT)- and oxygen-dependent manner, implying that a redox cycle might take place in the inactivation process. Reactive oxygen species detection experiments using a chemical indicator, together with electron spin resonance measurement, suggest that ROS can be generated by reaction of isoquinoline-1,3,4-trione derivatives with DTT. Oxygen-free radical scavenger catalase and superoxide dismutase eliciting the inactivation of caspase-3 by the inhibitors confirm that ROS mediates the inactivation process. Crystal structures of caspase-3 in complexes with isoquinoline-1,3,4-trione derivatives show that the catalytic cysteine is oxidized to sulfonic acid (-SO(3)H) and isoquinoline-1,3,4-trione derivatives are bound at the dimer interface of caspase-3. Further mutagenesis study shows that the binding of the inhibitors with caspase-3 appears to be nonspecific. Isoquinoline-1,3,4-trione derivative-catalyzed caspase-3 inactivation could also be observed when DTT is substituted with dihydrolipoic acid, which exists widely in cells and might play an important role in the in vivo inactivation process in which the inhibitors inactivate caspase-3 in cells and then prevent the cells from apoptosis. These results provide valuable information for further development of small molecular inhibitors against caspase-3 or other oxidation-sensitive proteins.     

Characterization of 73 kDa thiol protease from Serratia marcescens and its effect on plasma proteins

The 73-kDa protease (73K protease) was purified from a clinical isolate of Serratia marcescens kums 3958. The purified protease appeared homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence or absence of 2-mercaptoethanol. The protease is active in a broad pH range with maximum activity at pH 7.5-8.0. The protease appeared to be a thiol protease, since it was inhibited by sulfhydryl reactive compounds such as p-chloromercuribenzoic acid, fluorescein mercuric acetate (FMA), iodoacetamide, and N-ethylmaleimide, and the protease activity was enhanced by various reducing agents such as cysteine, glutathione, 2-mercaptoethanol, and dithiothreitol. The protease contained 2 mol of free sulfhydryl residues per mol of protease. When the protease was reacted with FMA, a maximum of 2 mol of FMA per mol of enzyme was found reacted, based on fluorescence quenching in which the enzyme inactivation was paralleled linearly with the loss of both SH groups. This indicates possible equal involvement of the two thiol groups for the enzyme activity. The inactivation of the protease by FMA was partially restored by a dialysis in the presence of cysteine or dithiothreitol. The protease was not inhibited by high molecular weight kininogen but was inhibited by alpha 2-macroglobulin. The protease bound stoichiometrically to alpha 2-macroglobulin with 1:1 molar ratio and 25% activity remained constant even after the addition of 4 molar excess of alpha 2-macroglobulin. The protease extensively degraded IgG, IgA, fibronectin, fibrinogen, and alpha 1-protease inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of jack bean urease by N-ethylmaleimide: pH dependence, reversibility and thiols influence

N-Ethylmaleimide (NEM) was studied as an inactivator of jack bean urease at 25 degrees C in 20 mM phosphate buffer, pHs 6.4, 7.4, and 8.3. The inactivation was investigated by incubation procedure in the absence of a substrate. It was found that NEM acted as a time and concentration dependent inactivator of urease. The dependence of urease residual activity on the incubation time showed that the activity decreased with time until the total loss of enzyme activity. The process followed a pseudo-first-order reaction. A monophasic loss of enzyme activity was observed at pH 7.4 and 8.4, while a biphasic reaction occurred at pH 6.4. Moreover, the alkaline pH promoted the inactivation. The presence of thiol-compounds, such as L-cysteine, glutathione or dithiothreitol (DTT), in the incubation mixture significantly slowed down the rate of inactivation. The interaction test showed that the decrease of inactivation was an effect of NEM-thiol interaction that lowered NEM concentration in the incubation mixture. The reactivation of NEM-blocked urease by DTT application and multidilution did not result in an effective activity regain. The applied DTT reacted with the remaining inactivator and could stop the progress of enzyme activity loss but did not cause the reactivation. This confirmed the irreversibility of inactivation. Similar results obtained at pH 6.4, 7.4 and 8.4 indicated that the mechanism of urease inactivation by NEM was pH-independent. However, the pH value significantly influenced the process rate.     

Inactivation of jack bean urease by N-ethylmaleimide: pH dependence,reversibility and thiols influence

N-Ethylmaleimide (NEM) was studied as an inactivator of jack bean urease at 25 °C in 20 mM phosphate buffer, pHs 6.4, 7.4, and 8.3. The inactivation was investigated by incubation procedure in the absence of a substrate. It was found that NEM acted as a time and concentration dependent inactivator of urease. The dependence of urease residual activity on the incubation time showed that the activity decreased with time until the total loss of enzyme activity. The process followed a pseudo-first-order reaction. A monophasic loss of enzyme activity was observed at pH 7.4 and 8.4, while a biphasic reaction occurred at pH 6.4. Moreover, the alkaline pH promoted the inactivation. The presence of thiol-compounds, such as L-cysteine, glutathione or dithiothreitol (DTT), in the incubation mixture significantly slowed down the rate of inactivation. The interaction test showed that the decrease of inactivation was an effect of NEM-thiol interaction that lowered NEM concentration in the incubation mixture. The reactivation of NEM-blocked urease by DTT application and multidilution did not result in an effective activity regain. The applied DTT reacted with the remaining inactivator and could stop the progress of enzyme activity loss but did not cause the reactivation. This confirmed the irreversibility of inactivation. Similar results obtained at pH 6.4, 7.4 and 8.4 indicated that the mechanism of urease inactivation by NEM was pH-independent. However, the pH value significantly influenced the process rate.     

A study of dithiothreitol inactivation of the enzyme rhodanese.

When air oxidized, partially inactivated rhodanese (EC 2.8.1.1) is treated with dithiothreitol (DTT) to regenerate the reduced essential sulfhydryl group there is an initial reactivation followed by an anomalous slower inactivation. Fully active enzyme shows only inactivation. The inactivated enzyme may be completely reactivated on long incubation with the substrate thiosulfate ion. None of the normal potentialities of DTT appear to be responsible for the inactivation. The results are interpreted in terms of disulfide formation between DTT and an essential enzymic sulfhydryl group with the resulting complex being stabilized by secondary interactions which are particularly favorable due to similarities between DTT and lipoic acid--a normal sulfur acceptor substrate.     

Binding of 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole to an Essential Cysteine Residue(s) in the Tonoplast H-ATPase from Mung Bean (Vigna radiata L.) Hypocotyls

Vacuolar-type H⁺-ATPase was solubilized from tonoplasts of mung bean (Vigna radiata L.) and purified on a Mono Q anion-exchange column by fast protein liquid chromatography. The purified enzyme was inactivated by the reactive adenine analog, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). This inactivation was reversed by addition of dithiothreitol (DTT). Inactivation by NBD-Cl was prevented by Mg-ADP, a competitive inhibitor of ATPase. [¹⁴C]NBD-Cl predominantly modified the 68-kilodalton subunit and the degree of ¹⁴C incorporation was decreased in the presence of Mg-ADP or upon subsequent addition of DTT. The loss of activity followed pseudo first-order kinetics with respect to NBD-Cl concentration, and double log plots of pseudo first-order rate constants versus reagent concentration yielded a straight line with a slope of 0.957. The NBD-modified/inactivated enzyme showed an absorbance maximum at 418 nanometers and a fluorescence emission peak at 515 nanometers. The absorption and fluorescence emission spectra of the NBD-modified enzyme were essentially the same as those of the model compound, N-acetyl-S-NBD cysteine. Absorbance by the modified enzyme at 418 nanometers disappeared upon addition of DTT, which coincided with the restoration of ATPase activity and the decrease in bound [¹⁴C]NBD-Cl. These findings show that NBD-Cl modifies an essential cysteine residue(s) at or near the catalytic site in the 68-kilodalton subunit of tonoplast H⁺-ATPase and that the modification closely correlates with the loss of ATPase activity.     

E. coli propionyl-CoA synthetase is regulated in vitro by an intramolecular disulfide bond

The E. coli propionyl-CoA synthetase (PCS) was cloned, expressed, purified, and analyzed. Kinetic analyses suggested that the enzyme preferred propionate as substrate but would also use acetate. The purified, stored protein had relatively low activity but was activated up to about 10-fold by incubation with dithiothreitol (DTT). The enzyme activation by DTT was reversed by diamide. This suggests that the protein contains a regulatory disulfide bond and that the reduction to two sulfhydryl groups activates PCS while the oxidation to a disulfide leads to its inactivation. This idea was tested by sequential mutagenesis of the 9 Cys in the protein to Ala. It was revealed that the C128A and C315A mutants had wildtype enzyme activity but were no longer activated by DTT or inhibited by diamide. The data obtained indicate that two Cys residues could be involved in redox-regulated system through formation of an intramolecular disulfide bridge in PCS.     

Mechanism of Inactivation of Bacteriophage J 1 by Dithiothreitol,Cysteine, Mercaptoethanol,or Thioglycollate

The mechanism of inactivation of a double-stranded DNA phage, phage J1 of Lactobacilluscasei, by reducing agents containing thiol group(s) other than glutathione was studied mainly with dithiothreitol (DTT).

Air bubbling, oxidizing agents, and transition metal ions enhanced the rate of phage inactivation by DTT. Partial oxidation of DTT resulted in a more rapid rate of phage inactivation. In contrast, nitrogen bubbling, reducing agents including high concentrations of DTT itself, chelating agents, and radical scavengers prevented phage inactivation. Fully oxidized DTT had no phagocidal effect. These results indicate that the inactivating effect of DTT requires the presence of molecular oxygen and is indirectly caused by free radicals involved in the mechanism of DTT oxidation. The target attacked by DTT in phage particle was not protein but DNA; DTT reacted with DNA to produce single-strand scissions in DNA, which were the cause of inactivation of phage.

This was true also for L-cysteine, 2-mercaptoethanol, and thioglycollate.

Possible mechanisms by which these thiols fail to inactivate phage at high thiol concentrations are also discussed.     

E. coli propionyl-CoA synthetase is regulated in vitro by an intramolecular disulfide bond

The E. coli propionyl-CoA synthetase (PCS) was cloned, expressed, purified, and analyzed. Kinetic analyses suggested that the enzyme preferred propionate as substrate but would also use acetate. The purified, stored protein had relatively low activity but was activated up to about 10-fold by incubation with dithiothreitol (DTT). The enzyme activation by DTT was reversed by diamide. This suggests that the protein contains a regulatory disulfide bond and that the reduction to two sulfhydryl groups activates PCS while the oxidation to a disulfide leads to its inactivation. This idea was tested by sequential mutagenesis of the 9 Cys in the protein to Ala. It was revealed that the C128A and C315A mutants had wildtype enzyme activity but were no longer activated by DTT or inhibited by diamide. The data obtained indicate that two Cys residues could be involved in redox-regulated system through formation of an intramolecular disulfide bridge in PCS.     

Dithiothreitol inactivation of frog epidermis tyrosinase.

Frog epidermis tyrosinase inactivation by dithiothreitol (DTT), both in the proenzyme and active forms, have been studied. Upon increasing DTT:enzyme-up to 1o(6):1 ratios and depending on the incubation period, two inactivation steps both in proenzyme and enzyme were observed. Enzyme lost its activity faster than proenzyme. Oxygen favoured inactivation. After dialysis of the DTT:protein (10(6):1) incubation medium, 20% of the original enzyme activity was recovered. However it decreased to 15% if the enzyme had been incubated with substrate. Conformational changes due to loss of activity were not shown on the fluorescence spectra.     

The role of disulfide bonds in the conformational stability and catalytic activity of phytase.

Previous studies have predicted five disulfide bonds in Aspergillus niger phytase (phy A). To investigate the role of disulfide bonds, intrinsic fluorescence spectra, far-ultraviolet circular dichroism (CD) spectra, and an enzyme activity assay were used to compare the differences of catalytic activity and conformational stability of phytase during denaturation in urea in the presence and absence of dithiothreitol (DTT). In the presence of 2 mM DTT, the inactivation and unfolding were greatly enhanced at the same concentration of denaturant. The fluorescence emission maximum red shift and decreases of ellipticity at 222 nm were in accord with the changes of catalytic activity. The kinetics of the unfolding courses were a biphasic process consisting of two first-order reactions in the absence of DTT and a monophasic process of a first-order reaction in the presence of DTT. The results suggested that the loss of enzymatic activity was most likely because of a conformational change, and that disulfide bonds played an important role in three-dimensional structure and catalytic activity.     

Reactive nitrogen species derived activation of rat liver microsomal glutathione S-transferase

The effect of reactive nitrogen species on rat liver microsomal glutathione S-transferase (MGST1) was investigated using microsomes and purified MGST1. When microsomes or the purified enzyme were incubated with peroxynitrite (ONOO(-)), the GST activity was increased to 2.5-6.5 fold in concentration-dependent manner and a small amount of the MGST1 dimer was detected. MGST1 activity was increased by ONOO(-) in the presence of high amounts of reducing agents including glutathione (GSH) and the activities increased by ONOO(-) or ONOO(-) plus GSH treatment were decreased by 30-40% by further incubation with dithiothreitol (DTT, reducing disulfide) or by sodium arsenite (reducing sulfenic acid). Furthermore, GSH was detected by HPLC from the MGST1 which was incubated with ONOO(-) plus GSH or S-nitrosoglutathione followed by DTT treatment. In addition, the MGST1 activity increased by nitric oxide (NO) donors such as S-nitrosoglutathione, S-nitrosocysteine or the non-thiol NO donor 1-hydroxy-2-oxo-3 (3-aminopropyl)-3-isopropyl was restored by the DTT treatment. Since DTT can reduce S-nitrosothiol and disulfide bond to thiol, S-nitrosylation and a mixed disulfide bond formation of MGST1 were suggested. Thus, it was demonstrated that MGST1 is activated by reactive nitrogen species through a forming dimeric protein, mixed disulfide bond, nitrosylation and sulfenic acid.     

Stabilization and protection of an enzymatic system, participating in the transformation of beta-carotene into retinal, during its isolation]

The authors studied the effect of dithiothreitol (DTT), carotenoids and protease inhibitors on stabilization and protection of the enzyme catalysing the conversion of beta-carotene into retinal during the enzyme isolation from the rabbit small intestine. The addition of 1 mM DTT into the homogenization mixture increased the activity of the enzyme 5 times compared with control. The additional introduction of 0.7 mg/ml soybean trypsin inhibitor or 2.10(-4) M phenylmethylsulfonyl fluoride increased the enzyme activity 2.1 and 1.2 times, respectively. Lutein, beta-carotene and lycopene at a concentration of 10 mg/ml increased the enzyme activity 2.1, 1.9 and 1.6 times respectively. The effects of DTT, lutein and the protease inhibitor depended on their concentrations and was of an independent additive character. The maximum activity of the isolated enzyme exceeded the control without DTT 15 times.