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Results for “Diethyl Pyrocarbonate”

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At least 19 recordsLinked to original sources

PubMed1989

Identification by proton nuclear magnetic resonance of the histidines in cytochrome b5 modified by diethyl pyrocarbonate.

Diethyl pyrocarbonate (DEP) is an electrophilic reagent that is used to modify reversibly the histidine residues of proteins. Unfortunately, the lability of the acylated histidine adduct usually does not permit the isolation and identification of the modified histidine. By use of 500-MHz proton NMR spectroscopy, it has been possible to identify the C-H resonances of the nonaxial histidines of trypsin-solubilized bovine, rabbit, and porcine cytochrome b5 and therefore observe the interaction of DEP with specific histidine residues of cytochrome b5. In addition, the pKa of the peripheral histidines of bovine and rabbit cytochrome b5 have been measured in D2O. In the bovine protein it was found that the histidines are modified sequentially with increasing DEP concentration in the order His-26 greater than His-15 greater than His-80. This order is maintained in the rabbit protein with the following additions: His-26 approximately His-27 greater than His-15 greater than or equal to His-17 greater than His-80. The relative reactivity of the peripheral histidines with DEP was rationalized by considering three of their characteristics: (1) the pKa of the histidine, (2) the fraction of the side chain exposed to the solvent, and (3) the hydrogen-bond interactions of the imidazole ring.

Animals↗

PubMed1991

Chemical modification of Pseudomonas ochraceae 4-hydroxy-4-methyl-2-oxoglutarate aldolase by diethyl pyrocarbonate.

Diethyl pyrocarbonate inactivates Pseudomonas ochraceae 4-hydroxy-4-methyl-2-oxoglutarate aldolase [4-hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase: EC 4.1.3.17] by a simple bimolecular reaction. The inactivation is not reversed by hydroxylamine. The pH curve of inactivation indicates the involvement of a residue with a pK of 8.8. Several lines of evidence show that the inactivation is due to the modification of epsilon-amino groups of lysyl residues. Although histidyl residue is also modified, this is not directly correlated to the inactivation. No cysteinyl, tyrosyl, or tryptophyl residue or alpha-amino group is significantly modified. The modification of three lysyl residues per enzyme subunit results in the complete loss of aldolase activity toward various 4-hydroxy-2-oxo acid substrates, whereas oxaloacetate beta-decarboxylase activity associated with the enzyme is not inhibited by this modification. Statistical analysis suggests that only one of the three lysyl residues is essential for activity. l-4-Carboxy-4-hydroxy-2-oxoadipate, a physiological substrate for the enzyme, strongly protects the enzyme against inactivation. Pi as an activator of the enzyme shows no specific protection. The molecular weight of the enzyme, Km for substrate or Mg2+, and activation constant for Pi are virtually unaltered after modification. These results suggest that the modification occurs at or near the active site and that the essential lysyl residue is involved in interaction with the hydroxyl group but not with the oxal group of the substrate.

Amino Acids↗

PubMed1975

Cyclization of uridine monophosphate by diethyl pyrocarbonate.

Reaction between diethyl pyrocarbonate and uridine 2'-phosphate or uridine 3'-phosphate leads to the formation in high yields of uridine 2':3'-cyclic phosphate. This reaction product was identified in experiments involving (a) ultraviolet spectrophotometry, (b) paper chromatography, (c) high voltage paper electrophoresis at both pH 3.5 and 7.4, (d) acid hydrolysis, and (e) digestion with pancreatic ribonuclease.

Alkaline Phosphatase↗

PubMed1991

On the role of histidine residues in cyclodextrin glycosyltransferase: chemical modification with diethyl pyrocarbonate.

Ethoxyformylation with diethyl pyrocarbonate of approximately 1.5 His residues per molecule of enzyme reduced the cyclising activity of both the alpha-cyclodextrin glycosyltransferase from Klebsiella pneumoniae strain M 5 al and the beta-cyclodextrin glycosyltransferase from Bacillus circulans strain 8 by greater than 90%. Pre-incubation with substrate protected the enzymes from ethoxyformylation. Digestion of starch by the modified enzymes resulted in a delayed formation of cyclodextrins (cyclomalto-oligosaccharides, CDs), but a marked increase in the production of reducing saccharides. Similarly, coupling of alpha CD and maltose and successive disproportionation yielded mainly glucose and malto-oligosaccharides. The results are discussed in the context of the role of conserved His residues for binding of substrate and the transfer reactions.

Amino Acid Sequence↗

PubMed1992

Effect of diethyl pyrocarbonate modification on spectral and steady-state kinetic properties of bovine heart cytochrome oxidase.

The histidine-specific reagent diethyl pyrocarbonate has been used to chemically modify bovine heart cytochrome oxidase. Thirty-two of sixty-seven histidine residues of cytochrome oxidase are accessible to modification by diethyl pyrocarbonate. Effects on the Soret and alpha bands of the heme spectrum indicate disturbance in the environment of one or both of the heme groups. However, diethyl pyrocarbonate modification does not alter the 830-nm absorbance band, suggesting that the environment of CuA is unchanged. Maximal modification of cytochrome oxidase by diethyl pyrocarbonate results in loss of 85-90% of the steay-state electron transfer activity, which can be reversed by hydroxylamine treatment. However, modification of the first 20 histidines does not alter either activity or the heme spectrum, but only when 32 residues have been modified are the activity and heme spectral changes complete. The steady-state kinetic profile of fully modified oxidase is monophasic; the phase corresponding to tight cytochrome c binding and low turnover is retained, whereas the high turnover phase is abolished. Proteoliposomes incorporated with modified oxidase have a 65% lower respiratory control ratio and 40% lower proton pumping stoichiometry than liposomes containing unmodified oxidase. These results are discussed in terms of a redox-linked proton pumping model for energy coupling via cytochrome oxidase.

Animals↗

PubMed1976

Purification of RNA from animal cells using diethyl-pyrocarbonate.

Extraction of RNA from animal cells by a method using diethyl-pyrocarbonate yielded 50-60% of the total RNA. RNA purified by a hot phenol-SDS method from adenovirus 2 infected cells showed about 9% homology with adenovirus DNA, and RNA purified by diethyl-pyrocarbonate-SDS showed over 7% hybridization. Profiles of RNA prepared by both methods were identical when studied by polyacrylamide gel electrophoresis.

Adenoviridae↗

PubMed1977

Modification of lactate oxidase with diethyl pyrocarbonate. Evidence for an active-site histidine residue.

1. Diethyl pyrocarbonate inactivated l-lactate oxidase from Mycobacterium smegmatis. 2. Two histidine residues underwent ethoxycarbonylation when the enzyme was treated with sufficient reagent to abolish more than 90% of the enzyme activity, but analyses of the inactivation showed that the modification of one histidine residue was sufficient to cause the loss of enzyme activity. The rates of enzyme inactivation and histidine modification were the same. 3. Substrate and competitive inhibitors decreased the maximum extent of inactivation to a 50% loss of enzyme activity and modification was decreased from 1.9 to 0.75-1.2 histidine residues modified/molecule of FMN. 4. Treatment of the enzyme with diethyl [(14)C]pyrocarbonate (labelled in the carbonyl groups) confirmed that only histidine residues were modified under the conditions used and that deacylation of the ethoxycarbonylhistidine residues by hydroxylamine was concomitant with the removal of the (14)C label and the re-activation of the enzyme. 5. No evidence was found for modification of tryptophan, tyrosine or cysteine residues, and no difference was detected between the conformation and subunit structure of the modified and native enzyme. 6. Modification of the enzyme with diethyl pyrocarbonate did not alter the following properties: the binding of competitive inhibitors, bisulphite and substrate or the chemical reduction of the flavin group to the semiquinone or fully reduced states. The normal reduction of the flavin by lactate was, however, abolished.

Binding, Competitive↗

PubMed1991

Kidney brush-border membrane transporters: differential sensitivity to diethyl pyrocarbonate.

The effects of the histidine modifier, diethyl pyrocarbonate (DEPC), on brush-border membrane transport systems were studied in rat kidney. DEPC caused a strong inhibition of sodium-dependent phosphate and D-glucose uptake. Phosphate uptake remained linear up to 10 s in control and DEPC-treated membrane vesicles. The D-glucose carrier was more sensitive than the phosphate carrier with half-times of inhibition being 4 and 7 min, respectively. Sodium-independent phosphate and D-glucose uptake remained unaffected by DEPC. Intravesicular volume and two enzyme activities endogenous to the luminal membrane (alkaline phosphatase and aminopeptidase M) remained unaffected by DEPC. Increasing the preincubation pH from 5 to 9 increased phosphate transport inhibition caused by DEPC from 73 to 88% in the presence of DEPC. Hydroxylamine was able to completely reverse phosphate uptake inhibition by DEPC (100%), but only partially reversed the D-glucose uptake inhibition (16%). Sodium or substrate (D-glucose or phosphate) in the preincubation media were unable to protect their respective carriers from DEPC. Sodium-dependent transport of L-glutamine, L-phenylalanine, L-leucine, L-alanine, L-glycine, beta-alanine and L-proline were inhibited at different levels ranging from 70 to 90%. Three transport processes were found insensitive to DEPC modification: L-glutamate, L-lysine and D-fructose. None of the amino acid transporters was protected against DEPC by sodium and/or their respective substrates. Sodium influx was inhibited by DEPC (47%) in the absence of any substrate. Our results show a differential sensitivity of sodium-dependent transporters to DEPC and suggest an important role for histidine residues in the molecular mechanisms of these transporters. More experiments are in progress to further characterize the residue(s) involved in these transport inhibitions by DEPC.

Amino Acids↗

PubMed1991

Modification of histidine 56 in adrenodoxin with diethyl pyrocarbonate inhibited the interaction with cytochrome P-450scc and adrenodoxin reductase.

Three histidine residues of bovine adrenodoxin, His-10, His-56, and His-62, were modified with diethyl pyrocarbonate. The order of the modification among the three histidines were monitored by measuring the proton NMR spectra. The modified adrenodoxin exhibited reduced affinity for adrenodoxin reductase as determined in cytochrome c reductase activity. In the presence of cholesterol, the modified adrenodoxin induced a high spin form of cytochrome P-450scc on complex formation in the same manner as native adrenodoxin. The spectral titration showed that adrenodoxin modified with diethyl pyrocarbonate exhibited a 5-fold higher Kd value than that of native adrenodoxin. These effects of the modification of adrenodoxin on the affinities for the redox partners were not proportional to the number of modified histidines determined by the optical absorbance change at 240 nm. Modification of adrenodoxin up to 2 histidine residues did not affect the affinity for the redox partners, but further modification on the third one resulted in an increase of apparent Km in cytochrome c reductase activity by 2-fold and of Kd for cytochrome P-450scc by 5-fold. The 1H NMR spectra of the modified adrenodoxin unequivocally demonstrated that histidine residues at His-10 and His-62 reacted more readily with diethyl pyrocarbonate than His-56 did, indicating that modification of His-56 was responsible for the reduction of binding affinities of adrenodoxin for redox partners. These results are consistent with the proposal that the residue of His-56 in adrenodoxin has an essential role in the electron transfer mechanism where adrenodoxin functions as a mobile shuttle.

Adrenodoxin↗

PubMed1977

Binding of triethyltin to cat haemoglobin and modification of the binding sites by diethyl pyrocarbonate.

Cat haemoglobin binds 2 mol of triethyltin/mol of haemoglobin. Pretreatment of the haemoglobin with diethyl pyrocarbonate at pH6.0 prevents binding to one site only, whereas photo-oxidation with Methylene Blue removes both sites. Pretreatment of rat haemoglobin with diethyl pyrocarbonate also leads to the loss of one binding site. The possibility is discussed that the two binding sites for triethyltin on both cat and rat haemoglobin have a different chemical nature.

Animals↗

PubMed1978

Inhibition of ribonuclease. Efficacy of sodium dodecyl sulfate, diethyl pyrocarbonate, protein ase K and heparin using a sensitive ribonuclease assay.

The effectiveness of several commonly used inhibitors of ribonuclease (RNAase) has been studied using the removal of radio-labelled leucine from leucyl-tRNA as a sensitive assay for RNAase activity. The inhibitors were tested under a variety of conditions, varying the temperature, the pH, and the source of RNAase. When each inhibitor is udes separately in the presence of pancreatic RNAase, sodium dodecyl sulfate (SDS) is the most effective; but during long exposures to temperatures above 0 degrees C considerable amounts of RNA are still degraded. Combination of inhibitors are more effective in preserving RNA; with this assay, a combination of SDS with diethyl pyrocarbonate is the most effective. Proteinase K acts as an inhibitor when used in combination with SDS; however, it has RNAase activity when used by itself. Diethyl pyrocarbonate, when used at the high range of concentrations employed by others for RNAase inhibition, reacts with RNA changing its charge. However, when diethyl pyrocarbonate is used in smaller amounts the effects on RNA are minimal, and when used in combination with SDS it effectively inhibits RNAase.

Diethyl Pyrocarbonate↗

PubMed1990

Modification of uridine phosphorylase from Escherichia coli by diethyl pyrocarbonate. Evidence for a histidine residue in the active site of the enzyme.

Uridine phosphorylase from Escherichia coli is inactivated by diethyl pyrocarbonate at pH 7.1 and 10 degrees C with a second-order rate constant of 840 M-1.min-1. The rate of inactivation increases with pH, suggesting participation of an amino acid residue with pK 6.6. Hydroxylamine added to the inactivated enzyme restores the activity. Three histidine residues per enzyme subunit are modified by diethyl pyrocarbonate. Kinetic and statistical analyses of the residual enzymic activity, as well as the number of modified histidine residues, indicate that, among the three modifiable residues, only one is essential for enzyme activity. The reactivity of this histidine residue exceeded 10-fold the reactivity of the other two residues. Uridine, though at high concentration, protects the enzyme against inactivation and the very reactive histidine residue against modification. Thus it may be concluded that uridine phosphorylase contains only one histidine residue in each of its six subunits that is essential for enzyme activity.

Binding Sites↗

PubMed1979

The reaction of rabbit muscle creatine kinase with diethyl pyrocarbonate.

The reaction of rabbit muscle creatine kinase with diethyl pyrocarbonate was studied. It was found that up to five of the sixteen histidine groups per enzyme subunit could be modified, and under the conditions employed, there was no evidence for formation of the disubstituted derivative of histidine. Evidence was obtained for small but significant amounts of modification of lysine and cysteine groups; tyrosine groups were not modified. Modification of the enzyme led to inactivation; this could be protected against by inclusion of substrates or, more effectively, by inclusion of the combination MgADP plus creatine plus nitrate, which is thought to produce a 'transition-stage-analogue' complex. Analysis of data on the rates of inactivation and the stoicheiometry of modification suggested that there was one essential histidine group per enzyme subunit, modification of which led to inactivation.

Adenosine Diphosphate↗

PubMed1991

Substrate-decreased modification by diethyl pyrocarbonate of two histidines in isocitrate lyase from Escherichia coli.

The inactivation of tetrameric 188-kDa isocitrate lyase from Escherichia coli at pH 6.8 (37 degrees C) by diethyl pyrocarbonate, exhibiting saturation kinetics, is accompanied by modification of histidine residues 266 and 306. Substrates isocitrate, glyoxylate, or glyoxylate plus succinate protect the enzyme from inactivation, but succinate alone does not. Removal of the carbethoxy groups from inactivated enzyme by treatment with hydroxylamine restores activity of isocitrate lyase. The present results suggest that the group-specific modifying reagent diethyl pyrocarbonate may be generally useful in determining the position of active site histidine residues in enzymes.

Amino Acid Sequence↗

PubMed1990

Effect of diethyl pyrocarbonate modification on the calcium binding mechanism of the sarcoplasmic reticulum ATPase.

Diethyl pyrocarbonate was used to modify histidyl residues on the sarcoplasmic reticulum ATPase. Difference spectra of the N-carbethoxyhistidyl derivative indicated that most all the histidyl residues on the enzyme had been modified. These residues could be divided into two populations on the basis of their reaction rate with the reagent. It could then be shown that enzyme inhibition followed modification of the slower reacting population. Reversal with hydroxylamine verified that the loss of activity was due specifically to histidyl modification. Using [32P]ATP as a substrate it was further determined that the modified ATPase could form a phosphoenzyme intermediate, but that the hydrolysis of this intermediate was inhibited. Size exclusion chromatography was used to obtain equilibrium binding curves for high affinity Ca2+ sites on the enzyme. With the normal ATPase a cooperative binding curve for two Ca2+ with a Hill coefficient of 1.8 was observed. With the modified ATPase binding to two independent sites was observed; however, the dissociation constants remained the same as in the cooperative mechanism (K1 = 14 microM; K2 = 0.5 microM). That is, modification had eliminated cooperativity without changing the site specific binding affinities. E-P formation was then shown to follow binding to the higher affinity of the two sites. This would be the second site to bind Ca2+ in a sequential, cooperative mechanism. A model is suggested in which the binding of Ca2+ to an initial site allows for the binding of a second Ca2+ to an occluded site, this second site being responsible for enzyme activation. Modification apparently allows the binding properties of both sites to be observed independently.

Adenosine Triphosphatases↗

PubMed1988

Essential histidine residues in dextransucrase: chemical modification by diethyl pyrocarbonate and dye photo-oxidation.

Treatment of Leuconostoc mesenteroides B-512F dextransucrase with diethyl pyrocarbonate (DEP) at pH 6.0 and 25 degrees or photo-oxidation in the presence of Rose Bengal or Methylene Blue at pH 6.0 and 25 degrees, caused a rapid decrease of enzyme activity. Both types of inactivation followed pseudo-first-order kinetics. Enzyme partially inactivated by DEP could be completely reactivated by treatment with 100 mM hydroxylamine at pH 7 and 4 degrees. The presence of dextran partially protected the enzyme from inactivation. At pH 7 or below, DEP is relatively specific for the modification of histidine. DEP-modified enzyme showed an increased absorbance at 240 nm, indicating the presence of (ethoxyformyl)ated histidine residues. DEP modification of the sulfhydryl group of cysteine and of the phenolic group of tyrosine was ruled out by showing that native and DEP-modified enzyme had the same number of sulfhydryl and phenolic groups. DEP modification of the epsilon-amino group of lysine was ruled out by reaction at pH 6 and reactivation with hydroxylamine, which has no effect on DEP-modified epsilon-amino groups. The photo-oxidized enzyme showed a characteristic increase in absorbance at 250 nm, also indicating that histidine had been oxidized, and no decrease in the absorbance at 280 nm, indicating that tyrosine and tryptophan were not oxidized. A statistical, kinetic analysis of the data on inactivation by DEP showed that two histidine residues are essential for the enzyme activity. Previously, it was proposed that two nucleophiles at the active site attack bound sucrose, to give two covalent D-glucosyl-enzyme intermediates. We now propose that in addition, two imidazolium groups of histidine at the active site donate protons to the leaving, D-fructosyl moieties. The resulting imidazole groups then facilitate the formation of the alpha-(1----6)-glycosidic linkage by abstracting protons from the C-6-OH groups, and become reprotonated for the next series of reactions.

Carbohydrates↗

PubMed1990

Chemical modification of chalcone isomerase by diethyl pyrocarbonate: histidine residues are not essential for catalysis.

Chalcone isomerase form soybean is inactivated by treatment with diethyl pyrocarbonate (DEP). The competitive inhibitor 4',4-dihydroxychalcone provides kinetic protection against inactivation by DEP with a binding constant at the site of protection in agreement with its binding constant at the active site. Very high concentrations of the competitive inhibitors 4',4-dihydroxychalcone or morin hydrate offer a 10- to 40-fold maximal protection, suggesting a second slower mechanism for inactivation which cannot be prevented by blockage of the active site. Blockage of the only cysteine residue in chalcone isomerase with p-mercuribenzoate does not affect the rate constant for DEP-dependent inactivation and indicates that the modification of the cysteine residue is not responsible for the activity loss observed in the presence of DEP. Treatment of inactivated enzyme with hydroxylamine does not restore catalytic activity, indicating that the modification of histidine or tyrosine residues is not responsible for the activity loss. All five histidines of chalcone isomerase are modified by DEP at pH 5.7 and ionic strength 1.0 M. The rate constant for the modification of the histidine residues of chalcone isomerase is close to that for the reaction of N-acetyl histidine with DEP, indicating that the histidine residues are quite accessible to the modifying reagent. The rate of histidine modification is the same in native enzyme, in urea-denatured enzyme, and in the presence of a competitive inhibitor. In the presence of the competitive inhibitor morin hydrate, all of the histidine residues of chalcone isomerase can be modified without significant loss in catalytic activity. These results demonstrate that the histidine residues of chalcone isomerase are not essential for catalysis and therefore cannot function as nucleophilic catalysts as previously proposed.

Binding Sites↗

PubMed1992

Chemical modification of prostaglandin H synthase with diethyl pyrocarbonate.

The role of histidine in catalysis by prostaglandin H synthase has been investigated using chemical modification with diethyl pyrocarbonate (DEPC), an agent that has been found to rather selectively derivatize histidine residues in proteins under mild conditions. Incubation of the synthase apoprotein with DEPC at pH 7.2 resulted in a progressive loss of the capacity for both cyclooxygenase and peroxidase catalytic activities. The kinetics of inactivation of the cyclooxygenase activity were dependent on the concentration of DEPC; a second-order rate constant of 680 M-1 min-1 was estimated for reaction of the apoenzyme at pH 7.2 and 0 degrees C. The kinetics of inactivation of the cyclooxygenase by DEPC exhibited a sigmoidal dependence on the pH, indicating that deprotonation of a group with a pKa of 6.3 was required for inactivation. The presence of the heme prosthetic group slowed, but did not prevent, inactivation by DEPC. The stoichiometry of histidine modification of apoenzyme during inactivation determined from absorbance increases at 242 nm agreed well with the overall stoichiometry of derivatized residues determined with [14C]DEPC, indicating that modification by DEPC was quite selective for histidine residues on the synthase. Although modification of several histidine residues by DEPC was observed, only one of the histidine residues was essential for cyclooxygenase activity. Modification of the holoenzyme with DEPC altered the EPR signal of the hydroperoxide-induced tyrosyl free radical from the wide doublet (35 G, peak-to-trough) found with the native synthase to a narrower singlet (28 G, peak-to-trough) quite like that found in the indomethacin-synthase complex. Reaction of the indomethacin-synthase complex with DEPC was found to increase the cyclooxygenase velocity by 9 times its initial value, to about one-third of the uninhibited value, without displacement of the indomethacin; the peroxidase was significantly inactivated under the same conditions. Histidyl residues in the synthase are thus likely to have important roles not only in cyclooxygenase and peroxidase catalysis but also in the interaction of the synthase with indomethacin.

Acetylation↗