The role of nick formation in delayed quinacrine mustard fluorescence in the C-heterochromatin of Apodemus argenteus.

Chromosomes stained with fluorochromes, including quinacrine mustard (QM), emit the brightest fluorescence immediately after exposure to excitation light, and the fluorescence gradually fades with an increase in exposure time. However, in the QM-stained chromosomes of the small Japanese field mouse Apodemus argenteus, most C-heterochromatic regions emit weak fluorescence immediately after exposure to blue light, and they become brightly fluorescent by prolonged exposure (delayed QM fluorescence). We proposed recently that the delayed QM fluorescence is somehow related to nicks produced in C-heterochromatic DNA by blue light irradiation. To test this possibility, we examined the chromosomal distribution of nicks by in-situ nick translation and changes, if any, in the QM fluorescence pattern after methylene blue (MB) -mediated photooxidation, which is considered to induce nicks in chromosomal DNA. It was found that C-heterochromatic regions fluoresced brightly without any delay after exposure to blue light, and that nicks increased considerably in the same regions after the MB-mediated photooxidation. It seems, therefore, that photooxidation and strand breaks in DNA (including nicks) are responsible for the induction of delayed QM fluorescence. Trypsin digestion, on the other hand, abolished delayed QM fluorescence. Thus, not only DNA but also chromosomal protein(s) are involved in the unusual sequence of QM fluorescence patterns in A. argenteus.

The delayed quinacrine mustard fluorescence from the C-blocks of Apodemus argenteus is due to the introduction of nicks into the DNA.

"Delayed QM-fluorescence" refers to the unusual kinetics of fluorescence from most of the C-heterochromatic regions of the chromosomes of the small Japanese field mouse Apodemus argenteus. When stained with quinacrine mustard (QM-stained), these C-heterochromatic regions emit weak fluorescence immediately after exposure to blue light (BL); they emit bright fluorescence within a few minutes; and the intensity of the fluorescence gradually decreases after maximum fluorescence has been recorded. To elucidate the mechanism of this phenomenon, we used acridine orange staining (AO-staining) and a modified version of the in situ nick-translation method. Focusing on the large C-heterochromatic region (C-block) of the X chromosome, we noted that AO-stained C-blocks emitted greenish fluorescence, while QM-stained and BL-exposed (QM-BL-processed) C-blocks emitted reddish fluorescence upon AO-staining after removal of QM. These findings suggested that the C-block DNA of A. argenteus might undergo a structural change, such as strand breaks, during QM-BL processing. Application of the modified in situ nick-translation method revealed the generation of an appreciable number of nicks in the C-block DNA by QM-BL processing. No such nick formation was observed in the C-blocks of three other mammalian species: Apodemus peninsulae, Microtus montebelli, and Urotrichus talpoides. Our findings support the hypothesis that nick formation due to exposure to BL might play a primary role in inducing delayed QM-fluorescence in the C-blocks of A. argenteus. On the basis of the present and earlier findings, we propose a probable mechanism for delayed QM-fluorescence in A. argenteus chromosomes.

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.

Two interacting binding sites for quinacrine derivatives in the active site of trypanothione reductase: a template for drug design.

Trypanothione reductase is a key enzyme in the trypanothione-based redox metabolism of pathogenic trypanosomes. Because this system is absent in humans, being replaced with glutathione and glutathione reductase, it offers a target for selective inhibition. The rational design of potent inhibitors requires accurate structures of enzyme-inhibitor complexes, but this is lacking for trypanothione reductase. We therefore used quinacrine mustard, an alkylating derivative of the competitive inhibitor quinacrine, to probe the active site of this dimeric flavoprotein. Quinacrine mustard irreversibly inactivates Trypanosoma cruzi trypanothione reductase, but not human glutathione reductase, in a time-dependent manner with a stoichiometry of two inhibitors bound per monomer. The rate of inactivation is dependent upon the oxidation state of trypanothione reductase, with the NADPH-reduced form being inactivated significantly faster than the oxidized form. Inactivation is slowed by clomipramine and a melarsen oxide-trypanothione adduct (both are competitive inhibitors) but accelerated by quinacrine. The structure of the trypanothione reductase-quinacrine mustard adduct was determined to 2.7 A, revealing two molecules of inhibitor bound in the trypanothione-binding site. The acridine moieties interact with each other through pi-stacking effects, and one acridine interacts in a similar fashion with a tryptophan residue. These interactions provide a molecular explanation for the differing effects of clomipramine and quinacrine on inactivation by quinacrine mustard. Synergism with quinacrine occurs as a result of these planar acridines being able to stack together in the active site cleft, thereby gaining an increased number of binding interactions, whereas antagonism occurs with nonplanar molecules, such as clomipramine, where stacking is not possible.

Alleviation of mutagenic effects of polycyclic aromatic agents (quinacrine mustard, ICR-191 and ICR-170) by caffeine and pentoxifylline.

Previous studies performed by others indicated that apart from its other biological effects, caffeine (CAF) may have a role in protection of organisms against cancer. However, biological mechanism of this phenomenon remained unknown. Recent studies suggested that caffeine can form stacking (pi-pi) complexes with polycyclic aromatic chemicals. Therefore, one might speculate that effective concentrations of polycyclic aromatic mutagens could be reduced in the presence of caffeine. Here we demonstrate that caffeine and another xanthine, pentoxifylline (PTX), effectively alleviate mutagenic action of polycyclic aromatic agents (exemplified by quinacrine mustard (QM), 2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine.2HCl (ICR-191) and 1,3,7-propanediamine-N-(2-chloroethyl)-N'-(6-chloro-2-methoxy-9-acridinyl)-N-ethyl.2HCl (ICR-170)), but not of aliphatic mutagens (exemplified by mechlorethamine), in the recently developed mutagenicity test based on bacterium Vibrio harveyi. Biophysical studies indicated that caffeine and pentoxifylline can form stacking complexes with the aromatic agents mentioned above. Molecular modeling also confirmed a possibility of stacking interactions between examined molecules.

Interactions of chlorophyllin with acridine orange, quinacrine mustard and doxorubicin analyzed by light absorption and fluorescence spectroscopy.

The present study was designed to estimate the ability of chlorophyllin (CHL) to interact with two acridine mutagens, quinacrine mustard (QM) and acridine orange (AO), and with the antitumor anthracycline doxorubicin (Dox). To this end, aqueous solutions of QM, AO or Dox during titration with CHL were subjected to spectrophotometry and spectrofluorimetry to detect possible interactions between these reagents. The data indicate that CHL forms complexes with AO, QM or Dox in these solutions. The presence of the complexes was manifested by a bathochromic shift of the absorption spectra, as well as by strong quenching of the fluorescence of each of these mutagens in the presence of CHL. CHL, thus, may serve as an interceptor of these mutagenic acridines in different in vivo or in vitro applications. Its ability to interact with Dox may potentially be utilized to detoxify patients overdosed with this or similar drugs.

Structural effects of quinacrine binding in the open channel of the acetylcholine receptor.

Noncompetitive inhibitors of the nicotinic acetylcholine (ACh) receptors suppress cation flux directly by binding in and blocking the open channel or indirectly by stabilizing closed states of the receptor. The lidocaine derivative QX-314 and the acridine derivative quinacrine act directly as open channel blockers, but can act indirectly as well. The binding site for quinacrine in the open channel of mouse-muscle ACh receptor was mapped in cysteine-substituted mutants of the alpha subunit expressed with wild-type beta, gamma, and delta subunits. In the open state, substituted cysteines in the inner half of the second membrane-spanning segment (M2), but not in the outer half, were protected by quinacrine from reaction with 2-aminoethyl methanethiosulfonate. In addition, an alkylating derivative, quinacrine mustard, affinity labeled a subset of the substituted cysteines in M2, but only in the open state. These results, mapped onto a model of the open channel surrounded by five alpha-helical M2s, imply that quinacrine binds midway down M2 in the same site previously mapped for QX-314. A cysteine substituted for a residue in the outer third of alphaM1, which reacted with 2-aminoethyl methanethiosulfonate only in the presence of ACh, reacted faster in the additional presence of quinacrine or QX-314. It is proposed that channel opening involves both the opening of the resting gate at the inner end of M2 and the removal of an obstruction formed by the outer end of M1 that retards diffusion of blockers into the closed channel. Blocker binding in the open channel causes a further change in structure.

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.

Molecular cytogenetic analysis of the highly repetitive DNA in the genome of Apodemus argenteus, with comments on the phylogenetic relationships in the genus Apodemus.

The DNA of Apodemus argenteus was digested with DraI, and the resultant DraI fragment of highly repetitive DNA was isolated and analyzed by DNA filter hybridization, cloning, sequencing, and fluorescence in situ hybridization (FISH). Southern blot hybridization and nucleotide sequencing revealed that most of the DraI fragment consisted of a 230-bp repeating unit and contained no sex-chromosome-specific nucleotide sequences. The DraI fragment included the CENP-B box-like sequence, with a strong homology to the human CENP-B box sequence. FISH revealed that the DraI fragment was specific to all pericentromeric C-band-positive regions, as well as to the C-block of the X chromosome. No hybridization signals were obtained from A. speciosus, A. peninsulae peninsulae, A.p. giliacus, A. agrarius, A. sylvaticus, A. semotus, or Mus musculus when the DraI fragment was used as probe. Peptide nucleic acid (PNA)-FISH using the CENP-B box-like sequence in the DraI fragments as probe suggested that this nucleotide sequence was also specific to all pericentromeric C-heterochromatic regions of A. argenteus chromosomes. Zoo-blot hybridization using DraI-digested genomic DNA from three species of Apodemus (namely, A. argenteus, A. speciosus, and A. peninsulae) and from Mus musculus strongly suggested that the consensus DraI fragment contained nucleotide sequences that were species-specific for A. argenteus. These results also suggest that A. argenteus is phylogenetically distant from other Apodemus species examined, as well as the possibility that the DraI fragment might be related directly to the delayed quinacrine mustard fluorescence of many pericentromeric C-heterochromatic regions of the chromosomes in A. argenteus.

Chlorophyllin protects cells from the cytostatic and cytotoxic effects of quinacrine mustard but not of nitrogen mustard.

Chlorophyllin (CHL), the sodium and copper salt of chlorophyll, is capable of inhibiting the mutagenic activity of many chemical compounds. Several mechanisms have been advanced to explain the antimutagenic activity of CHL, including its antioxidant properties and its ability to form complexes with mutagens. The present study was designed to reveal whether the heterocyclic aromatic nature of a potential mutagen is essential to its sensitivity to CHL. Toward this end, the inhibitory effect of CHL on two compounds of similar chemical reactivity (mustards), that either embodied an aromatic structure (quinacrine mustard; QM) or did not (nitrogen mustard; NM), were compared. Human leukemic HL-60 and breast carcinoma MCF-7 cells were treated with QM or NM in the absence or presence of various concentrations of CHL. Both QM and NM when administered for 1-2 h at micromolar concentrations exerted similar effects; both arrested cells in G2 phase of the cell cycle, induced apoptosis and reduced the clonogenicity of MCF-7 cells. The simultaneous addition of 0.22 M CHL to cultures receiving QM virtually abolished the QM-induced inhibition of cell growth and clonogenicity. In contrast, CHL had no effect on reducing the cytostatic or cytotoxic activity of NM. CHL alone, at a concentration of 0.22 M, had minimal effect on growth of HL-60 cells slightly perturbing their progression through G2. The results are consistent with the model that explains the inhibition of the activity of mutagens or antitumor drugs with aromatic structures by CHL as mediated by its ability to sequester these molecules within heterologous mutagen:CHL complexes that are maintained by stacking interactions. Therefore, excess of chlorophyll in the diet, by sequestering aromatic mutagens (or antitumor drugs with a heterocyclic structure, if taken orally), may inhibit their accessibility to cells, thereby reducing their activity.

In vivo biotinylation studies: specificity of labelling of reticulated platelets by thiazole orange and mepacrine.

Animal in vivo biotinylation studies have demonstrated that thiazole orange (TO) labels the youngest cells in the circulation. TO has since been widely used for the measurement of reticulated platelets. As recent findings suggest that at high concentrations TO also labels platelet dense granules non-specifically, the value of previous work is unclear. Mepacrine also labels platelet dense granules and can detect storage pool defects. In this study, a mouse in vivo biotinylation model was used to determine the specificity of TO and mepacrine staining on platelets recently released into the circulation. The mean life span of biotin/TO (low), biotin/TO (high) and mepacrine/TO dual-positive platelets was 1.4 d (SD 0.5), 2.2 d (SD 0.2) and 2.3 d (SD 0.3) respectively (n = 6) compared with a life span for biotin-positive platelets of 4.9 d (SD 1.6). TO (low), TO (high) and mepacrine labelled 8.0% (SD 3.1), 43.9% (SD 8.3) and 40.0% (SD 9.9) of the total platelet population respectively (results of 30 samples from six mice), which decreased to 6.8% (SD 3. 9), 26.6% (SD 6.9) and 25.7% (SD 10.6) after thrombin degranulation. The shorter life span and lack of thrombin sensitivity of TO (low)-positive platelets, suggests that TO (low) measures reticulated platelets specifically. The comparative life spans and thrombin sensitivity of TO (high) and mepacrine-positive platelets suggest that TO (high) labels platelet dense granules. These data also suggest that dense granules are lost during platelet ageing.

Separation of sperm through a 12-layer percoll column decreases the percentage of sperm staining with quinacrine.

Previous methods of enriching sperm with a higher percentage of Y-bearing sperm have been questioned because the claims that Y enrichment was present were based on quinacrine staining of the Y chromosome, and the enrichment was not confirmed by polymerase chain reaction (PCR) or fluorescent in situ hybridization (FISH) techniques. A technique was evaluated that theoretically could increase the percentage of X-bearing sperm by isolating a fraction of the "heaviest" sperm by passing them through 12 layers of discontinuous Percoll gradient. Initially 12 specimens were checked both before and then after separation with 12 layers of Percoll for percentage of Y sperm. The median for baseline Y percentage was 49% and after processing the percentage of Y dropped to 10%. An additional 19 specimens were checked after separation only. The median was 19%. The sample with the lowest preseparation % of quinacrine staining sperm was 45% and the highest was 54%. After 12-layer Percoll, the lowest percentage was 3% and the highest was 24%. There have been claims that quinacrine staining can falsely increase apparent Y-bearing sperm enrichment following certain separation procedures, e.g.. albumin separation, by nonspecific staining of autosomal chromosomes. If anything, then, it should falsely decrease X-bearing sperm enrichment. Thus, 12-layer Percoll separation may actually enrich for X-bearing sperm or possibly this procedure somehow nonspecifically inhibits the ability of quinacrine to stain the Y chromosome.

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.

Delayed response of QM- and DA/DAPI-fluorescence in C-heterochromatin of the small Japanese field mouse, Apodemus argenteus.

The small Japanese field mouse Apodemus argenteus has the diploid chromosome number of 46, carrying rather large centromeric C-heterochromatin in most of the 44 autosomes and a large amount of C-heterochromatin in the sex chromosomes: the largest subtelocentric X was heterochromatic in almost two-fifth (whole short arm and proximal part of the long arm) of its entire length and the medium-sized acrocentric Y was totally heterochromatic. The C-heterochromatin (C-positive) areas, other than those of the Y and smallest three pairs, had a unique property of "delayed QM-fluorescence", which has not been reported to-date, showing dull QM-fluorescence immediately after exposure to blue light (BL), but gradually turning to bright fluorescence in a few minutes. The fluorescence intensity gradually decreased after attaining its peak, and finally became extinct. A similar pattern of fluorescence was also obtained in DA/DAPI-stained-X chromosome C-heterochromatin, but not in autosomal C-heterochromatin. No such dull-to-bright transition of QM-fluorescence could be obtained by CMA staining, for which the C-positive areas were apparently negative even after overexposure to BL. These facts indicate that the C-positive areas of A. argenteus showing dull-to-bright transition of QM-fluorescence contain A-T rich DNA. The delayed QM-fluorescence was found only in A. argenteus, in thirteen mammalian species so-far examined. Furthermore, this unique property of QM-fluorescence could be artificially altered to non-delayed ordinary type of fluorescence by sequentially pretreating the fixed chromosomes with hydrochloride and barium hydroxide solutions. The cytological implication of the delayed fluorescence in the C-heterochromatin of A. argenteus is briefly discussed.

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.

Differential inhibition of the DNA binding of transcription factors NF kappa B and OTF-1 by nitrogen mustard and quinacrine mustard: transcriptional implications.

Nitrogen mustard (HN2) and quinacrine mustard (QM) both inhibited the binding of NF kappa B to the GC-rich consensus sequence in the HIV long terminal repeat (LTR), as assessed by gel-shift assays. QM also inhibited the binding of OTF-1 to the AT-rich octamer present in the H2B promoter whereas HN2 was inactive. Inhibition of the binding of transcription factors was due to the drug interaction with DNA, since it also occurred when transcription factors were added to DNA after removal of free drug. In Jurkat cells transfected with pI3CAT, where the chloramphenicol acetyltransferase (CAT) gene is under the control of the HIV LTR, both HN2 and QM inhibited CAT gene expression. However, in Jurkat cells transfected with plasmid -147, where the CAT gene is under the control of the H2B promoter, QM inhibited CAT expression but HN2 did not. These results were obtained at concentrations of HN2 or QM that inhibited total DNA and RNA synthesis to a similar extent. The present results suggest that the more selective pharmacological activity of HN2 (HN2 is an active antineoplastic agent whereas QM is inactive and very toxic) might be related to its preferential functional inhibition of GC-rich consensus sequence, possibly important in the regulation of genes involved in the malignant proliferation and behavior of some tumors.

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.

Use of [8-3H]guanine-labeled deoxyribonucleic acid to study alkylating agent reaction kinetics and stability.

Alkylation at the N7 position of guanine in DNA renders the C8-hydrogen acidic. This serves as the basis for an assay of guanine N7 alkylation using [8-3H]-guanine-labeled DNA. I modified the assay by preparing a high specific activity substrate in vitro and by replacing the distillation step with charcoal adsorption of substrate. Using the appearance of noncharcoal-adsorbable label as a measure of guanine-N7 alkylation I examined the reaction of DNA with dimethyl sulfate and mechlorethamine. The rate of reaction of dimethyl sulfate with the N7 position of guanine in DNA was constant over time, i.e., loss of label from DNA proceeded linearly with time. On the other hand, the rate of reaction of mechlorethamine with DNA increased with time, consistent with the initial formation of the reactive aziridinium ion. The assay can also be used to compare the reaction rates of various alkylating agents with DNA. Thus, the acridine mustards ICR-170 and quinacrine mustard were far more potent alkylating agents than mechlorethamine. Furthermore the assay may be used to determine the alkylating potency and stability of various alkylating agent preparations: while frozen solutions of acridine mustards in organic solvents retained alkylating activity for several months, different commercial preparations of quinacrine mustard had little or no alkylating activity.