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PubMed2001

Mapping Cu(II) binding sites in prion proteins by diethyl pyrocarbonate modification and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometric footprinting.

Although Cu(II) ions bind to the prion protein (PrP), there have been conflicting findings concerning the number and location of binding sites. We have combined diethyl pyrocarbonate (DEPC)-mediated carbethoxylation, protease digestion, and mass spectrometric analysis of apo-PrP and copper-coordinated mouse PrP23-231 to "footprint" histidine-dependent Cu(II) coordination sites within this molecule. At pH 7.4 Cu(II) protected five histidine residues from DEPC modification. No protection was afforded by Ca(II), Mn(II), or Mg(II) ions, and only one or two residues were protected by Zn(II) or Ni(II) ions. Post-source decay mapping of DEPC-modified histidines pinpointed residues 60, 68, 76, and 84 within the four PHGGG/SWGQ octarepeat units and residue 95 within the related sequence GGGTHNQ. Besides defining a copper site within the protease-resistant core of PrP, our findings suggest application of DEPC footprinting methodologies to probe copper occupancy and pathogenesis-associated conformational changes in PrP purified from tissue samples.

Amino Acid Sequence↗
PubMed1988

Detection of intercalation-induced changes in DNA structure by reaction with diethyl pyrocarbonate or potassium permanganate. Evidence against the induction of Hoogsteen base pairing by echinomycin.

Binding of the bis-intercalators echinomycin and N,N'-di(9-acridinyl)spermidine or the mono-intercalators 9-aminoacridine and ethidium to DNA induces hypersensitivity of adenines towards reaction with diethyl pyrocarbonate. It is proposed that this hyperreactivity is due to the DNA helix unwinding and extension induced by intercalation, thereby exposing N7 in the major groove, and not as previously suggested to the formation of Hoogsteen base pairing. Hypersensitivity of thymines towards oxidation with permanganate is also induced upon binding of these drugs (especially the bis-intercalators) to DNA. This thymine hyperreactivity is both sequence- and intercalator-dependent, thereby indicating the potential of KMnO4 as a useful probe for analyzing the structure of intercalator-DNA complexes in solution.

Base Composition↗
PubMed1987

Reaction of 5-enol-pyruvoylshikimate-3-phosphate synthase with diethyl pyrocarbonate: evidence for an essential histidine residue.

5-enol-Pyruvoylshikimate-3-phosphate synthase catalyzes the reversible condensation of phosphoenolpyruvate and shikimate 3-phosphate to yield 5-enol-pyruvoylshikimate 3-phosphate and inorganic phosphate. The enzyme is a target for the nonselective herbicide glyphosate (N-phosphonomethylglycine). Diethyl pyrocarbonate inactivated this enzyme with a second-order rate constant of 220 M-1 min-1 at pH 7.0 and 0 degrees C. The rate of inactivation is pH dependent and the pH inactivation rate data show the involvement of a group with a pKa of 6.8. Almost all of the original activity was recovered by treatment of the inactivated enzyme with hydroxylamine. The difference spectrum of the inactivated and native enzyme reveals a single peak at 242 nm but no trough at around 278 nm is observed. Complete inactivation required the modification of four histidine residues per molecule of the enzyme. However, statistical analysis of the residual activity and the extent of modification shows that among the four modifiable residues, only one is critical for activity. Furthermore, this inactivation is prevented by the substrates of the enzyme. The above results indicated that one histidine is located within or very close to the active site and may play an important role in catalysis.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2005

Diethyl pyrocarbonate, a histidine-modifying agent, directly stimulates activity of ATP-sensitive potassium channels in pituitary GH(3) cells.

The ATP-sensitive K(+) (K(ATP)) channels are composed of sulfonylurea receptor and inwardly rectifying K(+) channel (Kir6.2) subunit. These channels are regulated by intracellular ADP/ATP ratio and play a role in cellular metabolism. Diethyl pyrocarbonate (DEPC), a histidine-specific alkylating reagent, is known to modify the histidine residues of the structure of proteins. The objective of this study was to determine whether DEPC modifies K(ATP)-channel activity in pituitary GH(3) cells. Steady-state fluctuation analyses of macroscopic K(+) current at -120 mV produced power spectra that could be fitted with a single Lorentzian curve in these cells. The time constants in the presence of DEPC were increased. Consistent with fluctuation analyses, the mean open time of K(ATP)-channels was significantly increased during exposure to DEPC. However, DEPC produced no change in single-channel conductance, despite the ability of this compound to enhance K(ATP)-channel activity in a concentration-dependent manner with an EC(50) value of 16 microM. DEPC-stimulated K(ATP)-channel activity was attenuated by pretreatment with glibenclamide. In current-clamp configuration, DEPC decreased the firing of action potentials in GH(3) cells. A further application of glibenclamide reversed DEPC-induced inhibition of spontaneous action potentials. Intracellullar Ca(2+) measurements revealed the ability of DEPC to decrease Ca(2+) oscillations in GH(3) cells. Simulation studies also demonstrated that the increased conductance of K(ATP)-channels used to mimic DEPC actions reduced the frequency of spontaneous action potentials and fluctuation of intracellular Ca(2+). The results indicate that chemical modification with DEPC enhances K(ATP)-channel activity and influences functional activities of pituitary GH(3) cells.

Adenosine Triphosphate↗
PubMed1999

Identification of essential histidine residues in 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase: analysis by chemical modification with diethyl pyrocarbonate and site-directed mutagenesis.

The enzyme 3-deoxy-D-manno-octulosonic acid 8-phosphate (KDO 8-P) synthase from Escherichia coli that catalyzes the aldol-type condensation of D-arabinose 5-phosphate (A 5-P) and phosphoenolpyruvate (PEP) to give KDO 8-P and inorganic phosphate (P(i)) is inactivated by diethyl pyrocarbonate (DEPC). The inactivation is first-order in enzyme and DEPC. A second-order rate constant of 340 M(-1) min(-1) is obtained at pH 7.6 and 4 degrees C. The rate of inactivation is dependent on pH and the pH-inactivation rate data imply the involvement of an amino acid residue with a pK(a) value of 7.3. KDO 8-P synthase activity is not restored to the DEPC-inactivated enzyme following treatment with hydroxylamine. Complete loss of KDO 8-P synthase activity correlates with the ethoxyformylation of three histidine residues by DEPC. KDO 8-P synthase is protected against DEPC inactivation by PEP and partially protected against inactivation by A 5-P. To provide further evidence for the involvement or role of the histidine residues in the aldol-type condensation catalyzed by KDO 8-P synthase, all six histidines were individually mutated to either glycine or alanine. The kinetic constants for the three mutants H40A, H67G, and H246G were unaffected as compared to the wild type enzyme. In contrast, H241G demonstrates a >10-fold increase in K(M) for both PEP and A 5-P and a 4-fold reduction in k(cat), while H97G demonstrates an increase in K(M) for only A 5-P and a 2-fold reduction in k(cat). The activity of the H202G mutant was too low to be measured accurately but the data obtained indicated an approximate 400-fold reduction in k(cat). Circular dichroism measurements of the wild-type and mutant enzymes indicate modest structural changes in only the fully active H67G and H246G mutants. The H241G mutant is protected against DEPC inactivation by PEP and A 5-P to the same extent as the wild-type enzyme, suggesting that the functionally important H241 may not be located in the vicinity of the substrate binding sites. The H97G mutant is protected by PEP against DEPC inactivation to the same degree as the wild-type enzyme but is no longer protected by A 5-P. In the case of the H202G mutant, both A 5-P and PEP protect the mutant against DEPC inactivation but to different extents from those observed for the wild-type enzyme. The catalytic activity of the H97G mutant is partially restored (20% --> 60% of wild-type activity) in the presence of imidazole, while a minor amount of activity is restored to the H202G mutant (<1% --> 4% of wild-type activity) in the presence of imidazole.

Aldehyde-Lyases↗
PubMed1988

Effects of amiloride and diethyl pyrocarbonate on CSF HCO-3 and ventilation in hypercapnia.

Amiloride (10(-3) M), a Na+-H+ countertransport inhibitor, infused into the cisterna magna (10 microliter/min for 40 min) of ketamine-xylazine-anesthetized rabbits decreased the cerebrospinal fluid (CSF) HCO3- response to 3 h of hypercapnia [arterial PCO2 (PaCO2) = 60 Torr] by 21.6% (mean delta CSF [HCO3-]/delta PaCO2 0.232 vs. 0.296 mmol.l-1.Torr-1, P less than 0.05). Diethyl pyrocarbonate (DEPC, 10(-3) M), a histidine-blocking agent, infused into the cisterna magna decreased the CSF HCO3- response to hypercapnia by 25.3% (mean delta CSF [HCO3-]/delta PaCO2, 0.230 vs. 0.308 mmol.l-1.Torr-1, P less than 0.02). DEPC is known to inhibit the ventilatory response to hypercapnia (E. Nattie. Respir. Physiol. 64: 161-176, 1986) by a direct effect at the ventrolateral medulla (E. Nattie. J. Appl. Physiol. 61: 843-850, 1986). In this study amiloride had no significant effect on the ventilatory response to hypercapnia. The interpretation is that a Na+-H+ countertransport protein, perhaps with a histidine at a key location, is involved in CSF acid-base regulation and that amiloride appears to have no effects on the chemoreception process. DEPC appears to have effects on chemoreception and on CSF acid-base regulation.

Acid-Base Equilibrium↗
PubMed1998

Diethyl pyrocarbonate (DEPC) inhibits CO2 chemosensitivity in Helix aspersa.

Central CO2 chemoreceptors in poikilothermic vertebrates may not regulate ventilation at a particular pH setpoint; central chemoreceptor responses may more accurately reflect the relative charge state (alpha) of the imidazole of histidine. We have tested the alphastat hypothesis in the terrestrial, air breathing, pulmonate snail, Helix aspersa, by chemically modifying histidine residues in the central CO2 chemoreceptor area of this animal using diethyl pyrocarbonate (DEPC). After focal application of 20 mM DEPC to the central CO2 chemoreceptor region, the pneumostome, a respiratory, CO2 responsive organ in the snail, no longer responded to hypercapnic, acidotic stimulation of the central chemoreceptor area. However, pneumostomal responses to hypoxic stimulation of the pneumostome and to focal stimulation of the central chemoreceptor area with sodium nitroprusside, a respiratory stimulant in H. aspersa, remained intact after DEPC treatment. Furthermore, DEPC treatment of the central chemoreceptor area blocked pneumostomal responses to ammonia pre-pulse treatment, which changes intracellular pH, while extracellular pH is held constant. These results resemble mammalian responses to DEPC treatment and indicate that central chemoreceptor responses in H. aspersa may originate from changes in the alpha of intracellular histidine residues.

Ammonium Chloride↗
PubMed1988

Pirenzepine prevents diethyl pyrocarbonate inhibition of central CO2 sensitivity.

Application by pledget of the M1-antimuscarinic receptor agent pirenzepine (40 mM) to the rostral chemosensitive areas of the ventrolateral medulla in anesthetized, paralyzed, vagotomized, glomectomized, and servoventilated cats inhibited the slope of the integrated phrenic response to CO2 by 32.5% (P less than 0.03) and the maximum value by 21.1% (P less than 0.01). Similar application of the imidazole-histidine blocking agent diethyl pyrocarbonate (DEPC) decreased the slope by 40.3% (P less than 0.01) and the maximum value by 29.3% (P less than 0.05). Both responses confirm previous results. DEPC treatment decreased the effectiveness of subsequent pirenzepine application such that although slope and maximum were further decreased, the values were not significantly different from those after DEPC. Pirenzepine treatment prevented any subsequent DEPC inhibitory effect. The results raise the possibility that the inhibitory effects of DEPC on CO2 chemosensitivity are via muscarinic receptors and that muscarinic receptor involvement in CO2 chemosensitivity requires the presence of imidazole-histidine. Analysis by scintillation counting of successive 100-micron sections of medulla after rostral area application of [3H]pirenzepine indicated that the pirenzepine and DEPC effects are most probably within 2.0 mm of the ventral surface as measured from the midline, well away from the dorsal and ventral respiratory group neurons.

Animals↗
PubMed1998

False positive staining in the TUNEL assay to detect apoptosis in liver and intestine is caused by endogenous nucleases and inhibited by diethyl pyrocarbonate.

BACKGROUND: The terminal transferase uridyl nick end labelling (TUNEL) assay allows the easy demonstration of cell death as a result of apoptosis. However, when this assay is applied to liver tissue, the number of TUNEL positive cells is dependent on the time of incubation with proteinase K. AIM: To test whether false positive results are the result of the release of endogenous endonucleases by proteinase K and can be abolished by pretreatment with diethyl pyrocarbonate (DEPC). METHODS: Involution of hyperplastic ductules in bile duct ligated rats after biliary decompression by Roux-en-Y anastomosis and acute CCl4 intoxication were studied as models of apoptosis and necrosis, respectively. A standard TUNEL assay was applied to formalin fixed tissue sections mounted with cement. To inhibit putative endogenous endonucleases, tissue slides were pre-incubated with DEPC. RESULTS: In the standard TUNEL assay, the number of positive nuclei was highly dependent upon the length of time that sections were incubated with proteinase K. After pretreatment with DEPC, only cells that also exhibited morphological features of apoptosis stained positive. DEPC pretreatment abolished false positive staining in CCl4 induced hepatocyte necrosis and blocked interference by endogenous alkaline phosphatase in intestine. The method of gluing the tissue section to the glass slide was found to be of utmost importance because the effect of DEPC was abolished on silanised slides. CONCLUSIONS: False positive staining in the TUNEL assay in the liver is caused by the release of endogenous endonucleases as a result of proteinase treatment. This can be abolished by pretreatment of tissue slides with DEPC.

Animals↗
PubMed1978

An essential active-site histidine residue in human prostatic acid phosphatase. Ethoxyformylation by diethyl pyrocarbonate and phosphorylation by a substrate.

Human prostatic acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) is a dimeric (alpha 2) protein that catalyses the hydrolysis of phosphomonoesters. Several reports suggest that a phosphoenzyme intermediate is involved in the mechanism of acid phosphatase. Chemical modification studies and trapping experiments were therefore undertaken in order to ascertain the identity of the amino acid residue(s) involved in the formation of this intermediate. Human prostatic acid phosphatase is inactivated by diethyl pyrocarbonate (second-order rate constant of 7 M-1. min-1 at pH 6.2) with an accompanying increase in absorbance at 242 nm due to formation of ethoxyformylhistidyl derivatives. In the presence of competive inhibitors the rate of inactivation is decreased. Inactivation can be partially reversed by hydroxylamine. The pH curve of inactivation indicates the involvement of a residue having a pK alpha of 6.5. Direct evidence for the involvement of a histidine residue in the mechanism was obtained by trapping a covalent phosphohistidyl-enzyme intermediate. Incubation of the enzyme with p-nitrophenyl [32 P] phosphate leads to incorporation of 0.44 mol 32P/mol enzyme. The denatured phosphoenzyme,which was acid labile but base stable, was hydrolyzed in 3 M KOH and the radioactivity was found to cochromatograph with synthetic tau-phosphohistidine on Dowex-1 ion-exchange resin. These results are consistent with a catalytic mechanism involving histidine as a nucleophile in the formation of a covalents phosphoenzyme intermediate.

Acid Phosphatase↗
PubMed2003

Chemical modification of contractile 3-nm-diameter filaments in Vorticella spasmoneme by diethyl-pyrocarbonate and its reversible renaturation by hydroxylamine.

A peritrich ciliate possesses a zooid and a long stalk consisting of a bundle of 3-nm-diameter filaments. Glycerinated stalks can contract in the presence of free Ca(2+) and re-extend in the absence of free Ca(2+). In the present study, we demonstrated that histidine residue(s) played a critical role in spasmoneme contraction by using glycerinated stalk of Vorticella. Concentration-dependent inhibition of spasmoneme contraction was observed in the presence of reversible histidine-modifying reagent named diethyl-pyrocarbonate (DEPC). In addition, the contractility degree of DEPC-modified spasmoneme could be partially restored by hydroxylamine treatment. The 244nm absorption of modified spasmoneme protein(s) increased with rising DEPC concentration and decreased following the addition of hydroxylamine treatment. Adding Ca(2+) before DEPC modification could prevent the spasmoneme contraction from inhibition of DEPC. Those results suggested that histidine residues were actively involved in spasmoneme contraction. Ca(2+)-binding ability of spasmin was not inhibited by DEPC modification, which suggested that the essential histidine residues were not on the calcium-binding site of spasmin.

Animals↗
PubMed1988

Chemical modification of acyl-CoA:cholesterol O-acyltransferase. 1. Identification of acyl-CoA:cholesterol O-acyltransferase subtypes by differential diethyl pyrocarbonate sensitivity.

Acyl-CoA:cholesterol O-acyltransferase (EC 2.3.1.26) (ACAT) catalyzes the intracellular synthesis of cholesteryl esters from cholesterol and fatty acyl-CoA at neutral pH. Despite the probable pathophysiologic role of ACAT in vascular cholesteryl ester accumulation during atherogenesis, its mechanism of action and its regulation remain to be elucidated because the enzyme polypeptide has never been identified or purified. Present chemical modification results identify two distinct tissue types of ACAT, based on marked differences in reactivity of an active-site histidine residue toward diethyl pyrocarbonate (DEP) and acetic anhydride. The apparent Ki of the DEP-sensitive ACAT subtype, typified by aortic ACAT, was 40 microM, but the apparent Ki of the DEP-resistant ACAT subtype, typified by liver ACAT, was 1500 microM, indicating a 38-fold difference in sensitivity to DEP. Apparent Ki's of aortic and liver ACAT for inhibition by acetic anhydride were also discordant (less than 500 microM and greater than 5 mM, respectively). On the basis of the reversibility of inhibition by hydroxylamine, a neutral pKa for maximal modification, and acetic anhydride protection against DEP inactivation, DEP and acetic anhydride appear to modify a common histidine residue. Oleoyl-CoA provided partial protection against inactivation by DEP and acetic anhydride, suggesting that the modified histidine is at or near the active site of ACAT. Systematic investigation of ACAT activity from 14 different organs confirmed the existence of 2 subtypes of ACAT on the basis of their different reactivities toward DEP and acetic anhydride.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetates↗
PubMed1980

Effects of adenosine 5'-diphosphate on bovine glutamate dehydrogenase: diethyl pyrocarbonate modification.

Initial rate kinetic studies and reduced coenzyme binding studies with bovine glutamate dehydrogenase have shown that an enzyme-NAD(P)H-glutamate abortive complex is a major participant in the overall enzyme-catalyzed reaction. The allosteric regulator ADP is shown to activate the enzyme by destabilizing this abortive complex. Destabilization of this abortive complex with concomitant activation is also achieved by the ethoxyformylation of a single histidine residue per subunit in the hexameric enzyme. ADP, in addition to its activatory effects, is shown to (a) remove the nonlinearity from the Lineweaver-Burk plots which is attributable to negative cooperativity and (b) inhibit the enzyme as a competitive inhibitor with respect to coenzyme under the appropriate conditions. Ethoxyformylation with diethyl pyrocarbonate, which mimics the activatory effects of ADP, has no effect on the negative cooperativity shown by this enzyme. A model for the action of ADP is proposed in which ADP activates glutamate dehydrogenase by binding to a regulatory binding site and blocks the negative cooperativity by mimicking the natural coenzyme at the active site of the enzyme.

Adenosine Diphosphate↗
PubMed1992

Modification of vertebrate and algal prolyl 4-hydroxylases and vertebrate lysyl hydroxylase by diethyl pyrocarbonate. Evidence for histidine residues in the catalytic site of 2-oxoglutarate-coupled dioxygenases.

A search for conserved amino acid residues within the cDNA-derived amino acid sequences of 2-oxoglutarate-coupled dioxygenases revealed the presence of two distinct motifs, spaced 49-71 amino acids apart, toward the C-terminal regions of these proteins. Each of the two common motifs contains an invariant histidine residue at a conserved position. The 2-oxoglutarate-coupled dioxygenases function in diverse processes, including the post-translational hydroxylation of proline and lysine residues in vertebrate collagens and the biosynthesis of microbial cephalosporins, yet they have a common reaction mechanisms, which requires the binding of Fe2+, 2-oxoglutarate, O2 and ascorbate at the catalytic site. The two regions of homology, and specifically the identical histidines, potentially represent functionally important sites related to their catalytic activity. Modification of histidine residues by diethyl pyrocarbonate inactivated vertebrate and algal prolyl 4-hydroxylase and vertebrate lysyl hydroxylase, indicating that histidine residues function in the catalytic site of these 2-oxoglutarate-coupled dioxygenases. Inactivation was prevented by the presence of co-substrates, but not by the peptide substrate. It is proposed that the histidine residues in the conserved motifs may function as Fe(2+)-binding ligands.

Amino Acid Sequence↗
PubMed2005

Imidazole binding reagent diethyl pyrocarbonate (DEPC) inhibits avian intrapulmonary chemoreceptor discharge in vivo.

Data indicate that avian intrapulmonary chemoreceptors (IPC) transduce CO2 stimuli by sensing the products of CO2 hydration, [H+] and [HCO3-]. The alphastat regulation hypothesis of physiological pH sensitivity suggests that proteins sense [H+] through changes in the ionization state of imidazole groups (alphaIm). To test whether imidazole is involved with IPC CO2 sensitivity, we administered diethyl pyrocarbonate (DEPC) intravenously while recording from IPC exposed to varying levels of inspired CO2. At physiological pH, DEPC converts pH sensitive imidazole groups to pH-insensitive N-carbethoxyhistidyl residues. Single cell extracellular neural recordings were made from vagal filaments in anesthetized, unidirectionally ventilated Anas platyrhynchos. Without DEPC, IPC discharge rate was inversely proportional to inspired CO2 with characteristic dynamic responses to rapid CO2 alterations (n = 10). After DEPC treatment (> or = 15 mM), mean sensitivity of IPC discharge to static inspired CO2 levels was decreased 75% (P < 0.05), and mean peak dynamic IPC discharge rate was decreased 80% (P < 0.05). Additionally, we tested whether DEPC might alter IPC discharge by binding imidazole groups in the enzyme carbonic anhydrase (CA), but we found no effect on CA catalytic rate. We conclude that DEPC inhibits IPC CO2 signal transduction by modifying imidazole groups on acid-sensitive proteins other than CA, possibly membrane acid-base exchangers or ion channels. These data support the alphastat regulation hypothesis in IPC CO2 respiratory chemoreception and suggests a more direct link between H+ and membrane excitability.

Action Potentials↗