The nature of inactivation of rat muscle 5''-adenylate aminohydrolase by fluorodinitrobenzene.

1. The inactivation of rat skeletal muscle AMP deaminase by Dnp-F (1-fluoro-2,4-dinitrobenzene) is accompanied by the arylation of thiol, amino and phenolic hydroxyl groups. 2. The number of thiol groups that react with Dnp-F is about 12; this is the number that reacts with Nbs2 [5,5'-dithiobis-(2-nitrobenzoic acid)] and N-ethylmaleimide without loss of enzyme activity, and it appears to be the same thiol groups that all three reagents attack. 3. Dinitrophenylation of these reactive SH groups is not the cause of inactivation, since active N-ethylmaleimide-substituted enzyme is also inactivated by Dnp-F.4. Complete inactivation of the N-ethylmaleimide-treated AMP deaminase occurs when about six tyrosine and two lysine residues are dinitrophenylated. 5. Since the treatment of Dnp-enzyme with 2-mercaptoethanol restores much of the enzyme activity, inactivation of AMP deaminase by Dnp-F is probably largely due to modification of tyrosine residues. 6. The kinetic properties of the Dnp-enzyme indicate that a marked decrease in V occurs only after extensive enzyme modification. The decreased activity after slight inactivation results from modification of Km.     

Rapid-mixing studies of radiosensitivity with thiol-depleted mammalian cells

A moderate reduction in the non-protein thiol content of V79 379A Chinese hamster cells, obtained by pretreatment with buthionine sulphoximine (BSO), diethyl maleate (DEM) or N-ethyl maleimide (NEM), increase both the absolute radiosensitivity of the cells in hypoxia and the radiosensitizing effect of adding oxygen 7 ms after irradiation. Combined pretreatment of cells with BSO and NEM removes most of the non-protein thiol and some of the protein thiol; such treatment further increases the radiosensitivity of hypoxic cells but there is no further effect of adding oxygen 7 ms after irradiation. Addition of 2-mercaptoethanol to cells 7 ms after irradiation gives protection factors that increase with increasing severity of thiol depletion. Substantial radioprotection can still be observed when 2-mercaptoethanol is added 70 ms after irradiation of cells pretreated with BSO and NEM; there is no effect of adding 2-mercaptoethanol to such cells 50s after irradiation. These observations support the repair-fixation model of radiation damage and suggest that, in addition to the established role of non-protein thiol in chemical repair of radiation damage, other endogenous reducing agents such as protein thiol may be important in determining cellular radiosensitivity. A relatively long-lived thiol-modifiable component of radiation damage has been observed within hypoxic thiol-depleted cells.     

Repair of damage in double-stranded phi X174 (RF) DNA due to radiation-induced water radicals

Experiments in which the yields of radiation-induced OH and H radicals were varied, showed that both types of water radicals inactivate phi X174 RF DNA to about the same extent as measured by transfection of the (irradiated) DNA to E. coli wild-type spheroplasts. On the other hand, using spheroplasts prepared from E. coli strains, deficient in one of the proteins involved in excision DNA repair (uvrA- or uvrC-) or in post-replication repair (recA-), clear differences between damage originating from OH or H radical attack were found. Part of the radiation damage due to H radicals appeared to be repairable by an uvrA-gene-dependent repair mechanism, whereas this repair pathway does not play an important role in the case of OH radical damage. The reverse applies to uvrC-gene-dependent repair, which only affects OH radical damage (obtained under anoxic conditions), but has no influence on damage due to H radicals. Irradiation of double-stranded phi X174 (RF) DNA in the presence of oxygen however, yields damage--due to OH radicals only--which appeared not to be sensitive to either uvrC- or uvrA-gene-dependent repair. Furthermore, post-replication repair (recA) has only very little effect on the amount of inactivation by H or OH radicals, when irradiation is carried out under anoxic conditions. We did not find significant inactivation due to hydrated electrons, whether the biological activity was determined by use of wild-type spheroplasts or of strains deficient in excision or post-replication repair proteins.     

Effects of oxygen and sulphydryl-containing compounds on irradiated transforming DNA. II. Glutathione, cysteine and cysteamine

This paper extends our earlier observations on the effects of the sulphydryl (SH)-containing compound dithiothreitol (DTT) on the radiation response of Bacillus subtilis transforming DNA to three other SH-containing compounds-cysteamine, cysteine and glutathione (GSH). In general, all four compounds protect transforming DNA in a manner which is dependent on gassing conditions. In O2, the protection is consistent with the scavenging of OH radicals by the SH compounds, but in N2 there is additional protection which may be due to hydrogen atom donation from the SH compound to radiation-induced DNA lesions, a process which is blocked by O2. This additional protection in N2 results in an increase in the ratio of inactivation in the absence and presence of oxygen with increasing SH concentration to a maximum followed by a decrease at high SH concentrations. The maximum value of the ratio and the SH concentration at which it occurs depend on the SH compound. In particular, GSH appears to be significantly less efficient in the hydrogen-donation repair reaction with transforming DNA than are the other three SH compounds. Furthermore, on the basis of our results, we postulate the existence of a damage fixation process which occurs in the absence of O2, in competition with damage repair by SH compounds, and that this anoxic damage fixation occurs at a rate not less than 300 s-1. We also demonstrate here that the damage fixing reaction of O2 with transforming DNA radicals proceeds 200-fold faster than the competing repair reaction by hydrogen-donation from DTT.     

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.     

Interaction of glutathione transferase from horse erythrocytes with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole

7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole reacts with two thiol groups of the dimeric horse erythrocyte glutathione transferase at pH 5.0, with strong inactivation reversible on dithiothreitol treatment. The inactivation kinetic follows a biphasic pattern, similar to that caused by other thiol reagents as recently reported. Both S-methylglutathione and 1-chloro-2,4-dinitrobenzene protect the enzyme from inactivation. Analysis of the reactive SH group-containing peptide gives the sequence Ala-Ser-Cys-Leu-Tyr, identical with that of the peptide that contains the reactive cysteine 47 of the human placental transferase. In the presence of glutathione, the enzyme is not inactivated by this reagent, but it catalyzes its conjugation to glutathione. At higher pH values, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reacts with 2 tyrosines/dimer and lysines, as well as with cysteines. Reaction with lysine seems essentially without effect on activity; whether the reactive tyrosines are important for activity could not be determined using this reagent only. However, 2 tyrosines among the 4 that are nitrated by tetranitro-methane are important for activity.     

Stepwise modulation of ATPase activity, nucleotide trapping, and sliding motility of myosin S1 by modification of the thiol region with residues of increasing size

Rabbit muscle myosin S1 was modified either at SH1 alone or at both SH1 and SH2, using a series of alkylthiolating reagents of increasing size, designed for correlating gradually changing structural disturbances in the thiol region with functional impairments in the myosin head. The reagents were of the type H(CH(2))(n)()-S-NTB, (NTB = 2-nitro-5-thiobenzoate) (n = 1, 2, 5, 8, 9, 10, 11, and 12). Modification of only SH1 led to the expected activation of the Ca(2+)-ATPase, but only with small reagents, while reagents with n > or = 10 caused inhibition of the Ca(2+)-ATPase. Modification of both SH1 and SH2 showed the expected inhibition of Ca(2+)-ATPase but likewise allowed considerable residual Ca(2+)-ATPase activity if the residues were small. Trapping of the nucleotide, known to occur with cross-linking reagents, was seen also with monovalent reagents, provided their length exceeded n = 9 or 10. All S1 derivatives prepared in this study possessed an affinity for actin comparable to native S1 but lacked sliding motility in in vitro motility assays. The biochemical data of this study can be related to existing models of myosin S1 and recent structural data [Houdusse, A., Kalabokis, V. N., Himmel, D., Szent-Gy?rgyi, A. G., and Cohen, C. (1999) Cell 97, 459-470] by making the assumptions that modification at SH1 prevents the formation of the SH1 helix mandatory for the transmission of conformational energy and that mobility of the thiol region is a prerequisite for ATPase activity. Immobilization of the thiol region by residues of increasing size apparently leads to lower enzyme activity and, finally, to inhibition of nucleotide exchange.     

Deoxycytidylate hydroxymethylase: purification, properties, and the role of a thiol group in catalysis

Deoxycytidylate (dCMP) hydroxymethylase from Escherichia coli infected with a T-4 bacteriophage amber mutant has been purified to homogeneity. It is a dimer with a subunit molecular weight of 28,000. Chemical modification of the homogeneous enzyme with N-ethylmaleimide (NEM) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) leads to complete loss of enzyme activity. dCMP can protect the enzyme against NEM inactivation, but the dihydrofolate analogues methotrexate and aminopterin alone do not afford similar protection. Compared to dCMP alone, dCMP plus either methotrexate or aminopterin greatly enhances protection against NEM inactivation. DTNB inactivation is reversed by dithiothreitol. For both reagents, inactivation kinetics obey second-order kinetics. NEM inactivation is pH dependent with a pKa for a required thiol group of 9.15 +/- 0.11. Complete enzyme inactivation by both reagents involves the modification of one thiol group per mole of dimeric enzyme. There are two thiol groups in the totally denatured enzyme modified by either NEM or DTNB. Kinetic analysis of NEM inactivation cannot distinguish between these two groups; however, with DTNB kinetic analysis of 2-nitro-5-thiobenzoate release shows that enzyme inactivation is due to the modification of one fast-reacting thiol followed by the modification of a second group that reacts about 5-6-fold more slowly. In the presence of methotrexate, the stoichiometry of dCMP binding to the dimeric enzyme is 1:1 and depends upon a reduced thiol group. It appears that the two equally sized subunits are arranged asymmetrically, resulting in one thiol-containing active site per mole of dimeric enzyme.     

Inactivation of alcohol dehydrogenase by piroxicam-derived radicals

Alcohol dehydrogenase (ADH) was used as a marker molecule to clarify the mechanism of gastric mucosal damage as a side effect of using piroxicam. Piroxicam inactivated ADH during interaction of ADH with horseradish peroxidase and H₂O₂ (HRP-H₂O₂). The ADH was more easily inactivated under aerobic than anaerobic conditions, indicating participation by oxygen. Superoxide dismutase, but not hydroxyl radical scavengers, inhibited inactivation of ADH, indicating participation by superoxide. Sulfhydryl (SH) groups in ADH were lost during incubation of piroxicam with HRP-H₂O₂. Adding reduced glutathione (GSH) efficiently blocked ADH inactivation. Other SH enzymes, including creatine kinase and glyceraldehyde-3-phosphate dehydrogenase, were also inactivated by piroxicam with HRP-H₂O₂. Thus SH groups in the enzymes seem vulnerable to piroxicam activated by HRP-H₂O₂. Spectral change in piroxicam was caused by HRP-H₂O₂. ESR signals of glutathionyl radicals occurred during incubation of piroxicam with HRP-H₂O₂ in the presence of GSH. Under anaerobic conditions, glutathionyl radical formation increased. Thus piroxicam free radicals interact with GSH to produce glutathionyl radicals. Piroxicam peroxyl radicals or superoxide, or both, seem to inactivate ADH. Superoxide may be produced through interaction of peroxyl radicals with H₂O₂. Thus superoxide dismutase may inhibit inactivation of ADH through reducing piroxicam peroxyl radicals or blocking interaction of SH groups with O₂^-, or both. Other oxicam derivatives, including isoxicam, tenoxicam and meloxicam, induced ADH inactivation in the presence of HRP-H₂O₂.     

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)     

Radical-Induced Damage to Bovine Serum Albumin: Role of the Cysteine Residue

The reactions of cerium(IV) and the hydroxyl radical [generated from iron(ii)/H₂O₂] with bovine serum albumin (BSA) have been investigated by EPR spin trapping. With the former reagent a protein-derived thiyl radical is selectively generated; this has been characterized via the anisotropic EPR spectra observed on reaction of this radical with the spin trap DMPO. Blocking of the thiol group results in the loss of this species and the detection of a peroxyl radical, believed to be formed by reaction of oxygen with initially-generated, but undetected, carbon-centred radicals from aromatic amino acids. Experiments with a second spin trap (DBNBS) confirm the formation of these carbon-centred species and suggest that damage can be transferred from the thiol group to carbon sites in the protein. A similar transfer pathway can be observed when hydroxyl radicals react with BSA.

Further experiments demonstrate that the reverse process can also occur: when hydroxyl radicals react with BSA, the thiol group appears to act as a radical sink and protects the protein from denaturation and fragmentation through the transfer of damage from a carbon site to the thiol group. Thiol-blocked BSA is shown to be more susceptible to damage than the native protein in both direct EPR experiments and enzyme digestion studies. Oxygen has a similar effect, with more rapid fragmentation detected in its presence than its absence.     

Reactivity and specificity of alpha-dicarbonyl compounds towards essential aminoacyl residues of D-beta-hydroxybutyrate dehydrogenase from the inner mitochondrial membrane

The effects of a alpha-dicarbonyl chromophoric reagent: 4-hydroxy-3-nitrophenylglyoxal on the D-beta-hydroxybutyrate dehydrogenase have been compared to those of phenylglyoxal, a specific arginyl reagent in proteins. Both reagents inactivate irreversibly the enzyme. Kinetic experiments show that only one molecule of these reagents per molecule of enzyme is sufficient to inactivate the enzyme. The second order inactivation rate constant is more than 500 times higher with the chromophoric reagent than with phenylglyoxal. A pseudosubstrate (methylmalonate) in presence of coenzyme (NAD) strongly protects enzyme against inactivation by both reagents. Coenzyme alone has no effect on inactivation by phenylglyoxal while it protects whether inhibitor is the chromophoric reagent or N-ethylmaleimide: a thiol specific reagent. These results indicate: 1. That one arginyl residue is essential for D-beta-hydroxybutyrate dehydrogenase activity (experiments with phenylglyoxal). 2. That the presence of a nitro group on position 3 and a hydroxyl-group on position 4 strongly increase the reactivity of the alpha-dicarbonyl groups, but the specificity of the chemical reaction with arginyl residues seems to be lost for the benefit of cysteyl residues.     

Mechanisms of the irreversible inactivation of horseradish peroxidase caused by hydroxymethylhydroperoxide

Hydrogen peroxide efficiently protects horseradish peroxidase against inactivation by hydroxymethylhydroperoxide, whereas the hydrogen donor substrate guaiacol has little protective effect. These results, and direct studies of the effects of hydroxymethylhydroperoxide on peroxidase compound II, indicate that compound II and possibly also compound I are quite resistant to hydroxymethylhydroperoxide. The inactivation of peroxidase caused by hydroxymethylhydroperoxide seems to be due to a reaction of the peroxide with free Fe³⁺ peroxidase.Albumin, which protects thiol enzymes against hydroxymethylhydroperoxide, does not protect peroxidase. Neither does 2-mercaptoethanol, which also protects thiol enzymes, appear to hamper the inactivation reaction. This seems to indicate that the inactivation is not caused by a free radical released from hydroxymethylhydroperoxide.Hydroxymethylhydroperoxide inactivates peroxidase by attacking the hematin group, the ring of which is opened by way of at least three intermediates. The most stable intermediate is green (“compound IV”).H₂C₂ attacks the methemoglobin-peroxide compound faster than hydroxymethylhydroperoxide does. These reactions are much slower than the attack of hydroxymethylhydroperoxide on peroxidase.It is suggested that the inactivation of peroxidase takes place as a side reaction in the process of forming compound I from hydroxymethylhydroperoxide and peroxidase.     

Comparative effects of sulfhydryl reagents on membrane-bound and solubilized UDP-glucose:ceramide glucosyltransferase from Golgi membranes. Evidence for partial involvement of a thiol group in the nucleotide sugar binding site of the solubilized enzyme

We have studied the inactivation of membrane-bound and solubilized UDP-glucose:ceramide glucosyltransferase from Golgi membranes by various types of sulfhydryl reagents. The strong inhibition of the membrane-bound form by the non-penetrant mercurial-type reagents clearly corroborated the fact that in sealed and right-side-out Golgi vesicles the ceramide glucosyltransferase is located on the cytoplasmic face. No significant differences in the susceptibility to the various sulfhydryl reagents were noted when solubilized enzyme was assayed, showing that solubilization does not reveal other critical SH groups. The different results obtained must be interpreted with regard to several thiol groups, essential for enzyme activity. No protection by the substrate UDP-glucose against mercurial-type reagents was obtained indicating that these thiol groups were not located in the nucleotide sugar binding domain. A more thorough investigation of the thiol inactivation mechanism was undertaken with NEM (N-ethylmaleimide), an irreversible reagent. The time dependent inactivation followed first order kinetics and provided evidence for the binding of 1 mol NEM per mol of enzyme. UDP-Glucose protected partially against NEM inactivation, indicating that the thiol groups may be situated in or near the substrate binding domain. Inactivation experiments with disulfide reagents showed that increased hydrophobicity led to more internal essential SH groups which are not obviously protected by the substrate UDP-glucose, thus not implicated in the substrate binding domain, but rather related to conformational changes of the enzyme during the catalytic process.Abbreviations Chaps 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulfonate - Mops 4-morpholinepropanesulfonic acid - PC phosphatidylcholine - NEM N-ethylmaleimide - CPDS carboxypyridine disulfide (dithio-6,6-dinicotinic acid) - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - DTP dithiodipyridine - p-HMB para-hydroxymercuribenzoate - DTT dithiothreitol - BAL British anti-Lewisite (dimercaptopropanol) - Zw 3–14 Zwittergent 3–14     

Studies on regulatory functions of malic enzymes. VII. Structural and functional characteristics of sulfhydryl groups in NADP-linked malic enzyme from Escherichia coli W.

NADP-linked malic enzyme from Escherichia coli W contains 7 cysteinyl residues per enzyme subunit. The reactivity of sulfhydryl (SH) groups of the enzyme was examined using several SH reagents, including 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM). 1. Two SH groups in the native enzyme subunit reacted with DTNB (or NEM) with different reaction rates, accompanied by a complete loss of the enzyme activity. The second-order modification rate constant of the "fast SH group" with DTNB coincided with the second-order inactivation rate constant of the enzyme by the reagent, suggesting that modification of the "fast SH group" is responsible for the inactivation. When the enzyme was denatured in 4 M guanidine HCl, all the SH groups reacted with the two reagents. 2. Althoug the inactivation rate constant was increased by the addition of Mg2+, an essential cofactor in the enzyme reaction, the modification rate constant of the "fast SH group" was unaffected. The relationship between the number of SH groups modified with DTNB or NEM and the residual enzyme activity in the absence of Mg2+ was linear, whereas that in the presence of Mg2+ was concave-upwards. These results suggest that the Mg2+-dependent increase in the inactivation rate constant is not the result of an increase in the rate constant of the "fast FH group" modification. 3. The absorption spectrum of the enzyme in the ultraviolet region was changed by addition of Mg2+. The dissociation constant of the Mg2+-enzyme complex obtained from the Mg2+- dependent increment of the difference absorption coincided with that obtained from the Mg2+- dependent enhancement of NEM inactivation. 4. Both the inactivation rate constant and the modification rate constant of the "fast SH group" were decreased by the addition of NADP+. The protective effect of NADP+ was increased by the addition of Mg2+. Based on the above results, the effects of Mg2+ on the SH-group modification are discussed from the viewpoint of conformational alteration of the enzyme.     

Reactivity and pH dependence of thiol conjugation to N-ethylmaleimide: detection of a conformational change in chalcone isomerase

The reactivity of simple alkyl thiolates with N-ethylmaleimide (NEM) follows the Br?nsted equation, log kS- = log G + beta pK, with G = 790 M-1 min-1 and beta = 0.43. The rate constant for the reaction of the thiolate of 2-mercaptoethanol with NEM is 10(7) M-1 min-1, whereas the rate constant for the reaction of the protonated thiol is less than 0.0002 M-1 min-1. The intrinsic reactivity of the protonated thiol (SH) is over (5 X 10(10]-fold less than the thiolate (S-) and makes a negligible contribution to the reactivity of thiols toward NEM. The rate of NEM modification of chalcone isomerase was conveniently measured by following the concomitant loss in enzymatic activity. The pseudo-first-order rate constants for inactivation show a linear dependence on the concentration of NEM up to 200 mM and yield no evidence for noncovalent binding of NEM to the enzyme. Evidence is presented demonstrating that the modification of chalcone isomerase by NEM is limited to a single cysteine residue over a wide range of pH. Kinetic protection against inactivation and modification by NEM is provided by competitive inhibitors and supports the assignment of this cysteine residue to be at or near the active site of chalcone isomerase. The pH dependence of inactivation of the enzyme by NEM indicates a pK of 9.2 for the cysteine residue in chalcone isomerase. At high pH, the enzymatic thiolate is only (3 X 10(-5))-fold as reactive as a low molecular weight alkyl thiolate of the same pK, suggesting a large steric inhibition of reaction on the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unmasking of an essential thiol during function of the membrane-bound enzyme II of the phosphenolpyruvate beta-glucoside phosphotransferase system of Escherichia coli.

beta-Glucoside transport by phosphoenolpyruvate-hexose phosphotransferase system in Escherichia coli is inactivated in vivo by thiol reagents. This inactivation is strongly enhanced by the presence of transported substrates. In a system reconstituted from soluble and membrane-bound components, only the particulate component, the membrane-bound enzyme IIbgl appeared as the target of N-ethylmaleimide inaction. The same feature was found in the case of methyl-alpha-D-glucoside uptake via enzyme IIglc. It is shown that the sensitizing effect of substrates is specific and not generalized, methyl-alpha-D-glucoside only sensitizes enzyme IIglc and p-nitrophenyl-beta-D-glucoside only sensitizes enzyme IIbgl towards N-ethylmaleimide inactivation. The inactivation of enzyme IIbgl by thiol reagents is also promoted in vivo by fluoride inhibition of phosphoenolpyruvate synthesis. In toluene-treated bacteria, the presence of phosphoenolpyruvate protects against inactivation by thiol reagents of p-nitrophenyl-beta-D-glucoside phosphorylation. Both results suggest that the inactivator resistent form of enzyme IIbgl is an energized form of the enzyme.     

Glutamate decarboxylase from barley embryos and roots. General properties and the occurrence of three enzymic forms.

Glutamate decarboxylase in extracts of barley has a Km value for L-glutamate of 22 mM and is activated by the addition of pyridoxal phosphate by up to 3.5 times. Sucrose-density-gradient experiments indicate the presence of two enzyme forms with molecular weights 256000 and 120000. The lower-molecular-weight form appears to be relatively inactive and spontaneously associates to the higher-molecular-weight form on storage. The enzyme is inhibited by thiol reagents and the distribution of activity on density gradients is altered in favour of the lower-molecular-weight form by the presence of 2-mercaptoethanol. After removal of the 2-mercaptoethanol spontaneous association to the higher-molecular-weight form occurs. The presence of oxygen in the extraction buffer and in the water during imbibition leads to a relative increase in the higher-molecular-weight form compared with situations where oxygen is excluded. In contrast, glutamate decarboxylase in extracts of 3-day-old barley roots has a Km value for L-glutamate of 3.1 mM and is activated up to 10% by addition of pyridoxal phosphate. The root enzyme occurs as a single species with molecular weight 310000 and this is unaffected by 2-mercaptoethanol although thiol reagents do act as weak inhibitors. The molecular weight is also unaffected by the presence or absence of oxygen in the extraction buffers.     

Effect of thiol reagents on the activity of NADP-dependent malate dehydrogenase isolated from bovine adrenal cortex cytoplasm

NADP-dependent malate dehydrogenase was rapidly inactivated in the presence of mercurous chloride. Titration of malate dehydrogenase by 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) in a solution of 8 M urea revealed 18 SH groups per molecule of the enzyme. Eight sulphydryl groups reacted with DTNB in native malate dehydrogenase and their modification was not accompanied by a loss of the enzyme activity. The interaction of p-chloromercury benzoate (PCMB) with malate dehydrogenase resulted in a 70% decrease in the enzyme activity. The binding of the thiol reagents by the malate dehydrogenase molecule appreciably increased the Michaelis constant value for the substrate. In the presence of magnesium ions, NADP and malate did not affect the process of malate dehydrogenase modification by DTNB and did not protect the enzyme from the inactivation by PCMB. It is suggested from the data obtained that the sulphyryl groups are involved in maintaining the active conformation of the enzyme.