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European Journal of Biochemistry May 1985Cells possessing a known enzymic activity may be located by fluorescent probes designed to act as competitive inhibitors of this enzyme. We have prepared a series of...
Cells possessing a known enzymic activity may be located by fluorescent probes designed to act as competitive inhibitors of this enzyme. We have prepared a series of dansyl N-substituted guanidino derivatives which bind to the active centre of guanidinobenzoatase. 9-Aminoacridine also acts as a competitive inhibitor and behaves similarly to these guanidino derivatives. These fluorescent probes have been used to locate tumour cells possessing this enzyme in thin sections of fixed tissue by employing fluorescent microscopy.
Topics: Aminacrine; Animals; Binding Sites; Binding, Competitive; Carboxylic Ester Hydrolases; Carcinoma, Ehrlich Tumor; Dansyl Compounds; Endopeptidases; Fluorescent Dyes; Humans; Mice; Neoplasms, Experimental; Rabbits; Spectrometry, Fluorescence; Staining and Labeling; Urinary Bladder Neoplasms
PubMed: 2581779
DOI: 10.1111/j.1432-1033.1985.tb08889.x -
The Journal of General Physiology Apr 1985The time-, frequency-, and voltage-dependent blocking actions of several cationic drug molecules on open Na channels were investigated in voltage-clamped, internally...
The time-, frequency-, and voltage-dependent blocking actions of several cationic drug molecules on open Na channels were investigated in voltage-clamped, internally perfused squid giant axons. The relative potencies and time courses of block by the agents (pancuronium [PC], octylguanidinium [C8G], QX-314, and 9-aminoacridine [9-AA]) were compared in different intracellular ionic solutions; specifically, the influences of internal Cs, tetramethylammonium (TMA), and Na ions on block were examined. TMA+ was found to inhibit the steady state block of open Na channels by all of the compounds. The time-dependent, inactivation-like decay of Na currents in pronase-treated axons perfused with either PC, 9-AA, or C8G was retarded by internal TMA+. The apparent dissociation constants (at zero voltage) for interaction between PC and 9-AA with their binding sites were increased when TMA+ was substituted for Cs+ in the internal solution. The steepness of the voltage dependence of 9-AA or PC block found with internal Cs+ solutions was greatly reduced by TMA+, resulting in estimates for the fractional electrical distance of the 9-AA binding site of 0.56 and 0.22 in Cs+ and TMA+, respectively. This change may reflect a shift from predominantly 9-AA block in the presence of Cs+ to predominantly TMA+ block. The depth, but not the rate, of frequency-dependent block by QX-314 and 9-AA is reduced by internal TMA+. In addition, recovery from frequency-dependent block is not altered. Elevation of internal Na produces effects on 9-AA block qualitatively similar to those seen with TMA+. The results are consistent with a scheme in which the open channel blocking drugs, TMA (and Na) ions, and the inactivation gate all compete for a site or for access to a site in the channel from the intracellular surface. In addition, TMA ions decrease the apparent blocking rates of other drugs in a manner analogous to their inhibition of the inactivation process. Multiple occupancy of Na channels and mutual exclusion of drug molecules may play a role in the complex gating behaviors seen under these conditions.
Topics: Aminacrine; Animals; Axons; Cations, Monovalent; Cesium; Decapodiformes; Electrophysiology; Guanidines; Ion Channels; Pancuronium; Quaternary Ammonium Compounds; Sodium; Time Factors
PubMed: 2409221
DOI: 10.1085/jgp.85.4.603 -
The Biochemical Journal Feb 1985The synthesis of a new bifunctional compound in which two aminoacridine chromophores are linked by the bicyclic depsipeptidic backbone of des-N-tetramethylTriostin A is...
The synthesis of a new bifunctional compound in which two aminoacridine chromophores are linked by the bicyclic depsipeptidic backbone of des-N-tetramethylTriostin A is described. The molecule, bis-[(9-acridinyl)-D-seryl-L-alanyl-L-cysteinyl-L-valine] dilactone disulphide, structurally analogous to the antibiotic anti-tumour drug Triostin A, is shown to possess a high affinity to DNA and to act as a bis-intercalator on the basis of spectroscopic, viscosimetric and thermal-denaturation studies. This model constitutes the first attempt of a synergic association between a peptidic moiety that mimics a naturally occurring drug and aminoacridine, the two parts themselves each exhibiting a high affinity for the DNA target.
Topics: Aminacrine; Chemical Phenomena; Chemistry; DNA; Intercalating Agents; Models, Chemical; Oligopeptides; Spectrophotometry
PubMed: 3838469
DOI: 10.1042/bj2250829 -
The Biochemical Journal Jan 1985Endosome fractions were isolated from rat liver homogenates on the basis of the subcellular distribution of circulating ligands, e.g. 125I-asialotransferrin internalized...
Endosome fractions were isolated from rat liver homogenates on the basis of the subcellular distribution of circulating ligands, e.g. 125I-asialotransferrin internalized by hepatocytes by a receptor-mediated process. The distribution of endocytosed 125I-asialotransferrin 1-2 min and 15 min after uptake by liver and a monensin-activated Mg2+-dependent ATPase activity coincided on linear gradients of sucrose and Nycodenz. The monensin-activated Mg2+-ATPase was enriched relative to the liver homogenates up to 60-fold in specific activity in the endosome fractions. Contamination of the endosome fractions by lysosomes, endoplasmic reticulum, mitochondria, plasma membranes and Golgi-apparatus components was low. By use of 9-aminoacridine, a probe for pH gradients, the endosome vesicles were shown to acidify on addition of ATP. Acidification was reversed by addition of monensin. The results indicate that endosome fractions contain an ATP-driven proton pump. The ionophore-activated Mg2+-ATPase in combination with the presence of undegraded ligands in the endosome fractions emerge as linked markers for this new subcellular organelle.
Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Aminacrine; Animals; Asialoglycoproteins; Ca(2+) Mg(2+)-ATPase; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Centrifugation, Density Gradient; In Vitro Techniques; Liver; Monensin; Protons; Rats; Subcellular Fractions; Transferrin
PubMed: 2983664
DOI: 10.1042/bj2250051 -
The Journal of General Physiology Sep 1984The kinetics of 9-aminoacridine (9-AA) block of single Na channels in neuroblastoma N1E-115 cells were studied using the gigohm seal, patch clamp technique, under the...
The kinetics of 9-aminoacridine (9-AA) block of single Na channels in neuroblastoma N1E-115 cells were studied using the gigohm seal, patch clamp technique, under the condition in which the Na current inactivation had been eliminated by treatment with N-bromoacetamide (NBA). Following NBA treatment, the current flowing through individual Na channels was manifested by square-wave open events lasting from several to tens of milliseconds. When 9-AA was applied to the cytoplasmic face of Na channels at concentrations ranging from 30 to 100 microM, it caused repetitive rapid transitions (flickering) between open and blocked states within single openings of Na channels, without affecting the amplitude of the single channel current. The histograms for the duration of blocked states and the histograms for the duration of open states could be fitted with a single-exponential function. The mean open time (tau o) became shorter as the drug concentration was increased, while the mean blocked time (tau b) was concentration independent. The association (blocking) rate constant, kappa, calculated from the slope of the curve relating the reciprocal mean open time to 9-AA concentration, showed little voltage dependence, the rate constant being on the order of 1 X 10(7) M-1s-1. The dissociation (unblocking) rate constant, l, calculated from the mean blocked time, was strongly voltage dependent, the mean rate constant being 214 s-1 at 0 mV and becoming larger as the membrane being hyperpolarized. The voltage dependence suggests that a first-order blocking site is located at least 63% of the way through the membrane field from the cytoplasmic surface. The equilibrium dissociation constant for 9-AA to block the Na channel, defined by the relation of l/kappa, was calculated to be 21 microM at 0 mV. Both tau -1o and tau -1b had a Q10 of 1.3, which suggests that binding reaction was diffusion controlled. The burst time in the presence of 9-AA, which is the sum of open times and blocked times, was longer than the lifetime of open channels in the absence of drug. All of the features of 9-AA block of single Na channels are compatible with the sequential model in which 9-AA molecules block open Na channels, and the blocked channels could not close until 9-AA molecules had left the blocking site in the channels.
Topics: Acetamides; Aminacrine; Aminoacridines; Culture Techniques; Electrophysiology; Ion Channels; Kinetics; Neuroblastoma; Sodium; Time Factors
PubMed: 6090578
DOI: 10.1085/jgp.84.3.361 -
The Journal of Biological Chemistry Aug 1984The proton gradient (delta pH) and electrical potential (delta psi) across the neurosecretory vesicles were measured using the optical probes 9-aminoacridine and Oxanol...
The proton gradient (delta pH) and electrical potential (delta psi) across the neurosecretory vesicles were measured using the optical probes 9-aminoacridine and Oxanol VI, respectively. The addition of neurosecretory vesicles to 9-aminoacridine resulted in a rapid quenching of the dye fluorescence which was reversed when the delta pH was collapsed with ammonium chloride or K+ in the presence of nigericin. From fluorescence quenching data and the intravesicular volume, delta pH across the membrane was calculated. Mg2+ ATP caused a marked carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive change in the membrane potential measured using Oxanol VI (plus 100 mV inside positive), presumably due to H+ translocation across the neurosecretory vesicle membrane. Imposition of this membrane potential was responsible for the lysis of vesicles in the presence of permeant anions. The effectiveness of these anions to support lysis reflected the relative permeability of the anion which followed the order acetate greater than I- greater than Cl greater than F- greater than SO4- = isethionate = methyl sulfate. These data showed that the neurosecretory vesicles possess a membrane H+-translocating system and prompted the study of Mg2+-dependent ATPase activities in the vesicle fractions. In intact vesicles a Mg2+ ATPase appeared to be coupled to electrogenic proton translocation, since the enzyme activity was enhanced by uncoupling the electrical potential, using proton ionophores. Inhibition of this enzyme with dicyclohexylcarbodiimide also inhibited the carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive delta psi across the vesicle membrane caused by H+ translocation. A second Mg2+ ATPase was also found on the vesicle membranes which is sensitive to vanadate. Complete inhibition of this enzyme with vanadate had little effect on the proton ionophore-uncoupled ATPase activity or on the Mg2+ ATP-induced membrane potential change.
Topics: Adenosine Triphosphatases; Aminacrine; Ammonium Chloride; Animals; Ca(2+) Mg(2+)-ATPase; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cattle; Dicyclohexylcarbodiimide; Diffusion; Hydrogen-Ion Concentration; Nigericin; Pituitary Gland, Posterior; Potassium; Proton-Translocating ATPases; Spectrometry, Fluorescence; Valinomycin; Vanadium
PubMed: 6146615
DOI: No ID Found -
The Journal of Biological Chemistry Jul 1984Mitochondrial nicotinamide nucleotide transhydrogenase from beef heart was purified by a novel procedure involving fast protein liquid chromatography and characterized...
Energy-linked nicotinamide nucleotide transhydrogenase. Properties of proton-translocating mitochondrial transhydrogenase from beef heart purified by fast protein liquid chromatography.
Mitochondrial nicotinamide nucleotide transhydrogenase from beef heart was purified by a novel procedure involving fast protein liquid chromatography and characterized with respect to molecular and catalytic properties. The method is reproducible, gives highly pure transhydrogenase as judged by silver staining, and can be modified to produce large amounts of pure transhydrogenase protein suitable for e.g. sequencing and other protein chemical studies. Transhydrogenase purified by fast protein liquid chromatography is reconstitutively active and pumps protons as indicated by an extensive quenching of 9-aminoacridine fluorescence. Under conditions which generate a proton gradient in the absence of a membrane potential the activity of reconstituted transhydrogenase is close to zero indicating a complete and proper incorporation in the membrane and a preferential regulation of the enzyme by a proton gradient rather than a membrane potential. Treatment of reconstituted transhydrogenase with N,N-dicyclohexylcarbodiimide results in an inhibition of proton pump activity without an effect on uncoupled catalytic activity, suggesting that proton translocation and catalytic activities are not obligatory linked or that this agent separates proton pumping from the catalytic activity.
Topics: Aminacrine; Amino Acids; Aminoacridines; Animals; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cattle; Dicyclohexylcarbodiimide; Energy Metabolism; Fluorescent Dyes; Kinetics; Liposomes; Mitochondria; Mitochondria, Heart; NADH, NADPH Oxidoreductases; NADP Transhydrogenases; Proton-Translocating ATPases; Submitochondrial Particles
PubMed: 6234316
DOI: No ID Found -
FEBS Letters Apr 1984The DNA binding characteristics of a mono-, di- and trimeric derivative of 9-aminoacridine were studied. The length of the linking carboxamidoalkyl chains was selected... (Comparative Study)
Comparative Study
The DNA binding characteristics of a mono-, di- and trimeric derivative of 9-aminoacridine were studied. The length of the linking carboxamidoalkyl chains was selected to allow bis- or tris-intercalation according to the excluded-site model. Measurements of DNA unwinding angle using closed circular DNA showed that the trimeric derivative behaves as a tris-intercalating agent. Nevertheless the increase of DNA binding affinity on going from dimer to trimer was found to be relatively small. This is probably related to the large structural constraint for DNA binding of the trimeric derivative. The nature of the linking chain for the design of high-affinity DNA poly-intercalating agents appears therefore critical.
Topics: Aminacrine; Aminoacridines; Animals; Cattle; DNA; Fluorometry; Macromolecular Substances; Poly dA-dT; Viscosity
PubMed: 6714420
DOI: 10.1016/0014-5793(84)80302-6 -
The EMBO Journal Apr 1984New molecules with high and specific affinity for nucleic acid base sequences have been synthesized. They involve an oligodeoxynucleotide covalently attached to an...
New molecules with high and specific affinity for nucleic acid base sequences have been synthesized. They involve an oligodeoxynucleotide covalently attached to an intercalating dye. Visible absorption spectroscopy and fluorescence have been used to investigate the binding of poly(rA) to octadeoxythymidylates substituted by a 9-aminoacridine derivative in different positions along the oligonucleotide chain. The 9-amino group of the acridine dye was linked through a polymethylene bridge to the 3'-phosphate, the 5'-phosphate, the fourth internucleotidic phosphate or to both the 3'- and 5'-phosphates. Different interactions of the acridine dye were exhibited by these different substituted oligodeoxynucleotides when they bind to poly(rA). The interaction was shown to be specific for adenine-containing polynucleotides. The stability of these complexes was compared with that of oligodeoxynucleotides substituted by an alkyl group on the 3'-phosphate. The increase in stability due to the presence of the intercalating dye has been determined from the comparison of melting temperatures. These results are discussed with respect to the strategy of synthesis of a new class of molecules with high affinity and high specificity for nucleic acid base sequences.
Topics: Aminacrine; Aminoacridines; Base Sequence; Drug Stability; Fluorescence Polarization; Hot Temperature; Oligodeoxyribonucleotides; Oligonucleotides; Osmolar Concentration; Poly A; Spectrometry, Fluorescence; Spectrophotometry; Thymidine Monophosphate
PubMed: 6723628
DOI: 10.1002/j.1460-2075.1984.tb01887.x -
Proceedings of the National Academy of... Apr 1984We investigated the effects of the well-known mutagenic agents ethyl methanesulfonate (EtMes), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), and ICR-191 on colonies of the...
We investigated the effects of the well-known mutagenic agents ethyl methanesulfonate (EtMes), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), and ICR-191 on colonies of the Chinese hamster ovary line CHO cultured on a semisolid substrate. These agents induced heterogeneity in diameter and integrated optical density of colonies as determined by computer-assisted photography and subsequent analysis of the images of the colonies. When CHO colonies were exposed to agents such as urethane that are not known to be mutagenic in mammalian systems or to activation-requiring mutagens such as cyclophosphamide, there was no noticeable effect on the distribution of colony diameter and volume. Similarly, nonmutagenic agents such as dimethyl sulfoxide (Me2SO) also did not induce heterogeneity in colony diameter and integrated optical density. Our observations recommend the use of agar-grown mammalian cell colonies for predictive testing of chemical mutagens and carcinogens in a simple, in vitro mammalian cell assay. This assay system, unlike other mammalian cell culture assays, allows detection and measurement of the simultaneous effects of chemical mutagens on several genetic and non-genetic targets and, thus, may emulate more closely the potential hazards of these agents in vivo.
Topics: Aminacrine; Animals; Cell Division; Cell Line; Cell Survival; Clone Cells; Cricetinae; Cricetulus; Cyclophosphamide; Dimethyl Sulfoxide; Ethyl Methanesulfonate; Female; Methylnitronitrosoguanidine; Mutagenicity Tests; Mutagens; Mutation; Nitrogen Mustard Compounds; Ovary; Urethane
PubMed: 6585791
DOI: 10.1073/pnas.81.7.2112