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.     

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.     

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.     

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.     

Modification of the insulin receptor by diethyl pyrocarbonate: effect on insulin binding and action

Insulin binding to rat liver plasma membranes is inhibited in a time- and dose-dependent fashion by prior treatment of membranes with the histidine-specific reagent diethyl pyrocarbonate. If all receptors are occupied by unlabeled hormone during diethyl pyrocarbonate treatment, no inhibition of 125I-labeled insulin binding is observed folowing washout of unlabeled hormone and unreacted reagent. Scatchard analysis of the binding inhibtion due to diethyl pyrocarbonate reveals a loss in receptor number rather than a change in receptor affinity for hormone. Fat cells treated with diethyl pyrocarbonate exhibit a rightward shift in the dose-response relationship for insulin-stimulated glucose oxidation consistent with a loss in receptor number due to the reagent. The pH profile for inhibition of insulin binding by diethyl pyrocarbonate and the partial reversibility of this inhibition by hydroxylamine are consistent with modification of a histidine residue. These results suggest that a histidine residue at or near the receptor binding site is required for formation of the biologically relevant insulin - receptor complex.     

Modification of tomato andAspergillus niger pectinesterases with diethyl pyrocarbonate

The role of histidine residues in pectinesterases was evaluated by monitoring the sensitivity to modification with diethyl pyrocarbonate in the tomato andAspergillus niger enzymes. Different and incomplete losses of enzyme activity were obtained. Inactivation of the enzymes was proportional to the histidine content (two in the tomato T1 form, six in theAspergillus form), suggesting that accessible histidine residues do not have active-site functions in these pectinesterases, but contribute to the overall structural stability. Lack of His roles in common between the enzyme forms is in agreement with the structures of pectinesterases having no conserved His residues.     

The reaction of diethyl pyrocarbonate with pyruvate kinase (Short Communication)

Diethyl pyrocarbonate inactivates muscle pyruvate kinase with the substitution of 3-4 histidine residues per subunit. Phosphoenolpyruvate, ATP and ADP to a lesser extent, and Mg(2+) and pyruvate to a small extent, protect against inactivation.     

The reaction of a histidine residue in glutamate dehydrogenase with diethyl pyrocarbonate

1. One mol of diethyl pyrocarbonate will react with one mol of glutamate dehydrogenase polypeptide chains to form one mol of N(1)-carbethoxyhistidine. Reaction is prevented by NADH. 2. The 1:1 complex has an increased specific activity (1.4-2.0-fold). 3. The reason for the activation is discussed. The results are not consistent with NADH dissociation from the enzyme-glutamate-NADH complex being rate-limiting in the steady state measured. 4. The effects of modification on the properties of the enzyme were investigated. The effects of GTP and NAD(+) on the enzyme activity are unaltered by activation. NADH binding is unaltered and there is no apparent change in the molecular weight. However, the activated enzyme can still be further activated by ADP. K(s) for ADP is decreased fivefold.     

Ethoxyformylation of tubulin with [3H]diethyl pyrocarbonate: a reexamination of the mechanism of assembly inhibition

In this study we reexamined the basis for the profound inhibitory effects of low concentrations of diethyl pyrocarbonate (DEP) on tubulin's ability to assemble into microtubules [cf. Lee, Y. C., Houston, L. I., & Himes, R. H. (1976) Biochem. Biophys. Res. Commun. 70, 50-56]. Assembly inhibition at low DEP concentrations can be resolved into two components: a component reversible with hydroxylamine (attributed to monoethoxyformylation of histidyl residues) that contributes approximately 40% of the inhibition and a hydroxylamine-resistant component (attributed to ethoxyformylation of non-histidyl residues) that contributes approximately 60% of the inhibition. Comparisons between the extent of assembly inhibition associated with each component and the degree of residue modification argue for the involvement of a small number of highly reactive residues in the inhibition process. To identify these residues, tubulin was reacted with limiting concentrations of [3H]DEP and subjected to tryptic digestion and HPLC analysis. Only one moderately reactive histidyl residue was detected. This residue (approximately 2-3-fold more reactive than the bulk histidyl residues) eluted in an apparently large, hydrophobic fragment. We failed to detect any non-histidyl residues that were exceptionally reactive to [3H]DEP. However, we did observe that the N-terminal methionyl residues in native protein were ethoxyformylated at rates comparable to that of the bulk histidyl residues. In denatured protein these methionyl residues were ethoxyformylated to a much larger extent (approximately 3-4-fold) than the bulk histidyl residues. We suggest that the N-terminal methionyl residues in tubulin are partly buried or are in a salt-bridge interaction in native protein and that ethoxyformylation of these residues disrupts tubulin structure and interferes with microtubule assembly.     

Modification of system A amino acid carrier by diethyl pyrocarbonate

Sodium-dependent alanine transport in plasma membrane vesicles from rat liver was inactivated in a time- and concentration-dependent fashion by prior treatment of membranes with the acylating reagent diethyl pyrocarbonate (DEPC). Both components of Na+/alanine cotransport (systems A and ASC) were inhibited. Exposure of vesicles to p-bromophenacyl bromide and methyl p-nitrobenzenesulfonate, which share with DEPC reactivity against histidine residues, also led to inhibition of alanine transport through systems A and ASC. The presence of Na+ (100 mM NaCl) and L-alanine (10 mM) during exposure to vesicles to DEPC protected against inactivation of system A (but not system ASC) transport activity. This protective effect was specific and required the presence of L-alanine since the presence of L-phenylalanine alone (10 mM) or L-phenylalanine plus Na+ (100 mM NaCl) did not cause any detectable protection. This overall pattern of protection is opposite to that previously found against specific sulfhydryl reagents (i.e. N-ethylmaleimide), where protection of system ASC was nearly maximal. The pH profile for DEPC-dependent inhibition of system A transport activity suggests modification of amino acid residue(s) with a pKr of approximately 7, most likely histidine(s), in close parallel with the pH dependence of system A transport activity. Our results suggest the presence of critical histidine residues on the system A carrier that may be responsible for the pH dependence of system A transport activity.     

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.