Tight binding inhibitors--VI. Interactions of deoxycoformycin and adenosine deaminase in intact human erythrocytes and sarcoma 180 cells

The inactivation and reactivation of adenosine deaminase (ADA) by deoxycoformycin was studied in intact human erythrocytes and murine Sarcoma 180 cells in vitro. The second-order rate constant (k₁) for the association reaction between deoxycoformycin and intraerythrocytic ADA was calculated to be 5.1 × 10³M^?1 sec^?1. This is about 300 to 500-fold lower than the k₁ values determined either with hemolyzed human erythrocytes (k₁ = 1.4 × 10⁶ M^?1 sec^?1) or with partially purified human erythrocytic ADA (k₁ = 2.6 × 10⁶ M^?1 sec^?1). In intact erythrocytes only slight reactivation (<10 per cent) of the inhibited ADA (EI complex) was detectable over 24 hr, whereas with hemolysates about 50 per cent reactivation of the inhibited ADA was observed in about 25 hr (k₂ = 7.7 × 10^?6sec^?1). The k¹ values with intact and supernatant fractions from homogenized Sarcoma 180 cells were determined to be 1.1 × 10⁴M^?1 sec^?1 and 4.2 × 10⁶ M^?1 sec^?1 respectively. With intact Sarcoma 180 cells, negligible reactivation of ADA was seen during a 48-hr period. Preliminary studies indicate an important role for the erythrocytic nucleoside transport system on the apparent k₁ values and the rate of inactivation of ADA by deoxycoformycin in intact cells.     

Tight-binding inhibitors—IV. Inhibition of adenosine deaminases by various inhibitors

Three ADA (adenosine deaminase) inhibitors, DHMPR (1,6-dihydro-6-hydroxymethyl purine ribonucleoside); EHNA [erythro-9-(2-hydroxy-3 nonyl)adenine] ; and deoxycoformycin [(R)-3-(2-deoxy-β-d-erythro-pento-furanosyl)-3, 6,7,8-tetrahydroimidazo[4,5-d] [1,3-diazepin-8-ol] or Covidarabin, were compared with regard to their inhibitory behavior with ADAs from human erythrocytes and calf intestine. Marked differences in the times required for establishment of steady state between the enzyme and inhibitors were observed, e.g. DHMPR, virtually instantaneous; EHNA, 2–3 min; and deoxycoformycin, many hr. The parameters of the inhibition of human erythrocytic ADA by deoxycoformycin were as follows: the association rate constant (k₁) = 2.6 × 10⁶ M^?1 sec^?1 ; the dissociation rate constant of the enzyme-inhibitor complex (k₂) = 6.6 × 10^?6 sec^?1; K_i (from $\text{k}{}_2\text{k}{}_1\text{) = 2.5 × 10}{}^{\mathrm{?12}}\text{M}$ and K_i (from I₅₀) = 1.5 × 10^?11 M. The K_i values for EHNA and DHMPR, as determined by classical methods after attainment of steady state, were 1.6 × 10^?9 and 1.3 × 10^?6 M, respectively, for human erythrocytic ADA. The kinetic parameters for EHNA and calf intestinal ADA were as follows: K_i = 6.5 × 10^?9 M (by the method of I₅₀); k₁ = 0.7 × 10⁶ M^?1 sec^?1' and k₂ = 4.6 × 10^?3 sec^?1. On the basis of K_i values, the inhibitors. DHMPR, EHNA and deoxycoformycin (a transition state analog), were classified as readily reversible, semi-tight-binding and tight-binding inhibitors. The difficulties encountered in the kinetic analyses of different types of inhibitors and the methods for dealing with the problems of these inhibitors are discussed.     

Angiotensin II binding to zona glomerulosa cells from rabbit adrenal glands.

Tritiated angiotensin II binds in a highly specific manner to zona glomerulosa cells prepared from the adrenal cortex of male rabbits. The reaction is a time-dependent process which obeys second-order kinetics (k₁ = 2.4 × 10⁵ M^?1 sec^?1) and reaches saturation in 5–7 min. Dissociation of the angiotensin II-cell complex is rapid (t_{1 2} = 100 sec) and obeys first-order kinetics for the first 3 min (k_?1 = 6.9 × 10^?3 sec^?1).Increased binding was observed with NaCl concentrations from 0 to 40 × 10^?3 M; however, concentrations from 40 to 140 × 10^?3 M decreased binding. Neither MgCl₂ nor Cll₂ at concentrations of 0 to 4 × 10^?3 M alter the binding of angiotensin II to zona glomerulosa cells. A significant decrease in binding was observed with increasing concentrations of KCl (0 to 140 × 10^?3 M).Temperature studies indicate that initially binding is more rapid at 37° (37° > 25° > 0°). However, binding of angiotensin II decreases after 3 min at 37° and after 7 min at 25°. Binding at 0° did not reach a plateau in the 15-min period studied.     

N-Methylformycins. Reactivity with adenosine deaminase, incorporation into intracellular nucleotides of human erythrocytes and L 1210 cells and cytotoxicity to L 1210 cells.

The N-methyl derivatives of the C-nucleoside, formycin (7-amino-3(β-d-ribofurano-syl)pyrazolo[4, 3-d]pyrirnidine) were compared to formycin and adenosine with regard to their substrate activity with human erythrocytie adenosine deaminase (ADA), their ability to form intracellular nucleotides and their cytotoxicity to L1210 cells. Only 2-methylformycin (K_m = 6.1 mM, relative V_max = 396) and N^? -methylformycin (K_m = 0.1 mM, relative V_max = 3) showed substrate activity with ADA (corresponding kinetic parameters for adenosine were: K_m = 0.025 mM, relative V_max = 100). In contrast to previous hypotheses, these results suggest that the conformation (either syn or anti) of an adenosine analog is not a major factor in determining substrate activity with ADA. Neither 4-methylformycin nor 6-methylformycin formed their corresponding nucleotides when incubated with human erythrocytes, whereas both 1-methylfor-mycin and 2-methylformycin formed large amounts of their corresponding mono-, di- and triphosphate nucleotides. Inhibition of ADA by pretreatment of the erythrocytes with the potent ADA inhibitor, 2'-deoxycoformycin, had no effect on the incorporation of 1-methylformycin into erythrocytic nucleotides but greatly increased the incorporation of 2-methylformycin and N⁷-methylformycin. The conversion of both 1-methylformycin and 2-methylformycin into nucleotides was almost complete after 18 hr of incubation (in the presence of 2'-deoxycoformycin in the case of 2 methylformycin), whereas that of N⁷-methylformycin was only partially complete in the presence of 2'-deoxycoformycin. With both 1-methylformycin and N⁷-methylformycin, transient accumulation of the corresponding nucleoside 5'-monophosphate derivative was observed prior to the accumulation of the triphosphate nucleotide. Results, qualitatively similar to those found with erythrocytes, were obtained when the effects of 2'-deoxycoformycin on the incorporation of 1-methyl- and 2-methylformycins into the nucleotide pools of L 1210 cells in vitro were examined. Compounds capable of forming analog nucleotides in human erythrocytes or L1210 cells if deamination is prevented either by the molecular structure of the analog or by pretreatment of the cells with 2'-deoxycoformycin, also showed marked cytotoxicity to L1210 cells in culture, i.e. 1-methyl-, 2-methyl- and N⁷-methylformycin exhibited id₅₀ values of 0.5 to 2 μM, whereas 4-methyl- and 6-methylformyein were not significantly growth inhibitory. The potential usefulness of the various N-methyl derivatives of formycin (alone or in combination with an ADA inhibitor) as cytotoxic or antiviral agents is discussed.     

Reactivity of the hydroxyl radical with amygdalin in aqueous solution

Rate constants were determined for the reactions of hydroxyl radicals with amygdalin in aqueous solutions by means of the pulse radiolysis technique. The overall rate constant (phenyl group addition and H-atom abstraction) was 4.1 × 10⁹M^?1 sec^?1 at 22. The addition rate constant was 3.7 × 10⁹M^?1 sec^?1. The abstraction rate constant was in the order of 4 × 10⁸M^?1 sec^?1. This high reactivity is pertinent to models for protection, by amygdalin, against alloxan-induced diabetes.     

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.     

Radical formation during autoxidation of 4-dimethylaminophenol and some properties of the reaction products

4-Dimethylaminophenol (DMAP), after intravenous injection, rapidly forms ferrihaemoglobin and has been successfully used in the treatment of cyanide poisoning. Since DMAP produces many equivalents of ferrihaemoglobin, it was of interest to obtain further insight into this catalytic process. DMAP autoxidizes readily at pH regions above neutrality, a process which is markedly accelerated by oxyhaemoglobin. The resulting red-coloured product was identified as the N-N,-dimethylamino) phenoxyl radical by EPR spectroscopy. The same radical was also produced by pulse radiolysis and oxidation with ferricyanide. The 4-(N-N-dimethylamino)phenoxyl radical is quite unstable and decays in a pseudo-first order reaction (k = 0.4 sec^?1 at pH 8.5,22°) with the formation of p-benzoquinone and dimethylamine. This observed decay rate is identical with the rate of hydrolysis of N,N-dimethylquinonimine. When a solution containing the phenoxyl radical was extracted with ether, half the stoichiometric amount of DMAP was recovered. Hence it is apparent that the phenoxyl radical decays by disproportionation yielding DMAP and N,N-dimethylquinomine. The latter product then quickly hydrolyses. The equilibrium of this disproportionation reaction is far towards the radical side, and the pseudo-first order hydrolysis controls the radical decay rate. p-Benzoquinone rapidly reacts with DMAP (k₂ = 2 × 10⁴M^?1sec^?1) with the formation of the 4-N,N-dimethylamino)phenoxyl and the semiquinone radicals. This reaction explains the autocatalytic phenoxyl radical formation during the autoxidation of DMAP. DMAP is not oxidized by H₂O₂ or O₁₂^? but the 4-(N-N-dimethylamino)phenoxyl radical is very rapidly reduced by O₂^? (k₂ = 2 × 10⁸M^?1 sec^?1. In addition, the phenoxyl radical is quickly reduced by NAD(P)H or GSH with the formation of NAD(P)⁺ or GSSG. Since DMAP is also able to reduce two equivalents of ferrihaemoglobin (provided that the ferrohaemoglobin produced is trapped by carbon monoxide), electrophilic addition reactions of the phenoxyl radical seem unimportant in contrast to N,N-dimethylquinonimine. Hence, during the catalytic ferrihaemoglobin formation, DMAP is oxidized by oxygen which is activated by haemoglobin, and the phenoxyl radical oxidizes ferrohaemoglobin. This catalytic process is terminated by covalent binding of N,N-dimethylquinonimine to SH groups of haemoglobin (and GSH in red cells).     

Role of thymidine kinase in the inhibitory activity of 5-substituted-2'-deoxyuridines on the growth of human and murine tumor cell lines

Twenty-four 5-substituted 2'-deoxyuridines have been evaluated for their inhibitory effects on the growth of three human lymphoblast cell lines (Namalva, Raji and TK^? (thymidine kinase deficient) Raji) and these inhibitory effects were compared to those for two murine leukemia cell lines (L1210/0 and L1210/BdUrd). The latter was selected from the parental L1210/0 cell line by its ability to grow at high concentrations of 5-bromo-dUrd and could also be considered as TK^?. There was a close correlation between the inhibitory effects of the deoxyuridine analogs on Namalva, Raji and L1210 cells: the correlation coefficient (r) for log id₅₀ (median inhibitory dose) for L1210 cell growth, on the one hand, and log id₅₀ for Namalva or Raji cell growth, on the other hand, was 0.902 and 0.929, respectively. There was also a strong correlation (r = 0.936) between the log id₅₀ values for the two human lymphoblast cell lines. However, there was no significant correlation (r < 0.40) either between the log id₅₀ for the TK^? Raji cells and the parental TK⁺ Raji cells, or between the log id₅₀ for the TK^? L1210/BdUrd cells and the parental TK⁺ L1210/0 cells. We may conclude therefore, that (i) the murine leukemia L1210 cell system is predictive for the growth-inhibitory effects of 5-substituted 2'-deoxyuridines on human lymphoblast cell lines, and (ii) the antitumor cell activity of the 5-substituted 2'-deoxyuridines is, to a large extent, dependent on the thymidine kinase activity of the tumor cells.     

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.     

Pharmacokinetics of 125I-labelled venom from the scorpion Androctonus amoreuxi,Aud. and Sav.

Androctonus amoreuxi venom was radioiodinated using the chloramine T method of [¹²⁵I] iodide oxidation giving a spec. act. of 12·5 μCi/mg. Gel filtration on Sephadex G-50 and cellulose acetate electrophoresis of the labelled venom showed good correlation between protein concentration and radioactivity. Labelled venom was injected i.v. into rabbits anaesthetized with urethane and blood samples were withdrawn from a common carotid artery at times from 1 to 270 min after venom injection for measurement of radioactivity and determination of blood glucose. A biphasic blood level curve showing a rapid initial declining phase during the first 15 min followed by a slower declining phase was obtained. This behaviour is characteristic of a two-compartment open model. The rate constants between blood and tissues “k_CT”, tissues and blood “k_TC” and the disposition rate constant “k_d” were estimated to be 9·8 × 10^?2, 3·34 × 10^?2 and 6·67 × 10^?3, respectively. The values indicate a very rapid distribution of venom from blood to tissues with an estimated half-life of 5·6 min whereas the overall elimination half-life is 104 min. Steady-state distribution indicated that 75% of the injected venom was in the tissue compartment after equilibrium was reached. Significant rise in blood glucose was observed when equilibrium was reached and continued throughout the equilibrium phase. Tissue distribution of labelled venom showed major radioactivity to be in liver and kidney. Much less activity was found (in decreasing order) in thyroid, lungs, heart, uterus, intestine, ovary, diaphragm and spleen.     

Tight binding inhibitors--VIII. Studies of the interactions of 2'-deoxycoformycin and transport inhibitors with the erythrocytic nucleoside transport system

Studies were performed to extend earlier observations that the rate-limiting step in the inactivation of intraerythrocytic human adenosine deaminase (ADA) by 2'-deoxycoiormycin (dCF) is the nucleoside transport system (NTS). The NTS inhibitors 2-amino-6-[(2-hydroxy-5-nitrobenzyl)thio]-9-β-D-ribofuranosyl purine (HNBTGR), 6-[(4-nitrobenzyl)thio]-9-β-d-ribofuranosyl purine (NBMPR), 2-amino-6-[(4-nitrobenzyl)Seleno]-9-β-D-ribofuranosyl purine (NBSeGR), dipyridamole and the competitive permeant, uridine, all decreased the rate of ADA inactivation by dCF in a concentrationdependent manner. Lineweaver-Burk plots of $\text{1}\text{k}{}_{\mathrm{\gamma }}$ (where k_γ is the pseudo first-order rate constant for the inactivation of ADA) 1/dCF concentrations were linear, giving a K_m tor dCF tor the NTS of 6 × 10^?7 M. The maximal k_γ calculated by extrapolation to infinite dCF concentrations was 6 × 10^?3 per sec which corresponds to a $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of about 115 sec. Similar plots for experiments with the NTS inhibitors and uridine yielded classic patterns of competitive inhibition for NBMPR, HNBTGR, NBSeGR and uridine, whereas with dipyridamole a pattern of non-competitive inhibition was obtained. Dissociation or inhibition constants have been reported for several of these compounds (determined by other methods) and values similar to these were obtained. Inhibition by dipyridamole was non-competitive (k_l = 2.5 × 10^?7 M) and was of a bi-phasic nature with respect to time. Dipyridamole caused rapid and irreversible inhibition for the first 7–15 min with slow and progressive but reversible inhibition thereafter. These observations are consistent with the hypothesis that NBMPR, HNBTGR, NBSeGR and uridine interact with the same site on a macromolecular component of the NTS that forms ligands with dCF. The behavior of dipyridamole appears more complex and will require more extensive study.     

Effect of acetylcholine on monoamine oxidase

The addition of acetylcholine to rat brain monoamine oxidase in vitro resulted in its inhibition. This inhibition was found to be of the competitive type. Constants characterizing this inhibition, namely the binding constant K_I (5.8 × 10^?2 M), and bimolecular rate constant k_a (27 M^?1 min^?1) were determined.     

Involvement of tryptophan residues of lysozyme in its binding with cytosine arabinoside

Cytosine-1-β-d-arabinofuronaside, an antileukemic drug and hen egg white lysozyme (E.C. 3.2.1.17) form a 1:1 complex with K_a 1.2 × 10⁴ M^?1 at low drug concentrations. However, association was cooperative in nature at high concentrations with values of K_a = 1.8 × 10⁴ M^?1 and N = 2. Involvement of tryptophan in the drug protein complex was evident from fluorescence quenching and from the association of the drug with free tryptophan with a K_a = 1.5 × 10⁴ M^?1. Modification of tryptophan 108 reduced the binding by 89% suggesting a major role for this residue in the binding process. Oxidation of tryptophan 62 and acetylation of lysine residues also decreased the affinity of the drug to the protein by 55 and 66% respectively. Binding improved with increase in temperature and positive values for change in enthalpy and entropy were obtained. Ara-C inhibited lysozyme activity noncompetitively.     

Characterization and solubilization of the specific binding sites for d-α-tocopherol from human erythrocyte membranes

Previous work from our laboratory has demonstrated the presence of specific binding sites for d-α-tocopherol (vitamin E) in intact human erythrocytes [A. E. Kitabchi and J. Wimalasena, Biochim. biophys. Acta684, 300 (1982)]. The binding was time, temperature and cell concentration dependent. To localize the binding sites, red blood cells were further fractionated; greater than 90% of the tocopherol binding sites were localized on membranes. The washed membrane fraction from normal human erythrocytes has specific binding sites for d-α-tocopherol with properties suggestive of protein receptors. Two binding sites with K_a values of 3.31 × 10⁷ M^?1 and 1.51 × 10⁶ M^?1 were demonstrated, and solubilized d-α-tocopherol binding site complexes were resolved to major component with an M_r of 65,000 and a minor component with an M_r of 125,000.     

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.     

Synthesis and biologic studies of arabinosylcytosine 5'-methylphosphonate

Arabinosylcytosine (ara-C), a clinically useful antitumor agent, is ineffective against cells that have deleted deoxycytidine kinase, the enzyme necessary for conversion of ara-C to its active nucleotide form. To circumvent this resistance, arabinosylcytosine-5'-methylphosphonate (ara-CMeP) was synthesized as an analogue of ara-CMP that would be membrane-permeable, resistant to serum phosphatase attack, and resistant to nucleoside deaminase inactivation. Ara-CMP was inhibitory to leukemia P388 in vitro but required concentrations 90-fold greater than that of ara-C for comparable cell inhibition. Both ara-CMeP and ara-CMP were competitive inhibitors of dCMP kinase from leukemia L1210 with K_i values of 4.0 × 10^?3 and 4.4 × 10^?3 M respectively. However, ara-CMP is a substrate for dCMP kinase, whereas ara-CMeP was not. Thus, the inability of ara-CMeP to be phosphorylated precludes its usefulness as a functional analogue of ara-CMP.     

Successful chemoimmunotherapy of murine L1210 lymphatic leukemia with cyclophosphamide and mafosfamide-treated leukemia cells

Summary Balb/c × DBA/2 F₁ mice (CD2F₁ mice) bearing L1210 lymphatic leukemia (10 L1210 cells i.p. injected on day 0) were subjected to chemoimmunotherapy. They received 100 mg/kg of cyclophosphamide i.p. on day + 8 and 10⁶ or 10⁷ immunogenic L1210 cells treated in vitro with mafosfamide — ASTA Z7654 (L1210-Maf cells) i.p. or i.p. + s.c. on days 0, + 3, + 6, + 9, + 12 after the leukemia implantation.About 30% of leukemia-bearing mice receiving cyclophosphamide and L1210-Maf cells after L1210 inoculation were able to reject the leukemia (as compared with 0% after injection of L1210-Maf cells only or 5% after cyclophosphamide administration). Better results (54% of cured mice) were obtained if 10⁷ L1210-Maf cells were injected i.p. + s.c. beside cyclophosphamide. Biological response modifiers (BRM's): levamisole, BCG, bestatin did not improve these results in the doses used in the experiment.Augmentation of anti-L1210 therapeutic response is dependent on the administration of cyclophosphamide and L1210-Maf cels. Cyclophosphamide not only reduces the tumor burden but probably can potentiate the L1210-Maf dependent antitumor immunity as well.     

Tight-binding inhibitors-II. Non-steady state nature of inhibition of milk xanthine oxidase by allopurinol and alloxanthine and of human erythrocytic adenosine deaminase by coformycin.

The non-steady state nature of the inhibition of milk xanthine oxidase by allopurinol and alloxanthine was demonstrated, and the kinetic data presented are consistent with the known mechanisms of inhibitions by these inhibitors. With the use of human erythrocytic adenosine deaminase and its tight-binding inhibitor, coformycin, it was demonstrated that the classical methods of enzyme kinetics based on the steady state assumptions are grossly inadequate for determining the inhibition mechanisms or inhibition constants for tight-binding inhibitors. The application of the Ackermann-Potter plot, I₅₀. the Easson-Stedman plot (or Henderson plot), and the rates of association and dissociation of enzyme-inhibititor complex were presented and their usefulness was evaluated. The molar equivalency and the catalytic number of human erythrocytic adenosine deaminase were estimated at about 1.0 × 10^?10 mole/unit and 1.0 × 10⁴ min^?1 respectively. It was also demonstrated that the K_i, value of coformycin for this enzyme does not exceed 1.2 × 10^?10 M, and that the second-order rate constant for the association of the enzyme with coformycin is approximately 2 × 10⁶ M^?1 sec^?1. The biphasic nature of the dissociation of the deaminase-coformycin complex (EI complex) indicates that the EI complex undergoes a slow conformational change. The implications of these new kinetic approaches for the study of tight-binding inhibitors, including transition-state analogs, were discussed.     

Inhibition of superoxide anion production in non-stimulated guinea pig peritoneal exudate cells by anti-inflammatory drugs.

Inhibitory effects of anti-inflammatory drugs on the production of superoxide anion (·O₂^? by isolated non-treated guinea pig peritoneal exudate cells (PEC) was studied spectro-photometrically using NADH and lactate dehydrogenase (LDH). Values of ID₅₀ were; diclofenac sodium (2 × 10^?5M), indomethacin (3 × 10^?5M), oxyphenbutazone (8 × 10^?5M), fenamic acid (1 × 10^?4M), ibuprofen (1 × 10^?4M), benzydamine (3 × 10^?4M), aspirin (10^?3M<) and dexamethasone (10^?3M<). The mechanism of inhibition seemed to block plasma membrane associated NAD(P)H oxidase(s) activity which produces ·O₂^? ID₅₀ values of other drugs; superoxide dismutase (SOD, 2 × 10^?8M), cytochalasin B(1 × 10^?7M) and NEM (6 × 10^?6M). d-Mannitol radical scavenger), 1,3-diphenyl-isobenzofuran (singlet oxygen scavenger) and sodium azide (mitochondrial electron transport inhibitor and singlet oxygen scavenger) were negative.Superoxide radical itself or oxygen-centered radical(s) derived from ·O₂^? is supposed recently as a rate-limiting factor for prostaglandin (PG) synthetase. Whether the inhibition of non-steroidal anti-inflammatory drug (NSAID) on ·O₂^? production is linked directly to PG biosynthesis or not, ·O₂^? was already demonstrated in our laboratory to make a role for the development of rat carageenan foot oedema. It may serve as a new in vitro sceening method of NSAID, to check the inhibitory potency of a compound on ·O₂^? production by guinea pig PEC.     

Eigenschaften der Cholinesterasen in Geweben des Dorsches(Gadus callarias) und ihre Inaktivierung durchin vivo Einwirkung von Paraoxon und Tabun

The investigations presented in this paper were carried out in connection with possible contamination of the Baltic Sea by dumped tabun ammunition. By means of different substrates it was possible to demonstrate that most tissues of Gadus callarias (cod), (brain > myocardium ≧ skeletal muscle ? gills) contain only acetyleholinesterase (AChE, E.C.3.1.1.7.), whereas the liver also contains some butyrylcholinesterase (E.C.3.1.1.8.). Neither type of cholinesterase can be detected in erythrocytes and serum. The properties of the AChE of brain were characterized by determination of the substrate optimum (1 · 10^?3M ACh), the substrate inhibition (I₅₀ at 2·10^?2M ACh), the Michaelis constant (Km = 1 · 10^?4M ACh), and the dependence on pH and temperature (maximal activity at pH 8.5 and 37°C). The specific activity of AChE of brain or skeletal muscle is practically independent of the body weight. Exposure of living fish to concentrations of paraoxon or tabun between 1 · 10^?8 M and 1 · 10^?6M in sea water for 0.5 to 24 h yielded the following results: Both organophosphates were absorbed rapidly. Paraoxon inactivates the AChE of brain and of skeletal muscle with an exponential activity course at similar rates. The bimolecular rate constant (k ₂) hardly differs from the one measured in vitro (k ₂ in vivo 1.15 · 10⁴M^?1min^?1,in vitro 1.6 · 10⁴M^?1min^?1). Thein vitro reaction of tabun was probably falsified by its hydrolysis in sea water (k ₂ for AChE of brain is approximately 8 · 10³M^?1min^?1). The AChE of muscle was inactivated to a lesser degree by tabun than was cerebral AChE. The fish died when the activity of the AChE of the brain was lowered to a 5 % level by paraoxon or tabun. In spite of the extremely slow rate of reaction of cerebral AChE with either organophosphate it was possible to detect concentrations of 3 · 10^?8 - 1· 10^?7M ( 0.005 to 0.015 ppm. of paraoxon and tabun) by means of the AChE inactivation after 3 to 6 h of exposure.