Defective DNA cross-link removal in Chinese hamster cell mutants hypersensitive to bifunctional alkylating agents.

DNA repair-deficient mutants from five genetic complementation groups isolated previously from Chinese hamster cells were assayed for survival after exposure to the bifunctional alkylating agents mitomycin C or diepoxybutane. Groups 1, 3, and 5 exhibited 1.6- to 3-fold hypersensitivity compared to the wild-type cells, whereas Groups 2 and 4 exhibited extraordinary hypersensitivity (30- to 90-fold). Mutants from Groups 1 and 2 were exposed to 22 other bifunctional alkylating agents in a rapid assay that compared cytotoxicity of the mutants to the wild-type parental strain, AA8. With all but two of the compounds, the Group 2 mutant (UV4) was 15- to 60-fold more sensitive than AA8 or the Group 1 mutant (UV5). UV4 showed only 6-fold hypersensitivity to quinacrine mustard. Alkaline elution measurements showed that this compound produced few DNA interstrand cross-links but numerous strand breaks that were revealed by proteinase treatment. Therefore, the extreme hypersensitivity of mutants from Groups 2 and 4 appeared specific for compounds the main cytotoxic lesions of which were DNA cross-links. Mutant UV5 was only 1- to 4-fold hypersensitive to all the compounds. Repair kinetics of DNA interstrand cross-link production and removal was measured by alkaline elution for AA8 and mutants UV4 and UV5 after exposure to diepoxybutane. Although the initial number of cross-links was similar for the three cell lines, during 24-h incubation, the efficiency of removal of cross-links was lowest in UV4 and intermediate in UV5. These results suggest that the different levels of sensitivity of the five complementation groups to bifunctional alkylation damage are specifically related to different efficiencies of DNA cross-link removal. The phenotype of hypersensitivity to both UV radiation and cross-link damage exhibited by the mutants in Groups 2 and 4 appears to differ from those of the known human DNA repair syndromes.

Acetylcholine receptor. Binding properties and ion permeability response after covalent attachment of the local anaesthetic quinacrine.

Membrane vesicles rich in nicotinic acetylcholine receptor prepared from Torpedo californica electric tissue have been irreversibly modified with quinacrine mustard, an alkylating derivative of the local anaesthetic quinacrine. The reaction blocked the ion channel regulated by the acetylcholine receptor. Acetylcholine still bound to the modified membrane vesicles with KD approx. 10(-8). The number of binding sites was reduced by up to 50%. Stopped-flow experiments showed that in contrast to what had been found with the reversibly binding quinacrine no fluorescence changes caused by energy transfer from the irradiated protein to the fluorescent local anaesthetic occurred after addition of agonist. This indicates that the conformational changes associated with the activation of the ion channel are blocked by the covalent reaction with quinacrine mustard. Analysis of the membrane vesicles by SDS-polyacrylamide gel electrophoresis showed that all polypeptide chains assumed to be part of the receptor complex had reacted with the mustard. Even small components, probably lipids, migrating with the dye front, showed fluorescence.

Inhibition of the bovine-heart mitochondrial F1-ATPase by cationic dyes and amphipathic peptides.

The bovine heart mitochondrial F1-ATPase is inhibited by a number of amphiphilic cations. The order of effectiveness of non-peptidyl inhibitors examined as assessed by the concentration estimated to produce 50% inhibition (I0.5) of the enzyme at pH 8.0 is: dequalinium (8 microM), rhodamine 6G (10 microM), malachite green (14 microM), rosaniline (15 microM) greater than acridine orange (180 microM) greater than rhodamine 123 (270 microM) greater than rhodamine B (475 microM), coriphosphine (480 microM) greater than safranin O (1140 microM) greater than pyronin Y (1650 microM) greater than Nile blue A (greater than 2000 microM). The ATPase activity was also inhibited by the following cationic, amphiphilic peptides: the bee venom peptide, melittin; a synthetic peptide corresponding to the presence of yeast cytochrome oxidase subunit IV (WT), and amphiphilic, synthetic peptides which have been shown (Roise, D., Franziska, T., Horvath, S.J., Tomich, J.M., Richards, J.H., Allison, D.S. and Schatz, G. (1988) EMBO J. 7, 649-653) to function in mitochondrial import when attached to dihydrofolate reductase (delta 11.12, Syn-A2, and Syn-C). The order of effectiveness of the peptide inhibitors as assessed by I0.5 values is: Syn-A2 (40 nM), Syn-C (54 nM) greater than melittin (5 microM) greater than WT (16 microM) greater than delta 11,12 (29 microM). Rhodamines B and 123, dequalinium, melittin, and Syn-A2 showed noncompetitive inhibition, whereas each of the other inhibitors examined (rhodamine 6G, rosaniline, malachite green, coriphosphine, acridine orange, and-Syn-C) showed mixed inhibition. Replots of slopes and intercepts from Lineweaver-Burk plots obtained for dequalinium were hyperbolic indicating partial inhibition. With the exception of Syn-C, for which the slope replot was hyperbolic and the intercept replot was parabolic, steady-state kinetic analyses indicated that inhibition by the other inhibitors was complete. The inhibition constants obtained by steady-state kinetic analyses were in agreement with the I0.5 values estimated for each inhibitor examined. Rhodamine 6G, rosaniline, dequalinium, melittin, Syn-A2, and Syn-C were observed to protect F1 against inactivation by the aziridinium of quinacrine mustard in accord with their experimentally determined I0.5 values.(ABSTRACT TRUNCATED AT 400 WORDS)

Reconstitution of active acetylcholine receptor by hybridisation of binding site-blocked with ion channel-blocked acetylcholine receptor protein.

The nicotinic acetylcholine receptor regulates the ion permeability of the postsynaptic membrane. This report presents evidence that the transmitter binding site and the ion channel may be located on distinct subunits. By hybridisation of receptor complexes, in which the transmitter binding site was blocked with complexes in which the ion channel was irreversibly inhibited, we reconstituted active acetylcholine receptor complexes. The reconstituted system was similar to the native receptor in its ability to regulate the ion permeability of lipid vesicles in response to nicotinic cholinergic effectors.

Quinacrine mustard and lipophilic cations inhibitory to both vacuolar H(+)-ATPase and F0F1-ATP synthase.

Various lipophilic cations, such as quinacrine mustard and dequalinium, which are known to inhibit mitochondrial F1-ATPase, strongly inhibited vacuolar H(+)-ATPase purified from bovine adrenal chromaffin granules. Quinacrine mustard bound irreversibly to vacuolar H(+)-ATPase subunit A, and the 115 kDa accessory polypeptide and dithiothreitol had no effect. The binding was competitively inhibited by chlorpromazine and quinacrine, and these compounds specifically reduced the amount of labeling of subunit A. Quinacrine mustard also prevented the binding of [alpha-32P]ATP to subunit A but had no effect on the binding of [3H]N-ethylmaleimide to either subunit A or the 115 kDa accessory polypeptide. These results suggest that the binding site of quinacrine mustard in subunit A is not related to the N-ethylmaleimide-binding site(s), which is important for activity.

The modulation of the DNA-damaging effect of polycyclic aromatic agents by xanthines. Part I. Reduction of cytostatic effects of quinacrine mustard by caffeine.

Recently, accumulated statistical data indicate the protective effect of caffeine consumption against several types of cancer diseases. There are also reports about protective effect of caffeine and other xanthines against tumors induced by polycyclic aromatic hydrocarbons. One of the explanations is based on biological activation of such carcinogens by cytochromes that are also known for metabolism of caffeine. However, there is also numerous data indicating reverse effect on cytotoxicity of anticancer drugs that inhibit the action of topoisomerase I (e.g. Camptothecin or Topotecan) and topoisomerase II inhibitors (e.g. Doxorubicin, Mitoxantrone or mAMSA). In this work we tested the hypothesis that the caffeine protective effect is the result of sequestering of aromatic mutagens by formation of stacking (pi-pi) complexes. As the models for the study we have chosen two well-known mutagens, that do not require metabolical activation: quinacrine mustard(QM, aromatic, heterocyclic nitrogen mustard) and mechlorethamine (NM2, aliphatic nitrogen mustard). The flow cytometry study of these agents' action on the cell cycle of HL-60 cells indicated that caffeine prevents the cytotoxic action of QM, but not that of NM2. The formations of stacking complexes of QM with caffeine were confirmed by light absorption, calorimetric measurements and by molecular modeling calculation. Using the statistical thermodynamics calculations we calculated the "neighborhood" association constant (K(AC)=59+/-2M(-1)) and enthalpy change (DeltaH(0')=-116cal mol(-1)); the favorable entropy change of complex formation (DeltaS(0')=7.72cal mol(-1)K(-1), due to release of several water molecules, associated with components in the process of complex formation). The Gibbs' free energy change of QM-CAF formation is DeltaG(0')=-2.41kcal mol(-1). We were unable to detect any interaction between NM2 and caffeine either by spectroscopic or calorimetric measurement. In order to establish, whether the intercalation of QM plays any role in cytotoxic effect we tested, as a control, non-alkylatiatig, but also intercalating QM derivative-quinacrine (Q). The later had no cytostatic effect on HL-60 cell even at there order of higher concentration than QM or NM2 but, similar to QM forms (which we demonstrated) stacking complexes with caffeine (K(AC)=75+/-3M(-1)). These results strongly indicate, that the attenuating effect of caffeine on cytotoxic or mutagenic effects of some mutagens, is not the results of metabolic processes in the cells, but simply the physicochemical process of sequestering of aromatic molecules (potential carcinogens or mutagens) by formation of stacking complexes with them. The caffeine may then act as the "interceptor" of potential carcinogens (especially in the upper part of digesting track where its concentration can reach the concentration of mM level). There is, however, no indication either in the literature or in our experiments that xanthines can reverse the damage to nucleic acids when the damage to DNA has already occurred.

Sister chromatid exchanges--a sensitive assay of agents damaging human chromosomes.

Sister chromatid exchange frequencies in human lymphocyte chromosomes are greatly increased by alkylating agents, but ionizing radiation has little if any such effect. Scoring these exchanges may provide a useful technique for exploring the mechanisms of chromosome breakage and repair.

Adaptive enhancement and kinetics of nucleotide excision repair in humans.

An adaptive response, low doses of a mutagen rendering cells more able to subsequently cope with higher doses of that or a related challenging mutagen, enhances nucleotide excision repair in human fibroblasts. After fibroblasts were flashed with 20 J/m2 of UVC, the cyclopyrimidine dimer frequency at any single dinucleotide position remained unchanged for several hours before abruptly displaying first order kinetics of repair. These kinetics were determined by ligation-mediated PCR along exon 9 of the human p53 gene. When a chronic dose of quinacrine mustard (QM) preceded the UVC challenge, the duration of the cyclobutane pyrimidine dimer (CPD) repair lags were reduced by a factor of three and the kinetic half-lives for CPD repair were reduced by a factor of three. The observed repair kinetics are consistent with the following model. The UVC dose required (K(m)) to generate a substrate concentration which half-saturates the cell's repair capacity is 3 J/m2 for the high affinity (6-4) photoproducts and greater than 100 J/m2 for the low affinity cyclobutane dimers. After 20 J/m2 of UVC, the repair enzyme is saturated with (6-4) photoproducts; these competitively inhibit CPD repair by binding all available repair enzyme. After the (6-4)s are repaired, the CPD concentration is less than K(m)(CPD) and so CPD repair kinetics initiate with first order kinetics. QM-induced enhancement, by increasing the concentration, Vmax, of repair enzyme, shortens the duration of (6-4) saturation and increases the rate constant for cyclobutane dimer repair. The data exactly fit the expectations from Michaelis kinetics. Transcription coupled repair is less amenable to Michaelis interpretations and enhanced global repair was almost as rapid as the slightly enhanced transcription coupled repair. We infer that repair enhancement is unable to proportionally increase the number of matrix attachment sites necessary for transcription coupled repair. Understanding competitive inhibition between adduct classes and adaptive enhancement of Vmax is important to understanding the effects of high doses of mutagen mixtures.

Comparative mutagenicity of 4 DNA-intercalating agents in L5178Y mouse lymphoma cells.

The mutagenicity of 4 known intercalating agents acridine orange (AO), quinacrine mustard (QM), proflavin (PF) and ethidium bromide (EB) has been investigated in L5178Y mouse lymphoma cells. Methyl methanesulfonate (MMS) was used as a positive control in these studies. AO, QM and PF induced mutation in the excess thymidine- and thioguanine-selective systems. These 3 compounds were negative in the ouabain- and cytosine-arabinoside-selective systems while EB was positive only in the cytosine arabinoside system. It would appear that the EB-induced mutagenesis is different from that of AO, QM and PF though all are intercalating agents. Since the molecular origin of cytosine arabinoside mutants is unknown, further interpretation of the EB results is not possible.

Induction of sister-chromatid exchanges and exchange aberrations by UV light and quinacrine mustard in relation to chiasma formation in a standard line and two oligochiasmatic mutants of Vicia faba L.

The frequencies of sister-chromatid exchanges (SCE) and exchange aberrations (EA) in root tip cells were compared to chiasma formation in pollen mother cells of a standard line and two oligochiasmatic lines of Vicia faba L. SCE and EA were induced by UV light and quinacrine mustard. Between the lines SCE frequencies were not different. The background level of SCE was doubled after UV irradiation and 4 times higher after exposure to quinacrine mustard in all Vicia lines analysed. However, the induced frequencies of EA were found to be different under the same treatment conditions for the standard line and the oligochiasmatic mutants. Between the frequencies of induced EA and the frequencies of chiasmata a correlation could be shown. The relationship between the formation of SCE and EA due to the reduced ability of meiotic recombination in the mutants of Vicia faba is discussed.

[Genetic control of mitotic crossing over in yeasts. IV. The induction of mitotic crossing over by chemical mutagens with different mechanisms of action]. / Geneticheskii kontrol' mitoticheskogo krossingovera u drozhzhei. Soobshchenie IV. Induktsiia mitoticheskogo krossingovera khimicheskimi mutagenami raznogo mekhanizma deistviia.

The genetic effects of methyl methanesulphonate (MMS) and bifunctional quinacrine mustard (QM) have been studied in three diploid strains of the yeast Saccharomyces cerevisiae: T1, with normal radiosensitivity, T2 - the excision-deficient mutant (rad2 rad2) and T3 - the mutant defective in recombinational repair (rad54 rad54). The strain T3 was much more sensitive to the lethal action of MMS than T1, but T2 did not differ from T1. The strain T2 was more sensitive to QM than T1 and T3. Both mutagens induce mitotic crossing over in T2 at a higher frequency than T1. MMS is not able to induce mitotic crossing over in T3 and QM demonstrates a very low induction. Treatment of the strains T1 and T2 with MMS and T1 with QM induces mitotic crossing over during the first cell division more often than during the second one. In most cases, QM induces mitotic crossing over in cells of the strain T2 during the second division. We suppose that the damages of DNA induced by QM in the wild type cells can be excised, but in the rad2 cells the gaps in DNA appeared after replication. In both cases, single-strand breaks of DNA are the main reason for mitotic crossing over.

Inhibition of heparin-induced tau filament formation by phenothiazines, polyphenols, and porphyrins.

Tau protein is the major component of the intraneuronal filamentous inclusions that constitute defining neuropathological characteristics of Alzheimer's disease and other tauopathies. The discovery of tau gene mutations in familial forms of frontotemporal dementia has established that dysfunction of the tau protein is sufficient to cause neurodegeneration and dementia. Here we have tested 42 compounds belonging to nine different chemical classes for their ability to inhibit heparin-induced assembly of tau into filaments in vitro. Several phenothiazines (methylene blue, azure A, azure B, and quinacrine mustard), polyphenols (myricetin, epicatechin 5-gallate, gossypetin, and 2,3,4,2',4'-pentahydroxybenzophenone), and the porphyrin ferric dehydroporphyrin IX inhibited tau filament formation with IC(50) values in the low micromolar range as assessed by thioflavin S fluorescence, electron microscopy, and Sarkosyl insolubility. Disassembly of tau filaments was observed in the presence of the porphyrin phthalocyanine. Compounds that inhibited tau filament assembly were also found to inhibit the formation of Abeta fibrils. Biochemical analysis revealed the formation of soluble oligomeric tau in the presence of the inhibitory compounds, suggesting that this may be the mechanism by which tau filament formation is inhibited. The compounds investigated did not affect the ability of tau to interact with microtubules. Identification of small molecule inhibitors of heparin-induced assembly of tau will form a starting point for the development of mechanism-based therapies for the tauopathies.

Simultaneous staining of sister chromatid exchanges and Q-bands in human chromosomes after treatment with methyl methane sulphonate, quinacrine mustard, and quinacrine.

Human peripheral lymphocyte chromosomes were stained simultaneously for sister chromatid exchanges (SCEs) and Q-banding. No effect of treatment with MMS, QM, and Q on the distribution of SCEs in chromosomes was found compared with controls. The SCEs were distributed between chromosomes roughly according to metaphase length, with the shorter chromosomes underrepresented. The majority of SCEs were located to pale bands, while a few occurred in bright bands and at interfaces between pale and bright bands. A greater frequency than expected of SCEs had occurred at identical sites in homologous chromosomes. This frequency was significantly increased after treatment with MMS.

Characterization of the catalytic and noncatalytic ADP binding sites of the F1-ATPase from the thermophilic bacterium, PS3.

Two classes of ADP binding sites at 20 degrees C have been characterized in the F1-ATPase from the thermophilic bacterium, PS3 (TF1). One class is comprised of three sites which saturate with [3H]ADP in less than 10 s with a Kd of 10 microM which, once filled, exchange rapidly with medium ADP. The binding of ADP to these sites is dependent on Mg2+. [3H]ADP bound to these sites is removed by repeated gel filtrations on centrifuge columns equilibrated with ADP free medium. The other class is comprised of a single site which saturates with [3H]ADP in 30 min with a Kd of 30 microM. [3H]ADP bound to this site does not exchange with medium ADP nor does it dissociate on gel filtration through centrifuge columns equilibrated with ADP free medium. Binding of [3H]ADP to this site is weaker in the presence of Mg2+ where the Kd for ADP is about 100 microM. [3H]ADP dissociated from this site when ATP plus Mg2+ was added to the complex while it remained bound in the presence of ATP alone or in the presence of ADP, Pi, or ADP plus Pi with or without added Mg2+. Significant amounts of ADP in the 1:1 TF1.ADP complex were converted to ATP in the presence of Pi, Mg2+, and 50% dimethyl sulfoxide. Enzyme-bound ATP synthesis was abolished by chemical modification of a specific glutamic acid residue by dicyclohexylcarbodiimide, but not by modification of a specific tyrosine residue with 7-chloro-4-nitrobenzofurazan. Difference circular dichroism spectra revealed that the three Mg2+ -dependent, high affinity ADP binding sites that were not stable to gel filtration were on the alpha subunits and that the single ADP binding site that was stable to gel filtration was on one of the three beta subunits. It has also been demonstrated that enzyme-bound ATP is formed when the TF0.F1 complex containing bound ADP was incubated with Pi, Mg2+, and 50% dimethyl sulfoxide.

DNA sequence selectivity of guanine-N7 alkylation by nitrogen mustards.

Nitrogen mustards alkylate DNA primarily at the N7 position of guanine. Using an approach analogous to that of the Maxam-Gilbert procedure for DNA sequence analysis, we have examined the relative frequencies of alkylation for a number of nitrogen mustards at different guanine-N7 sites on a DNA fragment of known sequence. Most nitrogen mustards were found to have similar patterns of alkylation, with the sites of greatest alkylation being runs of contiguous guanines, and relatively weak alkylation at isolated guanines. Uracil mustard and quinacrine mustard, however, were found to have uniquely enhanced reaction with at least some 5'-PyGCC-3' and 5'-GT-3' sequences, respectively. In addition, quinacrine mustard showed a greater reaction at runs of contiguous guanines than did other nitrogen mustards, whereas uracil mustard showed little preference for these sequences. A comparison of the sequence-dependent variations of molecular electrostatic potential at the N7-position of guanine with the sequence dependent variations of alkylation intensity for mechlorethamine and L-phenylalanine mustard showed a good correlation in some regions of the DNA, but not others. It is concluded that electrostatic interactions may contribute strongly to the reaction rates of cationic compounds such as the reactive aziridinium species of nitrogen mustards, but that other sequence selectivities can be introduced in different nitrogen mustard derivatives.

Study of the mechanism of MF1 ATPase inhibition by fluorosulfonylbenzoyl inosine, quinacrine mustard, and efrapeptin using intermediate 18O exchange as a probe.

The mitochondrial F1-ATPase (MF1) is known to be largely or totally inhibited by combination or reaction with one fluorosulfonylbenzoyl inosine (FSBI), quinacrine mustard, or efrapeptin per enzyme. Measurements were made with 18O in attempt to ascertain if the weak catalytic activity remaining after exposure to excess of these reagents was due to retention of some activity by the enzyme modified by these inhibitors. Any such activity could have different characteristics that might be revealed by the distribution of [18O]Pi isotopomers formed from [gamma-18O]ATP. The MF1 inhibited by FSBI showed progressive appearance of two new catalytic pathways as inhibition proceeded. Both pathways appeared to be operative in the enzyme after one beta subunit per enzyme had been modified by FSBI. A high exchange pathway showed no detectable change as ATP concentration was lowered. The lower exchange pathway showed an increase in the amount of exchange with lowering of the ATP concentration, similar to the cooperative behavior observed with the unmodified enzyme. With excess ATP more product was formed by the low exchange pathway, showing that compulsory alternation between two catalytic sites was not retained. The behavior can be explained by the ability of the modified beta subunit to undergo binding changes similar to those occurring in catalysis, with the other two beta subunits catalyzing sluggish hydrolysis by different pathways because of the asymmetry introduced by the modification. Inhibition by quinacrine mustard also resulted in the appearance of two new pathways, somewhat similar to those from FSBI inhibition. In contrast, activity remaining with excess efrapeptin present showed only one pathway like that of the native enzyme. This can be attributed to a low equilibrium concentration of free enzyme and total inhibition of MF1 combined with efrapeptin.

DNA sequence selectivity of guanine-N7 alkylation by nitrogen mustards is preserved in intact cells.

Nitrogen mustard alkylating agents react with isolated DNA in a sequence selective manner, and the substituent attached to the drug reactive group can impose a distinct sequence preference. It is not clear however to what extent the observed DNA sequence preferences are preserved in intact cells. The highly reiterated sequence of human alpha DNA has been used to determine the sites of guanine-N7 alkylation following treatment of cells with three nitrogen mustards, mechlorethamine, uracil mustard and quinacrine mustard, known to react in isolated DNA with distinctly different sequence preferences. Alpha DNA from drug treated cells was extracted, purified, end-labeled, and a 296 base pair, singly end-labelled, fragment isolated. Following the quantitative conversion of alkylation sites to strand breaks the fragments were separated on DNA sequencing gels. Clear differences were observed between the alkylation patterns of the three compounds, and the selectivities were qualitatively similar to those predicted and observed in the same sequence alkylated in vitro. In particular the unique preferences of uracil and quinacrine mustards for 5'-PyGC-3' and 5'-GT/GPu-3' sequences, respectively, were preserved in intact cells suggesting that the pattern of sequence dependent reactivity is not grossly affected by the nuclear milieu.

Quinacrine mustard inactivates the bovine heart mitochondrial F1-ATPase with the modification of the beta subunit.

The bovine mitochondrial F1-ATPase is reversibly inhibited by quinacrine. The concentration of quinacrine which causes 50% inhibition at pH 7.0 is estimated to be 580 microM. Lineweaver-Burk plots constructed from kinetic data collected at pH 7.0 with equimolar concentrations of Mg2+ and ATP in the reaction mixtures indicate competitive inhibition by quinacrine. Uncompetitive inhibition by quinacrine is indicated by Lineweaver-Burk plots constructed from kinetic data obtained at pH 7.0 with variable ATP concentrations and a constant Mg2+ concentration of 3.0 mM. A KI of 440 microM was calculated from a replot of 1/Vmaxi versus quinacrine concentration using the intercepts of the Lineweaver-Burk plots constructed from the data obtained at a fixed concentration of 3.0 mM Mg2+. Quinacrine mustard is a potent inactivator of the F1-ATPase. The pseudo-first order rate constant, k1, for the inactivation of the enzyme by 500 microM quinacrine mustard is 0.161 min-1 at pH 7.0 and 23 degrees C. Under the same conditions in the presence of 5.0 mM ATP, 5.0 mM ADP, or 5.0 mM Mg2+ plus 5.0 mM ADP, k1 is, respectively: 0.082 min-1, 0.136 min-1, or 0.075 min-1. In the presence of 1.0 mM chlorpromazine or 5.0 mM quinacrine under the above conditions, k1, is 0.089 min-1 and 0.037 min-1, respectively. Free Mg2+ has no effect on the rate of inactivation of the enzyme by quinacrine mustard. The rate of inactivation of the F1-ATPase by quinacrine mustard as a function of pH revealed an apparent pK alpha of 8.0. Examination of the resolved subunits for fluorescence after inactivating the enzyme with quinacrine mustard demonstrated that only the beta subunit was labeled.

Comparison of in vitro and in vivo histamine methylation by tissues.

A method for determining the histamine-methylating enzyme (HME) using crude enzyme, and minute quantities of the substrate, was applied to tissues of mice, guinea-pigs and rats. Since high levels of endogenous histamine can affect the results, tissue homogenates were dialyzed prior to incubation. Findings were compared with in vivo data on methylating ability of individual tissues; most of this in vivo data is published but a new test of guinea-pig tissues was made using amodiaquine as an inhibitor. The correlation was good, better than that obtained by other procedures. It was observed that dialysis caused an increase in HME for some guinea-pig tissues, but a loss for some mouse tissues. Possible explanations are considered. Quinacrine N-mustard, a derivative of a known HME inhibitor, was tested in mice; it altered the distribution of injected 14C-histamine but showed no evidence of HME inhibition.

Comparative pharmacokinetics of DNA lesion formation and removal following treatment of L1210 cells with nitrogen mustards.

The kinetics of formation and removal of DNA interstrand crosslinks (ISC), DNA-protein crosslinks (DPC), and single strand breaks (SSB) by several nitrogen mustards were compared in order to determine the degree to which lesion selectivity may vary. The kinetic measurements using DNA alkaline elution methodology were obtained in mouse L1210 cells treated with mechlorethamine (HN2), phenylalanine mustard (L-PAM), uracil mustard (UM), 6-methyl-UM, and quinacrine mustard (QM). The ISC or DPC challenge delivered to cells was gauged on the basis of the kinetics as either total ISC or DPC produced, or as the area under the lesions versus time curve (AUC). By either measure (excepting QM), ISC correlated well with loss of colony survival, whereas DPC did not. The ISC/DPC ratio may therefore be a useful index of lesion selectivity. This ratio was significantly greater for 6-methyl-UM than for HN2. The ratio was also greater for L-PAM than for HN2 but only when gauged by AUC; this was attributable to an unusually slow rate for ISC removal in the case of L-PAM. The preferential reaction of UM at some 5'-GC-3' sites in purified DNA had suggested that UM might produce ISC with increased efficacy. UM, however, was somewhat less efficacious in ISC production than was 6-methyl-UM, which lacked selectivity for alkylation at 5'-GC-3'. QM was the only compound that produced detectable SSB, and the SSB were so numerous that ISC could not be quantitated.(ABSTRACT TRUNCATED AT 250 WORDS)