Effects Of Diethyl Pyrocarbonate Research Articles

Involvement of Histidine Residue His382 in pH Regulation of MCT4 Activity.

Monocarboxylate transporter 4 (MCT4) is a pH-dependent bi-directional lactate transporter. Transport of lactate via MCT4 is increased by extracellular acidification. We investigated the critical histidine residue involved in pH regulation of MCT4 function. Transport of lactate via MCT4 was measured by using a Xenopus laevis oocyte expression system. MCT4-mediated lactate transport was inhibited by Zn2+ in a pH physiological condition but not in an acidic condition. The histidine modifier DEPC (diethyl pyrocarbonate) reduced MCT4 activity but did not completely inactivate MCT4. After treatment with DEPC, pH regulation of MCT4 function was completely knocked out. Inhibitory effects of DEPC were reversed by hydroxylamine and suppressed in the presence of excess lactate and Zn2+. Therefore, we performed an experiment in which the extracellular histidine residue was replaced with alanine. Consequently, the pH regulation of MCT4-H382A function was also knocked out. Our findings demonstrate that the histidine residue His382 in the extracellular loop of the transporter is essential for pH regulation of MCT4-mediated substrate transport activity.

Transmembrane domain histidines contribute to regulation of AE2-mediated anion exchange by pH

Activity of the AE2/SLC4A2 anion exchanger is modulated acutely by pH, influencing the transporter's role in regulation of intracellular pH (pH(i)) and epithelial solute transport. In Xenopus oocytes, heterologous AE2-mediated Cl(-)/Cl(-) and Cl(-)/HCO(3)(-) exchange are inhibited by acid pH(i) or extracellular pH (pH(o)). We have investigated the importance to pH sensitivity of the eight histidine (His) residues within the AE2 COOH-terminal transmembrane domain (TMD). Wild-type mouse AE2-mediated Cl(-)/Cl(-) exchange, measured as DIDS-sensitive (36)Cl(-) efflux from Xenopus oocytes, was experimentally altered by varying pH(i) at constant pH(o) or varying pH(o). Pretreatment of oocytes with the His modifier diethylpyrocarbonate (DEPC) reduced basal (36)Cl(-) efflux at pH(o) 7.4 and acid shifted the pH(o) vs. activity profile of wild-type AE2, suggesting that His residues might be involved in pH sensing. Single His mutants of AE2 were generated and expressed in oocytes. Although mutation of H1029 to Ala severely reduced transport and surface expression, other individual His mutants exhibited wild-type or near-wild-type levels of Cl(-) transport activity with retention of pH(o) sensitivity. In contrast to the effects of DEPC on wild-type AE2, pH(o) sensitivity was significantly alkaline shifted for mutants H1144Y and H1145A and the triple mutants H846/H849/H1145A and H846/H849/H1160A. Although all functional mutants retained sensitivity to pH(i), pH(i) sensitivity was enhanced for AE2 H1145A. The simultaneous mutation of five or more His residues, however, greatly decreased basal AE2 activity, consistent with the inhibitory effects of DEPC modification. The results show that multiple TMD His residues contribute to basal AE2 activity and its sensitivity to pH(i) and pH(o).

Evidence for allosteric regulation of pH-sensitive System A (SNAT2) and System N (SNAT5) amino acid transporter activity involving a conserved histidine residue.

System A and N amino acid transporters are key effectors of movement of amino acids across the plasma membrane of mammalian cells. These Na+-dependent transporters of the SLC38 gene family are highly sensitive to changes in pH within the physiological range, with transport markedly depressed at pH 7.0. We have investigated the possible role of histidine residues in the transporter proteins in determining this pH-sensitivity. The histidine-modifying agent DEPC (diethyl pyrocarbonate) markedly reduces the pH-sensitivity of SNAT2 and SNAT5 transporters (representative isoforms of System A and N respectively, overexpressed in Xenopus oocytes) in a concentration-dependent manner but does not completely inactivate transport activity. These effects of DEPC were reversed by hydroxylamine and partially blocked in the presence of excess amino acid substrate. DEPC treatment also blocked a reduction in apparent affinity for Na+ (K0.5Na+) of the SNAT2 transporter at low external pH. Mutation of the highly conserved C-terminal histidine residue to alanine in either SNAT2 (H504A) or SNAT5 (H471A) produced a transport phenotype exhibiting reduced, DEPC-resistant pH-sensitivity with no change in K0.5Na+ at low external pH. We suggest that the pH-sensitivity of these structurally related transporters results at least partly from a common allosteric mechanism influencing Na+ binding, which involves an H+-modifier site associated with C-terminal histidine residues.

Slowing of ERG current deactivation in NG108-15 cells by the histidine-specific reagent diethylpyrocarbonate

The aim of this study was to explore and characterize the effect of the histidine-specific reagent diethylpyrocarbonate (DEPC) on the ERG ( ether-à-go-go related gene) channels of whole-cell voltage-clamped NG108-15 neuroblastoma x glioma hybrid cells. The channels were fully activated by long depolarizing prepulses. Hyperpolarizing pulses elicited K + inward currents which deactivated after reaching a peak. DEPC (0.26–2.1 mM, externally applied for 5–12 min) irreversibly decreased τ −1, the rate constant of deactivation. At a pulse potential of −120 mV τ −1 decreased on average by 60%. The effect can be described as a −25 mV shift of the τ −1( V) curve. The activation curve and the curve relating steady-state current to pulse potential were shifted by similar amounts. The decrease of τ −1 was the same at 0.26 and at 2.1 mM, but developed faster at the higher concentration. The slowing of deactivation was only seen when the cells were held at a potential of −20 mV. At this potential it developed with a time constant of 47 s. At more negative holding potentials (−40 or −70 mV) only a slight reduction of the peak occurred. The observations suggest preferential binding of DEPC to the open and inactivated channel states. The DEPC effect can possibly be explained by the reaction of DEPC with histidine residues or other amino acids in the external loops of the channel. However, dichloro-(2,2′:6′,2″-terpyridine)-platinum (II) dihydrate (DTPD), another histidine-specific reagent, markedly decreased the peak current without affecting τ −1. Therefore, the possibility that (some of) the effects of DEPC and DTPD are unrelated to their property as histidine-specific reagents cannot be excluded.

Modification of surface histidine residues abolishes the cytotoxic activity of Clostridium difficile toxin A

Clostridium difficile toxin A displays both cytotoxic and enterotoxic activities. It has recently been demonstrated that toxin A exerts its cytotoxic effect by the glucosylation of the small GTP-binding proteins of the Rho family. Diethyl pyrocarbonate, at pH 7.0, was used to chemically modify exposed histidine residues on toxin A. Modification of toxin A with diethyl pyrocarbonate abolished both its cytotoxic activity and the ability of the toxin to bind Zn-Sepharose gel. Treatment of toxin A with [ 14C]-diethyl pyrocarbonate revealed concentration dependent labelling of histidine residues on the toxin molecules. The effects of diethyl pyrocarbonate could be reversed by hydroxylamine treatment. These data suggest the modified histidine residues on toxin A are critical to its cytotoxic activity. Histidine modification had no effect on the glucosyl transferase enzyme activity of toxin A. However, modification abolished the ‘cold’ binding of toxin to bovine thyroglobulin in an ELISA and reduced ligand binding activity in a rabbit erythrocyte haemagglutination assay. The data suggest that the histidine residues may be crucial to the receptor-binding activity of toxin A. Exposed histidines on toxin A are available for zinc chelation, and these have been exploited in the development of a novel purification protocol for toxin A using zinc-chelating chromatography.

Single-channel currents from diethylpyrocarbonate-modified NMDA receptors in cultured rat brain cortical neurons.

The role of histidine residues in the function of N-methyl-D-aspartate (NMDA)-activated channels was tested with the histidine-modifying reagent diethylpyrocarbonate (DEP) applied to cells and membrane patches from rat brain cortical neurons in culture. Channels in excised outside-out patches that were treated with 3 mM DEP for 15-30 s (pH 6.5) showed an average 3.4-fold potentiation in steady state open probability when exposed to NMDA and glycine. Analysis of the underlying alterations in channel gating revealed no changes in the numbers of kinetic states: distributions of open intervals were fitted with three exponential components, and four components described the shut intervals, in both control and DEP-modified channels. However, the distribution of shut intervals was obviously different after DEP treatment, consistent with the single-channel current record. After modification, the proportion of long shut states was decreased while the time constants were largely unaffected. Burst kinetics reflected these effects with an increase in the average number of openings/burst from 1.5 (control) to 2.2 (DEP), and a decrease in the average interburst interval from 54.1 to 38.2 ms. These effects were most likely due to histidine modification because other reagents (n-acetylimidazole and 2,4,6-trinitrobenzene 1-sulfonic acid) that are specific for residues other than histidine failed to reproduce the effects of DEP, whereas hydroxylamine could restore channel open probability to control levels. In contrast to these effects on channel gating, DEP had no effect on average single-channel conductance or reversal potential under bi-ionic (Na+:Cs+) conditions. Inhibition by zinc was also unaffected by DEP. We propose a channel gating model in which transitions between single- and multi-opening burst modes give rise to the channel activity observed under steady state conditions. When adjusted to account for the effects of DEP, this model suggests that one or more extracellular histidine residues involved in channel gating are associated with a single kinetic state.

Inhibition of ligand binding to thromboxane A 2/prostaglandin H 2 receptors by diethylpyrocarbonate : Protection by receptor ligands and reversal by hydroxylamine

The potential of histidines to modulate the binding of agonists and antagonists to human platelet thromboxane A 2 (TXA 2) receptors was investigated. TXA 2 receptors were purified from crude platelet membranes via affinity and wheat germ lectin chromatography. Radioligand binding studies were conducted using the TXA 2, mimetic [ 125I]BOP ( I-BOP = [1S-(lα,2β(5 Z),3α(1E,3 R ∗),4α)]-7-[3-(3- hydroxy -4-(4′- iodophenoxy)-1- butenyl)7- oxabicyclo-[2.2.1] heptan-2- yl]-5- heptenoic acid) and the TXA, receptor antagonist [ 125I]SAP (I-SAP = 7-[(1 R,2 S,3 S,5 R)-6,6-dimethyl-3-(4-iodobenzenesulfonylamino)-bicyclo-[3.1.1] hept-2-yl]-(5Z)-heptenoic acid). The histidine modifying reagent diethylpyrocarbonate (DEPC) produced a concentration (30–100 μM) dependent inhibition of binding of both [ 125I]BOP and [ 125I]SAP. DEPC treatment significantly ( P < 0.05, N = 6) decreased the affinity of the receptor for [ 125I]SAP ( K d = 2.4 ± 0.4 and 5.4 ± 0.4 nM, control and DEPC, respectively) without significantly decreasing the B max. The effects of DEPC were reversed by hydroxylamine. The inhibition of [ 125I]BOP and [ 125I]SAP binding produced by DEPC was reduced significantly by prior incubation of the purified receptors with the TXA 2 receptor agonist U-46619 or the TXA 2 receptor antagonist SO 29548. The results strongly support the notion that one or more histidines reside in a domain that can modulate ligand binding to the TXA 2 receptor.

Modification of Histidine Residues of Photosystem II by Diethyl Pyrocarbonate Inhibits the Electron Transfer between the Primary (QA) and Secondary (QB) Quinone Acceptors

The effect of diethyl pyrocarbonate (DEPC) on the photosynthetic electron transport was investigated in isolated spinach thylakoids by partial electron transport rate and thermolumi- nescence measurements. Incubation of thylakoids at pH 6.5 with 5 mм DEPC for 15 min resulted in a considerable inhibition of electron transport from water to dichlorophenolindo- phenol. The inhibition was only partially releaved by addition of the donor, diphenylcarbazide indicating the effect of DEPC both on the donor and acceptor sides of PS II. In the thermoluminescence glow curve DEPC-treatment abolished the B band (S2QB - radiative charge recombination) at 30 °C with a concomitant appearance of the Q band (S2QA - charge recombination) at 10 °C. This suggests that in isolated thylakoids possessing an active water-splitting system DEPC affects the electron transfer from QA to QB but does not inhibit the electron transport from manganese to QA during the S,→S2 transition of the water-splitting system. At the acceptor side of PS II the targets of DEPC are probably the histidines which are coordinated to the non-heme iron. Illumination of thylakoids at -80 °C following DEPC addition after two preflashes at 5 °C resulted in the replacement of the A(AT) thermoluminescence band at -30 °C with a band appearing at -15 °C. This observation can be explained by the effect of DEPC on a donor side histidine component participating in the generation of the A(AT) band. Consequently, in the interpretation of results obtained by DEPC treatment of PS II, both the donor and acceptor side effects of DEPC should be considered.

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.

Differential inhibition of estrogen and antiestrogen binding to the estrogen receptor by diethylpyrocarbonate

Diethylpyrocarbonate differentially inhibited the specific binding, in lamb uterine cytosol, of estradiol (inhibition ~90% with 4mM reagent) and 4-hydroxytamoxifen (inhibition ≲50% with 4–16 mM reagent), a potent triphenylethylene antiestrogen. Saturation analysis experiments indicated that the effects of diethylpyrocarbonate were due to progressive but differing decreases in the concentration of binding sites for the two ligands, with no apparent change in the affinity constants. However, competitive binding and dissociation experiments evidenced that steroidal and nonsteroidal estrogens still bound, but with very low affinities, to diethylpyrocarbonate-modified receptor (> 1000-fold decrease in affinity) whereas the affinities of triphenylethylene antiestrogens were much less affected (< 10-fold decrease). Both ligands prevented the inactivation of the estrogen receptor by diethylpyrocarbonate, estradiol being more efficient than 4-hydroxytamoxifen. These data indicate that the action of diethylpyrocarbonate results in the formation of two populations of estrogen receptor that are quantitatively nearly equivalent: the first does not bind estrogens or antiestrogens; the second does not bind estrogens significantly but still interacts with antiestrogens at a high affinity. The simplest interpretation is that these two populations arise from mutually exclusive modifications by diethylpyrocarbonate of at least two aminoacid residues located at or close to the ligand binding site; modification of one residue totally prevents the binding of estrogens and antiestrogens; the modification of the second impairs only the binding of estrogens. Considering that (i) hydroxylamine, which specifically reverses the diethylpyrocarbonate-induced modification of histidine and tyrosine residues, restored a large part (>80%) of the estradiol- and 4-hydroxytamoxifen-binding capacity of diethylpyrocarbonate-inactivated cytosol, and that (ii) similar differential inhibition of estrogen and antiestrogen binding was observed following the action of tetranitromethane, it is likely that these residues are histidine(s) and/or tyrosine(s). These results evince a marked difference in the interaction of estrogens and triphenylethylene antiestrogens with the estrogen receptor, which could account for the altered activation of the receptor by triphenylethylene antiestrogens. Consequently, the screening of ligands with modified steroid receptors could be a useful method for distinguishing between potential hormone agonists and antagonists.

Modification of trypsin-solubilized cytochrome b5, apocytochrome b5, and liposome-bound cytochrome b5 by diethylpyrocarbonate

The interactions of diethylpyrocarbonate (DEP) with the various forms of cytochrome b 5 were studied to gain a better understanding of the factors that influence the extent of modification of the axial histidines of cytochrome b 5. Very low concentrations of DEP were able to decrease the heme binding capacity of apocytochrome b 5. Moreover, it was shown that two additional histidines, presumed to be the axial ligands (His 39 and 63), were modified in the apo but not the holo form of a given preparation of cytochrome b 5. Trypsin-solubilized bovine cytochrome b 5 was resistant to the effects of DEP. A 200-fold molar excess of DEP displaced only 15% of the heme in the trypsin-solubilized protein in contrast to an 84% displacement of the heme in the detergent-solubilized protein. However, detergent-solubilized cytochrome b 5 which had been incorporated into phospholipid vesicles exhibited the same reactivity with DEP as did the trypsin-solubilized protein. This is attributed to the fact that the two resistant preparations of cytochrome b 5 are monomeric in their respective environments while detergent-solubilized cytochrome b 5 is known to exist as an octamer in aqueous solutions. Our studies suggest that dissociation of the octamer to the monomer results in a conformational change that decreases the reactivity of the axial ligands of the hydrophilic heme-containing domain of cytochrome b 5. Examination of the cytochrome b 5 molecule by computer graphics indicates that a tunnel leads from the surface of the molecule to axial histidine 63 and that axial histidine 39 is buried.

Zinc inhibition of potassium efflux in depolarized frog muscle and its modification by external hydrogen ions and diethylpyrocarbonate treatment.

Efflux of 42K+ was measured in frog sartorius muscles equilibrated in hyperosmotic depolarizing solutions. At the internal potentials obtained, K+ passes mainly through the inward rectifier potassium channels. Inhibition of K+ efflux by external Zn2+ (0.25 to 15 mM) differs in three significant ways from inhibition by Ba2+. (1) The dose-response relation does not correspond to action at a single site. (2) The Zn2+-sensitivity of K+ efflux does not depend on [K+]0 at constant internal potential. (3) Zn2+ inhibition is reduced by hydrogen ions, while Ba2+ inhibition is unaffected. Further, the Ba2+-sensitivity of K+ efflux is not altered by a half-inhibiting Zn2+ concentration, suggesting that the two ions do not interact at a common site. The histidine-modifying reagent diethylpyrocarbonate (DEPC) reduces Zn2+ inhibition. After DEPC treatment Zn2+ inhibition is further reduced by low pH. DEPC has little effect on Ba2+ inhibition. Zn2+ inhibition is not altered by treatment with the sulfhydryl reagents 5,5'-dithio-bis(2-nitrobenzoic acid) or dithiothreitol. The results can be described by either of two models in which two sites can bind Zn2+ and one or both of the sites may also bind H+. When both sites bind Zn2+, K+ efflux is inhibited, and a third site may then bind H+. The effects of DEPC can be accounted for by a decrease in H+ affinity of the first two sites by a factor of 50, and a decrease in Zn2+ affinity of these sites and of the H+ affinity of the third site by about one order of magnitude.

Involvement of histidine and tryptophan residues of glutamine binding protein in the interaction with membrane-bound components of the glutamine transport system of Escherichia coli.

We treated the glutamine binding protein with diethyl pyrocarbonate (DEPC) and N-bromosuccinimide (NBS) to modify respectively the sole histidine and tryptophan residues and examined the effect of these modifications on the ability of the binding protein to bind glutamine as well as the ability to restore glutamine transport in membrane vesicles of Escherichia coli. Under the conditions used, both DEPC and NBS markedly inhibited the ability to restore glutamine transport in vesicles without any significant effect on glutamine binding. Moreover, saturating quantities of glutamine had no protective effect on the inactivation of the binding protein by DEPC or NBS. Fluorometric measurement and amino acid analysis indicate that the inactivation of the binding protein in restoring vesicle transport by NBS can be attributed to the oxidation of a single tryptophan residue. Similar analysis and the inability of hydroxylamine to reverse the effect of DEPC indicate that the effects of DEPC can probably be attributed to alterations of the sole histidine and/or one or more lysine residues of the binding protein. We conclude that the glutamine binding protein possesses at least two largely nonoverlapping functional domains, one responsible for glutamine binding and the other for the interaction with the other components of the glutamine transport system.

Chemical modification of histidine residues of rabbit hemopexin

Treatment of rabbit hemopexin with bromoacetic acid (BrAc) or with diethylpyrocarbonate (DEP) modified histidine residues and produced a concomitant decrease in the protein's ability to form a low-spin hemichrome complex with deuteroheme (ferrideuteroporphyrin IX). Deuteroheme bound to hemopexin before treatment decreased the extent of inactivation by either reagent. After exposure of deuteroheme-hemopexin to 0.16 m BrAc at pH 6.9 for 120 h, 10–11 of the 16 histidine residues of hemopexin were carboxymethylated, but 90–95% of the deuteroheme-hemopexin complex remained intact. Under the same conditions, 12 histidine residues of apo-hemopexin were carboxymethylated, and 95% of the protein's ability to form its normal hemichrome complex with heme (ferriprotoporphyrin IX) was abolished. The alkylated apo-protein, however, did retain a potential to interact with deuteroheme. The apparent dissociation constants for the complexes of metal-free deuteroporphyrin and deuteroheme with BrAc-treated apo-hemopexin were both about 10 −6 m and nearly equal to that of the native deuteroporphyrin-hemopexin complex, as assessed by quenching of tryptophan fluorescence. Approximately 10 histidyl residues of the deuteroheme-hemopexin complex, but only about 4 residues of the apo-protein, were modified by DEP before heme-binding was appreciably affected. The effects of DEP on hemopexin were reversed by hydroxylamine at neutral pH, indicating that ethoxyformylation of histidine residues caused the observed inactivation of hemopexin. This and the results of BrAc treatment suggest that hemopexin contains several easily accessible histidine residues which are not critical for its interaction with heme. The conformation-sensitive positive ellipticity at 231 nm of hemopexin was affected by carboxymethylation and ethoxyformylation. Treatment with BrAc had only a small effect on the intrinsic ellipticity of apo-hemopexin, but eliminated the increase in ellipticity produced by interaction of unmodified hemopexin with heme. Treatment with DEP, on the other hand, decreased both intrinsic and extrinsic ellipticity. These results provide further evidence that the heme-hemopexin complex involves histidyl-heme iron coordination. In addition, they show that formation of the histidyl-heme complex not only greatly enhances the strength of the heme-hemopexin interaction but also is important for triggering conformational changes in the protein.

Effects of diethyl pyrocarbonate on the isolation and recovery of bound polysomes from a particulate fraction of rat liver

The conventional treatment of rat liver postmitochondrial supernatant with detergents permits the recovery of a roughly equal mixture of undegraded free and bound polysomes [ 1 ] , whereas similar treatment of the pellet containing mitochondria, lysosomes and the bulk of bound polysomes leads to the recovery of largely degraded polysomes [2,3] in spite of the use of naturally-occurring nuclease inhibitor [4]. This presumably is due to detergent-mediated activation or release of nucleases that are refractory to nuclease inhibitor [5]. Intact bound polysomes can be prepared from purified rough membrane fractions; however, the whole procedure requires about 2 days of centrifugation and polysome recovery is low [l]. Recently, diethyl pyrocarbonate (DEP), a potent inhibitor of nucleases, has been employed for the preparation of undegraded polysomes and RNA from higher plants and bacteria [6-81. The inactivation of pancreatic ribonuclease by DEP in vitro is believed to result from the formation of intraand intermolecular peptide-like bonds which cause conformational changes and yield polymeric proteins [9]. It was, therefore, of interest to determine whether mitochondrial and lysosomal nucleases could be inactivated by DEP while still in their latent state, and thereby permit isolation of undegraded bound polysomes from a crude particulate fraction of rat liver. In this report we provide evidence that the nuclease activities of the more rapidly sedimenting fraction of

The Effects of Diethyl Pyrocarbonate on the Stability and Activity of Plant Polyribosomes

The Effects of Diethyl Pyrocarbonate on the Stability and Activity of Plant Polyribosomes

Effects of diethyl pyrocarbonate on tobacco mosaic virus and its RNA

Effects of diethyl pyrocarbonate on tobacco mosaic virus and its RNA

Effects of diethyl pyrocarbonate on barley seeds

1. 1. Barley seeds (karyopses) were treated with solutions of diethyl pyrocarbonate (DEP), and the biological effects during germination were observed. 2. 2. With increasing concentration of DEP, an increasing fraction of the seeds shows complete inhibition of germination. The surviving seedlings show practically no inhibition of growth rate. 3. 3. On the cellular level the following effects were observed: Chromosome breaks (low frequency), no exchanges, cells with single-stranded chromosomes, haploid cells, aneuploid cells, tripolar spindles, chromosome contraction. In a previous communication a certain sterility, but no mutation, was reported. The former may have some relation to haploid and aneuploid cells. 4. 4. Post-irradiation treatment with DEP was found to increase the frequencies of γ-ray and neutron induced chromosome breaks, and to decrease the frequency of γ-ray induced two-hit effects. Neutron induced two-hit effects were not affected. These effects are similar to those of such antimetabolites as Myleran, FUDR, and aminopterin. 5. 5. The results are briefly discussed considering the biochemical effects of DEP: a general reactivity towards proteins leading to enzyme inactivation, whereas nucleic acids are left intact under the same conditions of treatment.

Effects of diethyl pyrocarbonate and methyl methanesulfonate on nucleic acids and nucleases.

Effects of diethyl pyrocarbonate and methyl methanesulfonate on nucleic acids and nucleases.