Dansylarginine N-(3-ethyl-1,5-pentanediyl)amide: A potent and selective fluorescent inhibitor of butyrylcholinesterase

Interactions between dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA) and the cholinesterases were examined by the techniques of enzyme kinetics and fluorescence spectroscopy. When tested with partially purified enzyme preparations, DAPA was a potent inhibitor of butyrylcholinesterase $\text{(}\text{IC}{}_{50}\text{= 2 × 10}{}^{\mathrm{?7}}\text{M}\text{)}$ but not of acetylcholinesterase $\text{(}\text{IC}{}_{50}\text{= 4 × 10}{}^{\mathrm{?4}}\text{M}\text{)}$. For a detailed study of the effects of DAPA on butyrylcholinesterase (BuChE), the enzyme was purified to homogeneity from horse serum, with the aid of affinity chromatography on N-methyl acridinium. The kinetics of the inhibition of purified BuChE by DAPA were complex, having both competitive and non-competitive features, and it was not possible to estimate K_i unambiguously. Spectroscopic measurements showed that the fluorescence of the dansyl moiety was strongly affected by the binding to BuChE. With excitation at 330 nm, total fluorescence emission from bound DAPA (at 450 nm and above) was 21-fold greater than from free DAPA. In a titration experiment, this enhancement of fluorescence intensity was used to calculate that each monomer of BuChE has two apparently independent DAPA-binding sites with a K_d of 4.5 × 10^?7 M. Further measurements showed that the fluorescence emission of bound DAPA was markedly blue-shifted (to 502 nm from 570 nm in free solution) and that the fluorescence lifetime of this form was greatly prolonged (to 24 nsec from 2.7 nsec). These observations indicate that the high affinity binding sites on BuChE lock DAPA in a highly non-polar environment.     

Analog specific aberrancies in antifolate inhibition of L1210 cell dihydrofolate reductase

During studies with L1210 cells and a variety of folate analogs, large discrepancies were revealed between data on membrane transport, on inhibition of dihydrofolate reductase in cell-free extracts, and on inhibition of growth in culture for 10-oxa-, 10-benzyl- and 10-phenethyl-aminopterin, and for 3-deaza, 10-methyl-aminopterin. While aminopterin, 10-methyl (methotrexate)-, 10-ethyl- and 10-propyl-aminopterin were tight binding inhibitors (K_i: 2–3 × 10^?12M) of dihydrofolate reductase in cell-free extracts from L1210 cells, the other four analogs were only weak competitive inhibitors (K_i = 3–300 × 10^?8M). Similar differences among analogs were observed for inhibition of dihydrofolate reductase in cell-free extracts from Sarcoma 180 and Ehrlich cells, but not for this enzyme in microbial cell-free extracts. There were only small differences in the transport of all of the analogs by L1210 cells. Inhibition of L1210 cell growth in culture by 10-oxa-, 10-benzyl- and 10-phenethyl-aminopterin and by 3-deaza, 10-methyl-aminopterin, in contrast to the other analogs, was several orders of magnitude greater than that predicted from the data on dihydrofolate reductase inhibition. The extent of binding of 10-oxa-, 10-benzyl- and 10-phenethyl-aminopterin, and of 3-deaza and 10-methyl-aminopterin to dihydrofolate reductase in intact L1210 cells, in contradistinction to that seen for the cell-free enzyme preparations, approached that observed for methotrexate; these estimates of drug-enzyme interaction in situ were more predictive of the extent of inhibition by these analogs of L1210 cell growth in culture.     

Antitumor effects of bis-thiosemicarbazones— inhibition of deoxyribonucleic acid synthesis in vitro

A group of bis-thiosemicarbazones was evaluated for potential antitumor activity, using the L1210 murine leukemia in cell culture. Drug levels required to inhibit DNA synthesis by 50 per cent, under standard conditions, were determined. The most potent of the agents examined had the structure X[CH₂CR₁=NNHCSNHR₂]₂ where X = C or S and R₁ = R₂ = CH₃. Optimal activity was also obtained with R₁ = H and R₂ = CH₃ only when X = S. The most potent derivatives inhibited DNA synthesis by 50 per cent within 10 min at 10^?6 M levels (id₅₀). Metal chelates of several compounds tested were extremely potent inhibitors of DNA synthesis ($\text{id}{}_{50}\text{= 10}{}^{\mathrm{?7}}\text{M or less}$). Insolubility in water and short duration of action in vivo may limit effectiveness of the bis-thiosemicarbazones.     

Effects of bis(6-mercaptopurine-9-beta-D-ribofuranoside)-5',5"-phosphate and its butyryl derivative on mouse leukaemia L1210 and a 6-mercaptopurine-resistant subline in culture

Bis(6-mercaptppurine-9-β-d-ribofuranoside)-5′-5″′-monophosphate (bis(MPR)P) and its butyryl derivative, bis(O²,$\text{O}{}^3\text{-}\text{dibutyryl}\text{-6-}\text{mercaptopurine}\text{-9-β-}\text{d}\text{-}\text{ribofuranoside}$)-5′,5″′-monophosphate (bis(dibutyrylMPR)P) were synthesized from 6-mercaptopurine-9-β-d-ribofuranoside (MPR). Bis(MPR)P (ec₅₀ = 0.014 μM) and MPR (ec₅₀ = 0.022 μM) were essentially equivalent in their growth inhibitory activities against L1210/0 cell cultures, whilst bis(dibutyrylMPR)P (ec₅₀ = 1.1 μM) was considerably less effective. L1210/MPR cells grew normally in the presence of 1 mM MPR but were inhibited by bis(MPR)P (ec₅₀ = 580 μM) and (bis(dibutyrylMPR)P (ec₅₀ = 42 μM). Bis(dibutyrylMPR)P was less readily broken down to MPR by enzymes in the serum component of the culture medium than was bis(MPR)P, and leukaemia cells did not contribute to the extracellular degradation of the acylated derivative. The delayed cytotoxic effects of bis(MPR)P and bis(dibutyrylMPR)P on L1210/0 cells were those of the MPR breakdown product. Exposure to bis(MPR)P resulted in delayed cytotoxicity in L1210/MPR cultures whilst bis(dibutyrylMPR)P produced only acute growth inhibition and no delayed effect on the MPR-resistant subline. MPR was incorporated into DNA of L1210/0 cells as 6-thioguanine deoxyribonucleotide whilst bis(MPR)P was not incorporated into L1210/MPR cell DNA. These results suggested that the ultimate mechanisms of action of bis(MPR)P and bis(dibutyrylMPR)P in L1210/ MPR cells may have been different from that of MPR in sensitive L1210/0 cells and therefore might not represent true circumvention of resistance to MPR.     

Determination of extraction constant,true partition coefficient and formation constant of ion-pair complexes of quaternary ammonium salts,tetrabutylammonium bromide and isopropamide iodide.with some organic anions by a solvent extraction technique

A new method of determining the extraction constant (K_e, the true partition coefficient (TPC) and the formation constant (K_f) of ion-pairs, was developed by the solvent extraction technique. K_e and TPC were estimated from the reciprocals of the intercept and the slope of the regression line obtained by plotting

$\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{APC ? dA}\text{vs}\text{B}\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{dA}\text{APC ? dA}\text{+}\text{A}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{+}\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}$

in the following equation.

$\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{APC ? dA}\text{=}\text{1}\text{K}{}_{\mathrm{e}}\text{+}\text{B}\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{dA}\text{APC ? dA}\text{+}\text{A}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{+}\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{x}\text{1}\text{TPC}$

where [A^T_W] and [B^T_W] are the total concentrations of the cationic compound A and that of the anionic compound B in the aqueous phase respectively, APC is the apparent partition coefficient of A, dA is the partition coefficient of cation A⁺. K_f, which is expressed by K_e/TPC, was then calculated. These constants were determined for the ion-pair extraction of tetrabutylammonium bromide and isopropamide iodide with 4 organic anions, i.e. benzoic acid, p-toluenesulfonic acid, salicylic acid and taurodeoxycholic acid. This new method might be applicable to other ion-pairs without further assumptions except that the molar ratio of the ion-pair formation be 1 : 1.     

2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl]quinazoline (tmq), a potent non-classical folate antagonist inhibitor--I effect on dihydrofolate reductase and growth of rodent tumors in vitro and in vivo.

Seventeen non-classical 2,4-diamino-6-[(anilino)methyl]quinazoline antifolates were tested as inhibitors of dihydrofolate reductase from L1210 leukemia cells and from human leukemia cells (acute lymphocytic leukemia). Several potent inhibitors of this enzyme were found, some with I₅₀ values of 10^?9 M, thus displaying activity comparable to that of methotrexate. In general, the potency of dihydrofolate reductase inhibition correlated with the inhibition of cell growth in vitro against L1210 cells. Two of these compounds, compound 14 (2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl]quinazoline; TMQ, JB-11, NSC 249008) and compound 3 (2,4-diamino-5-chloro-6-[(3,4-dichloroanilino)methyl]quinazoline; NSC 208652), were further evaluated against murine tumors in vivo and both showed a broad spectrum of antitumor effects. The results of these studies encourage further evaluation of these compounds, in particular compound 14, as possible anti-neoplastic agents in the treatment of human disease.     

Adenosine receptor interactions and anxiolytics

$\text{[}{}^3\text{H}\text{]-N}{}^6\text{-}\text{cyclohexyladenosine}$ and [³H]-1,3-diethyl-8-phenylxanthine label the A₁ subtype of adenosine receptor in brain membranes. The affinities of methylxanthines in competing for A₁ adenosine receptors parallel their potencies as locomotor stimulants. The adenosine agonist N⁶-(-phenylisopropyl) adenosine is a potent locomotor depressant. Both diazepam and $\text{N}{}^6\text{-(}\text{l}\text{-}\text{phenylisopropyl}\text{)}\text{adenosine}$ cause locomotor stimulation in a narrow range of subdepressant doses. Combined stimulant doses of the two agents depress motor activity, as do larger doses of either one, given separately.Evidence supporting and against the hypothesis that some of the actions of benzodiazepines are mediated via the adenosine system is reviewed. A number of compounds interact with both systems, probably because of physico-chemical similarities between adenosine and diazepam. It is concluded that of the four classic actions of benzodiazepines, the sedative and muscle relaxant (but not anxiolytic or anticonvulsant) actions could possibly be mediated by adenosine.     

Kinetic studies on the metabolism of ethylmorphine by isolated hepatocytes from adult rats

Hepatocytes of adult rats were isolated by infusion of a hyaluronidase collagenase mixture. High yields of cells excluding trypan blue were obtained. These cells, in Hank's buffer containing rat serum and 0.1% glucose, $\text{N-}\text{demethylate}\text{[}{}^3\text{H-CH}{}_3\text{-N]}$ethylmorphine. The formaldehyde initially formed is further metabolized to tritiated water. Fifteen per cent of the original metabolic activity was observed after 21 hr at 37° in 5% CO₂-air, and cumulative metabolism is linear for up to 90 min under these conditions. The K_m for the N-demethylation of [³H-CH₃-N]ethylmorphine is 50 μM, 20 per cent of the value observed for this reaction by musomal preparations. An active transport of the substrate into the cell is postulated to account for this difference.     

Correlation of biologic data with physico-chemical properties among the vinca alkaloids and their congeners.

Displacement of [³H]vinblastine binding to tubulin by other Vinca alkaloid derivatives has been demonstrated to be a competitive process, allowing for determination of the association constant of each drug. Correlation of LD₅₀ data and anti-P-388 activity was found with log P and log K_a, according to the equations: $\text{log}\text{LD}{}_{50}\text{= 0.129 (}\text{log}\text{P)}{}^2\text{? 0.522}\text{log}\text{P ? 0.479}\text{log}\text{K}{}_{\mathrm{a}}\text{+ 4.652}\text{log}\text{P ? 388 = 0.222 (}\text{log}\text{P)}{}^2\text{? 1.059}\text{log}\text{P ? 0.520}\text{log}\text{K}{}_{\mathrm{a}}\text{+ 5.366}$. Vincristine and desacetylvinblastine were the two most active agents in this series. That the latter drug had significant biologic activity was of considerable interest, since it is known to be a human metabolite.     

Antiviral, antitumor, and thymidylate synthetase inhibition studies of 5-substituted styryl derivatives of 2'-deoxyuridine and their 5'-phosphates

2′-Deoxyuridine derivatives containing styryl, 3-nitrostyryl, 4-nitrostyryl, and phenylethyl groups substituted at the 5-position of the pyrimidine ring have been evaluated for their effects on vaccinia and herpes simplex virus replication (in primary rabbit kidney cell cultures) and mouse leukemia L-1210 cell culture growth. 5-Phenylethyl-2′-deoxyuridine inhibited herpes simplex (type 1 and 2) virus-induced cytopathogenicity by 50 per cent at a dose (id₅₀) of 10–30 μg/ml. It was inactive against tumor cell growth. The corresponding styryl derivative showed an id₅₀ of 30–70 μg/ml for herpes simplex virus, 20 μg/ml for vaccinia virus, and 280 μg/ml for L-1210 cell growth. 5(E)-(3-Azidostyryl)-2′-deoxyuridine 5′-phosphate inhibited vaccinia replication with an IC₅₀ of 20 μg/ml and L-1210 cell culture growth with an id₅₀ of 80 μg/ml. The nucleotides of these compounds were all potent reversible inhibitors of thymidylate synthetase (Lactobacillus casei) with the following $\text{K}{}_{\mathrm{i}}\text{K}{}_{\mathrm{m}}$ ratios: 3-nitrostyryl, 0.035; 4-nitrostyryl, 0.05; 3-azidostyryl, 0.06; styryl, 0.08; and phenylethyl, 0.31. The photodecomposition of the azidostyryl derivative, a photoaffinity labeling reagent for thymidylate synthetase, was examined at two wavelengths.     

Kinetic studies on the deethylation of ethoxybenzamide: A comparative study with isolated hepatocytes and liver microsomes of rat

Kinetic parameters (K_m and V_max) of ethoxybenzamide deethylation in isolated rat hepatocytes and liver microsomes were compared. Adjustment of cofactors in microsomal deethylation, such as NADPH and Mg²⁺, to give optimum conditions, and appropriate correction of the apparent kinetic parameters for nonspecific binding and microsomal yield resulted in good agreement among the kinetic parameters of isolated hepatocytes [$\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{= 0.0863 μmole · min}{}^{\mathrm{?1}}\text{· (g liver)}{}^{\mathrm{?1}}$ and K_m = 0.459 mM] and microsomes [V_max = 0.124 μmoles · min^?1 · (gliver)^?1 and K_m = 0.378 mM].     

Effect of polychlorinated biphenyls on the immune responses of rhesus monkeys and mice.

The relationship between lipid peroxidation, glutathione (GSH) content, and CCl₄-induced toxicity was investigated in rat hepatocytes isolated by a collagenase-perfusion technique. Two chemical initiators of lipid peroxidation, ferric ions complexed with adenosine diphosphate ($\text{ADP}\text{Fe}{}^{\mathrm{3+}}$) and diethyl maleate, were studied for comparison. CCl₄ caused a reduction of intracellular K⁺ and release of alanine aminotransferase (ALT) into the medium, but no evidence of lipid peroxidation, as measured by the absorbance of thiobarbituric acid (TBA)-reacting materials and lipid-extract diene conjugation. $\text{ADP}\text{Fe}{}^{\mathrm{3+}}$ caused lipid peroxidation, but only a small loss of K⁺. Diethyl maleate caused a greater amount of lipid peroxidation and cell damage than did $\text{ADP}\text{Fe}{}^{\mathrm{3+}}$. Neither response appeared to be related to the GSH content, which was reduced by diethyl maleate, but not by $\text{ADP}\text{Fe}{}^{\mathrm{3+}}$, and by CCl₄ only at the highest dose. The results suggest that lipid peroxidation is not a requisite step in CCl₄-induced toxicity in isolated hepatocytes.     

Similar activities of nerve growth factor and its homologue proinsulin in intracellular hydrogen peroxide production and metabolism in adipocytes: Transmembrane signalling relative to insulin-mimicking cellular effects

Generation of hydrogen peroxide in adipocyte plasma membrane and its intracellular metabolism and regulatory role have been shown by Mukherjee and co-workers to be a major effector system for insulin [Fedn Proc.35, 1694 (1976); Archs Biochem. Biophys.184, 69 (1977); Biochem. Pharmac.27, 2589 (1978); Fedn Proc.37, 1689 (1978); and Biochem. Pharmac.29, 1239 (1980)]. The possible involvement of this mechanism in the action of structurally similar polypeptides having some insulin-like metabolic effects was investigated. The β-subunit of nerve growth factor (2.5 S NGF, mol. wt 13,500) which has a striking structural homology with proinsulin and has been reported to exert certain insulin-like metabolic effects in its own target tissues (e.g. growing neurites and sympathetic ganglia), and the insulin-derived polypeptides, desalanine-insulin and desoctapeptide-insulin, as well as proinsulin, were examined for their effects on rat adipocytes, employing the technique of formate oxidation. Both NGF and proinsulin caused increased [¹⁴C]formate oxidation, showing similar intrinsic activities, up to a maximum of 140–160% of the basal rate; insulin increased the rate to 190–210% of the basal rate. The relative potencies of the hormones toward H₂O₂ formation and stimulation of the pentose phosphate pathway activity were: insulin $\text{(}\text{EC}{}_{50}\text{: 2.5 × 10}{}^{\mathrm{?11}}\text{M}\text{)}$, desalanine-insulin $\text{(}\text{EC}{}_{50}\text{: 2.5 × 10}{}^{\mathrm{?10}}\text{M}\text{)}$ , proinsulin $\text{(}\text{EC}{}_{50}\text{: 8 × 10}{}^{\mathrm{?9}}\text{M}\text{)}$, and NGF $\text{(}\text{EC}{}_{50}\text{: 10}{}^{\mathrm{?9}}\text{M}\text{)}$. The biologically inactive derivative, desoctapeptide-insulin, did not stimulate glucose oxidation, although it caused a small increase in formate oxidation, with an $\text{EC}{}_{50}\text{of}\text{5 × 10}{}^{\mathrm{?7}}\text{M}$, indicating a suboptimal level of H₂O₂ formation in the elevation of the hexose monophosphate shunt activity. 3-Amino-1,2,4,-triazole (50 mM), which irreversibly decomposes the peroxidatic compound II of the catalase: H₂O₂ complex, inhibited formate oxidation to a greater extent in the hormone-treated cells than in the control cells, whereas sodium azide, an inhibitor of the hemoprotein, catalase, completely inhibited it. The abilities of the polypeptides to stimulate H₂O₂ formation correlated with their abilities to promote lipogenesis from [U-¹⁴C]-D-glucose, as expected of insulin. The cellular GSH/GSSG ratio increased concomitantly with the stimulation of glucose oxidation via the shunt, indicating a tight coupling between these processes. The results confirm that the hydrogen peroxide production is a common basis of the metabolic actions of growth-promoting polypeptide hormones or mitogens beyond their respective receptors.     

Mechanisms by which quipazine,a putative serotonin receptor agonist,alters brain 5-hydroxyindole metabolism

Quipazine (2-[1-piperazinyl] quinoline maleate) was shown to increase serotonin and decrease 5-hydroxyindoleacetic acid concentrations in whole brain, several brain regions, and the spinal cord of rats 1 hr after its administration (10 mg/kg, i.p.). In animals with transected spinal cords, quipazine induced stronger activation of extensor reflexes than 5-hydroxytryptophan, chlorimipramine, or Lilly 110140. This response could be blocked by methiothepin. In slices of rat cerebral cortex, quipazine inhibited the uptakes of [³H]-serotonin (EC₅₀ = 10^?6 M) and [³H]-norepinephrine ($\text{EC}{}_{50}\text{= 2 × 10}{}^{\mathrm{?6}}\text{m}$); it was equipotent with Lilly 110140 in inhibiting serotonin uptake, but less potent than chlorimipramine ($\text{EC}{}_{50}\text{= 10}{}^{\mathrm{?7}}\text{m}$). Quipazine administration to rats did not inhibit monoamine oxidase activity, and actually elevated brain tryptophan levels. These observations suggest that the effects of quipazine on brain serotonin and 5-hydroxyindoleacetic acid concentrations could have been caused by direct activation of central serotonin receptors (which would secondarily decrease impulse flow along serotonergic neurones), or by the inhibition of serotonin reuptake, or by both mechanisms.     

Effects of exposure to ozone on susceptibility to experimental tuberculosis

Exposure of mice to 1.96 $\text{mg}\text{m}{}^3$ ozone (O₃) 3 $\text{h}\text{day}$, 5 days/week, for up to 8 weeks beginning at 1 or 2 weeks after challenge with Mycobacterium tuberculosis R 1Rv resulted in significant enhancement of bacterial titers in the lungs at 5 through 8 weeks after challenge when compared to mice exposed to filtered air. Exposure to lower concentrations of O₃ did not produce any significant changes compared to controls.Exposure of guinea pigs to 2.9 $\text{mg}\text{m}{}^3$ O₃ for 3 h immediately after challenge with M. tuberculosis resulted in a suppression of the cutaneous delayed hypersensitivity response, without affecting the serum hemagglutination antibody titers. However, exposure of guinea pigs to 0.98 $\text{mg}\text{m}{}^3$ O³ 3 $\text{h}\text{day}$ for 5 days, initiated within 3 h after the infectious challenge, enhanced hemagglutination antibody titers initially, but the delayed hypersensitivity reaction did not differ from controls.     

Growth inhibitory,transport and biochemical properties of the γ-glutamyl and γ-aspartyl peptides of methotrexate inL1210 leukemia cells in vitro

Both methotrexate-γ-glutamate and methotrexate-γ-aspartate are equivalent to metho-trexate as inhibitors of L1210 cell dihydrofolate reductase. However, the initial influx of both peptides into L1210 cells during transport studies is substantially lower than that of methotrexate. The apparent K_m tor influx of methotrexate-γ-glutamate and methotrexate-γ-aspartate is 15-fold and 100-fold greater than methotrexate respectively. Efflux measurements, which were possible only for methotrexate-γ-glutamate, showed a similar rate for this peptide and methotrexate. The intracelluiar accumulation and subsequent metabolism to methotrexate of methotrexate-γ-glutamate, but not of methotrexate-γ-aspartate, were confirmed by bioautographic analysis of cell extracts. After correction for extracellular cleavage of both peptides mediated by enzymes in calf serum supplementing the culture medium, the relative growth (L1210 cell)-inhibitory potency for the three agents was 1:18:210 for methotrexate, methotrexate-γ-glutamate and methotrexate-γ-aspartate respectively. Both the relative inhibitory potency and the difference in absolute inhibitory concentration among the three agents were predictable solely from the data on the influx of each measured during transport studies. Methotrexate-γ-aspartate is apparently more resistant to enzymic cleavage than is methotrexate-γ-glutamate.     

Effect of exposure to nitrogen dioxide on t and b cells in mouse spleens

Effects of exposure to nitrogen dioxide (NO₂) on thymus-derived (T) and bursa-derived (B) cells were studied in albino mice. The mice were exposed continuously 24 $\text{h}\text{day}$ to 940 $\text{μg}\text{m}{}^3$ NO₂ and to 188 $\text{μg}\text{m}{}^3$ NO₂ with daily 3-h peaks 5 $\text{days}\text{week}$ of either 470, 940 or 1880 $\text{μg}\text{m}{}^3$ NO₂. After 1, 3, 6, 9 and 12 months of exposure, single-cell suspensions were prepared from spleen and incubated with phytohemagglutinin (PHA) or bacterial lipopolysaccharide (LPS). The PHA and LPS responses from mice exposed to NO₂ were generally depressed when compared with those in mice exposed for the same time periods to filtered air.     

Interaction of [3H]flunitrazepam with the benzodiazepine receptor: Evidence for a ligand-induced conformation change

The interaction of [³H]flunitrazepam with benzodiazepine receptors in rat brain homogenates was studied in the presence of 2 μM endogenous GABA at 0° at pH 7.2. Equilibrium binding experiments showed a dominant component of high affinity with an equilibrium dissociation constant K = 0.86 ± 0.07 nM which accounted for 75% of total binding and another component of lower affinity (K ? 30 nM). The dissociation kinetics of the [³H]flunitrazepam complex at the high affinity site were strictly monophasic with a rate constant k_off = (7.7 ± 0.3) × 10^?4/sec. The association kinetics with the high affinity sites were studied with ligand concentrations [L]₀ in large excess over binding sites. The kinetics were in accordance with a single exponential with a reaction rate τ^?1. In the higher concentration range [L]₀ ? 10 nM, τ^?1 as a function of [L]₀ deviated from linearity and started to level off. The data are compatible with a two-step mechanism where R and L rapidly combine to form a pre-complex RL which then slowly isomerizes to the final complex C:

where $\text{K}{}_1\text{= (}\text{[}\text{R}\text{][}\text{L}\text{]}\text{([}\text{RL}\text{]}\text{)}$ and $\text{[}\text{RL}\text{]}\text{[}\text{C}\text{]}\text{=}\text{k}{}_{\mathrm{?2}}\text{k}{}_2\text{= k}{}_2$. Nonlinear parameter estimation yielded K₁24.2 ± 7.1 nM, k₂ = (2.8 ± 0.5) × 10^?2/sec and k_?2 = (9 ± 2) × 10^?4/sec. The isomerization step might reflect a ligand-induced conformation change of the high affinity site which is involved in the potentiation of GABA-ergic transmission produced by the benzodiazepines.