5-Ethyl-2′-deoxyuridine-5′-monophosphate inhibition of the thymidylate synthetase from escherichia coli

The synthesis of 5-ethyl-2′-deoxyuridine-5-monophosphate (EtdUrdMP) and an improved method for the purification of deoxythymidylate synthetase (TdRMP synthetase) from E. coli are described. TdRMP synthetase was inhibited competitively by EtdUrdMP (K₁ = 2·2 × 10^?5 M). Under similar conditions the K_i for deoxythymidine monophosphate (TdRMP) was 2·0 × 10^?5 M. The possible role of EtdUrdMP in the virostatic activity of the nucleoside 5-ethyl-2′-deoxyuridine (EtdUrd) is considered.     

Effect of a new potent H2-receptor antagonist 3[[[2-[(diaminomethylene)amino]-4-thiazolyl]methyl]thio]-N2-sulfamoylpropionamidine (YM-11170) on gastric mucosal histamine-sensitive adenylate cyclase from guinea pig

The effect of 3[[[2-[(diaminomethylene)amino]-4-thiazolyl]methyl]thio]-N²-sulfamoylpro-pionamidine (YM-11170), a new thiazole H₂-receptor antagonist bearing propionamidine at the terminus of a side chain, on histamine-sensitive adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1] of gastric mucosa from the guinea pig was studied and compared with that of cimetidine. YM-11170 displaced the concentration-stimulation curve of histamine-sensitive adenylate cyclase to the right with a pA₂ of 7.65 (K_i, = 2.25 × 10^?8M). Stimulation of gastric adenylate cyclase by 0.1 mM histamine was competitively inhibited by YM-11170 and cimetidine in a dose-dependent manner, with ic₅₀ values of 5.9 × 10^?7M and 1.4 × 10^?5M respectively. Hippocampal histamine-sensitive adenylate cyclase in the presence of 0.1 mM histamine was also competitively inhibited by YM-11170 with an ic₅₀ of 1.1 × 10^?7 M. YM-11170 did not affect Gpp(NH)p-, NaF-, PGE₂-stimulated or basal activity of the gastric adenylate cyclase. These data, together with other results, indicate that YM-11170 is a highly selective and potent H₂-receptor antagonist which competes with histamine at the receptor site on the histamine-sensitive adenylate cyclase.     

Characterization of adenosine deaminase from the malarial parasite, Plasmodium lophurae, and its host cell,the duckling erythrocyte

Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) was purified and characterized from the malarial parasite, Plasmodium lophurae, and its host cell, the duck (Anas domesticus) erythrocyte, using chromatofocusing (Pharmacia) and adenosine affinity columns. Gel filtration of the enzymes gave molecular weights of 33,800 (P. lophurae) and 36,500 (duck erythrocyte); both enzymes had broad pH optima (pH 6.8 to 8.0), similar stabilities when stored as crude lysates, and like K_m values with adenosine: 2.74 ± 0.88 × 10^?5 M (parasite) and 1.74 ± 0.27 × 10^?5 M (erythrocyte). The P. lophurae adenosine deaminase had a pI of 5.37 ± 0.09, and the duck erythrocyte enzyme had a pI of 4.72 ± 0.09, as determined by chromatofocusing. The parasite enzyme exhibited a specific activity in the crude lysate that was an average 60-fold higher than that of the erythrocyte enzyme. The pattern of elution from the adenosine affinity column, as well as kinetic studies with three adenosine analogs, revealed distinct differences in the binding characteristics of the two enzymes. The P. lophurae adenosine deaminase was weakly retarded by the affinity column, whereas the duck erythrocyte enzyme was strongly retarded. With 9-β-d-arabinosyladenine as substrate, the K_m values were similar (2.29 ± 0.98 × 10^?4 M for P. lophurae and 1.10 ± 0.21 × 10^?4 M for the duck erythrocyte). Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) was a potent inhibitor of the duck erythrocyte enzyme with 100% inhibition at 1.3 μM, whereas the parasite adenosine deaminase was not inhibited at 422 μM even when incubated for 24 hr. Inhibitor studies with coformycin, a tight-binding inhibitor, resulted in K_i values of 7.14 × 10^?11 M for P. lophurae and 1.86 × 10^?10 M for the duck erythrocyte. The molar equivalencies, E_t, and catalytic numbers, k₃, were slightly different for both enzymes. The E_t values were 2.80 × 10^?10 M (P. lophurae) and 3.13 × 10^?10 M (duck erythrocyte); the k₃ values were 5.18 × 10³ min^?1 and 4.36 × 10³ min^?1 respectively.     

Effects of adenosine analogs and adenine nucleotides on adenosine 5'-diphosphate-induced rat platelet aggregation

Various adenosine analogs and adenine nucleotides have been tested as inhibitors of ADP-induced aggregation of rat platelets. The potent inhibitors of human platelet aggregation, adenosine, 2-fluoroadenosine, 2-chloroadenosine, carbocyclic adenosine and N⁶-phenyl adenosine, had little effect on rat platelet aggregation (0–30 per cent inhibition). The effects of adenosine or its analogs on ADP-induced aggregation of cross-species platelet-rich plasmas (PRPs) (human platelets suspended in rat plasma or rat platelets in human plasma) were similar to those with the native PRPs, indicating that these species differences were due to intrinsic factors in the platelets and not in the plasma. When these analogs were tested in the presence of the cyclic AMP phosphodiesterase inhibitor papaverine, strong inhibiton of rat platelet ADP-induced aggregation was seen. 2′-Deoxyadenosine and 3′-deoxyadenosine were not inhibitory to ADP-induced aggregation of rat PRP even in the presence of papaverine. Adenosine 5′-tetraphosphate strongly inhibited both human and rat platelet aggregation. AMP, like adenosine, did not inhibit rat platelet aggregation but became strongly inhibitory in the presence of papaverine. This inhibitory effect was abolished by preincubating rat PRP with an adenylate cyclase inhibitor, 2′, 5′-dideoxyadenosine or adenosine deaminase. In the later case, however, if the adenosine deaminase inhibitor 2′-deoxycoformycin was included in the incubation mixture, the inhibition by AMP plus papaverine was similar to adenosine plus papaverine. About 50 per cent of [¹⁴C]AMP was converted to [¹⁴ C]adenosine in rat platelet-free plasma or PRP after a 10-min incubation. α,β-Methylene-ADP and β,γ-methylene-ATP (200 μM) inhibited rat platelet aggregation by 50 and 64 per cent, respectively. Cyclic AMP phosphodiesterase of rat and human platelets gave comparable K_m, and V_max values (K_m 0.53 and 0.21μM and V_max 6.0 and 6.7 pmoles/min/10⁷ platelets, respectively).     

The mechanisms of action of three fluorine substituted cytosine analogs: implications for cancer chemotherapy

The mechanisms of action in DNA synthesis of three fluorine substituted cytosine analogs have been studied in human phytohaemagglutinin-stimulated lymphocytes. 5-Fluorocytidine (over a wide range of concentrations) and 5-fluorocytosine (at high concentration) inhibited ³H-deoxyuridine incorporation into DNA without inhibiting ³H-thymidine incorporation. 5-Fluorocytidine caused reduction in the free cell concentration of TTP. In contrast, ara-fluorocytosine markedly inhibited both ³H-deoxyuridine and³H-thymidine incorporation into DNA but did not cause a consistent change in thymidine triphosphate concentration. These results indicate that 5-fluorocytidine, and to a lesser extent, 5-fluorocytosine block de novo TMP synthesis in human cells. It seems likely that they are both metabolized to 5-fluorodeoxyuridinc monophosphate which inhibits the enzyme TMP synthetase. On the other hand, the results suggest that ara-fluorocytosine has an action identical to that of cytosine arabinoside, inhibition of DNA polymerase. It is postulated that cells resistant to cytosine arabinoside which have high cytidine deaminase levels might be selectively sensitive to 5-fluorocytidine and to 5-fluorocytosine.     

Effect of ryanodine on skeletal muscle reticulum calcium adenosine triphosphatase (CaATPase)

Ryanodine activates the CaATPase of a heavy (2000–8000 g) sub-unit of skeletal muscle sarcoplasmic reticulum, with K_A (for half-maximal activation) of 1·9 × 10^?5 M in the presence of 5 × 10^?3 M MgATPand 10^?6 M freeCa.K_A is only 3·7 × 10^?6 M when the muscle fraction is exposed to ryanodine in the absence of Mg, and K_A in the presence of 5 mM free Mg is 3.3 × 10^?5M, with Mg competitively inhibiting the effect of the alkaloid. Ryanodine does not affect ADP-ATP exchange rate or the steady state level of phosphoprotein, but activates CaATPase by altering Ca permeability and decreasing intravesicular free Ca. The effect of ryanodine increases with temperature, is not affected by extensive dialysis and appears irreversible, with 1 mole of drug bound per 10⁷ g of membrane protein; the drug has little effect on standard fragmented sarcoplasmie reticulum (FSR) preparations.     

Choline and diethylcholine transport into a cholinergic clone of neuroblastoma cells.

The transport of choline and diethylcholine has been investigated in a cholinergic clone (S20F₃) of mouse neuroblastoma cells. Choline transport was linear for the first 20 min of incubation and was temperature dependent at low concentrations (< 1 × 10^?5 M). Diethylcholine transport was linear for the first 10 min of incubation and was also temperature dependent at low concentrations. High affinity (K_m < 1 × 10^?6 M) and low affinity (K_m > 1 × 10^?5 M) components of transport were found for both compounds. The transport system had a greater apparent affinity (lower K_m) for choline than for diethylcholine (3-fold), but maximal transport velocities were about equal. Each compound competitively inhibited the other's high affinity transport. Hemicholinium (1 × 10^?5 M) slightly inhibited high affinity choline transport but triethylcholine (1 × 10^?6?1 × 10^?4 M) did not. Choline transport was also found to be dependent on the pH and pCO₂ of the medium.     

Pyrimidine acyclonucleosides,inhibitors of uridine phosphorylase

A new class of nucleoside analogs, the pyridimine acyclonucleosides, are competitive inhibitors of uridine phosphorylase but have no effect on thymicline phosphorylase, uridine kinase or thymidine kinase. The most potent of the series is acyclothymidine [5-methyl-1-(2′-hydroxyethoxymethyl)uracil] with a K_i value of 3 μM. K_i values of less than 30 μM were estimated for other analogs substituted at the 5-position of the pyrimicline ring. Extracts of xenografts of six human tumors were assayed for tissue levels of uricline phosphorylase and thymicline phosphorylase and for inhibition of 5-fluoro-2′-deoxyuridine (FUdR) phosphorolytic activity by acyclouridine [1-(2′-hydroxyethoxymethyl) uracil]. FUdR cleavage was inhibited most in those tissues in which the ratio of thymidine phosphorylase to uricline phosphorylase was low. Potential usage of these uricline phosphorylase inhibitors with the chemotherapeutic agent FUdR is discussed.     

Inhibition of methylation of nuclear ribonucleic acid in L1210 cells by tubercidin, 8-azaadenosine and formycin

The adenosine analogs tubercidin (7-deazaadenosine), formycin (7-amino-3-[β-d-ribofuranosyl] pyrazolo[4,3-d]pyrimidine) and 8-azaadenosine were examined for their effects on the synthesis and methylation of nuclear RNA in L1210 cells in vitro. Total RNA and DNA synthesis was affected to the greatest extent by tubercidin (IC₅₀ = 7 × 10^?6M) and to an insignificant degree by 8-azaadenosine and formycin; however, the effects of the latter two drugs, but not of tubercidin, were potentiated by 2'-deoxycoformycin, an inhibitor of adenosine deaminase. In the presence of 2'-deoxycoformycin, RNA synthesis was inhibited by 40 per cent at 1 × 10^?4 M 8-azaadenosine and by 50 per cent at 2 × 10^?4 M formycin, while DNA synthesis was inhibited less extensively. Alkaline hydrolysis of nuclear RNA labeled with [¹⁴C]uridine and l-[methyl-³H]methionine showed preferential inhibition of base methylation in mononucleotides, but not of 2′-O-methylation in dinucleotides, for all three drugs. This differential effect persisted to varying degrees in ?18S and 4S nuclear RNA separated by electrophoresis. The reduction in base methylation in 4S RNA was associated with seven of the eight methylated nucleosides in 4S RNA separated by two-dimensional thin-layer chromatography. These results indicate that tubercidin, 8-azaadenosine and formycin can preferentially inhibit the base methylation of nuclear RNA relative to its synthesis.     

Guanosine 5′-monophosphate reductase from Leishmania donovani: A possible chemotherapeutic target

GMP reductase was highly purified from promastigotes of Leishmania donovani by chromatography on a single DEAE-cellulose column. Bimodal substrate saturation curves resulted in a 1/ν versus 1/[GMP] plot that curved downward above 40 μM GMP. The kinetic constants were, therefore, obtained with GMP below this concentration. The K′_m for GMP was 21 μM at pH 6.9. The enzyme was very sensitive to activation by GTP. At 20 μM GMP, a maximum of 600% activation occurred at 100 μM GTP. Half-maximal activation occurred at 8 μM GTP. GTP at 100 μM did not affect the K′_m for GMP but did increase its V′_max by 7-fold. Xanthosine monophosphate (XMP) and IMP analogs served equally well as competitive inhibitors versus GMP. The inhibition by the analogs and the activation by GTP were mutually antagonistic processes. The inhibition by the IMP analogs, allopurinol nucleotide and thiopurinol nucleotide is of chemotherapeutic interest because these compounds were shown previously to be produced in Leishmania from the anti-leishmanial agents allopurinol and thiopurinol. These nucleotides were 100- and 20-fold, respectively, more potent inhibitors of GMP reductase from L. donovani than of the corresponding enzyme from human erythrocytes.     

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.     

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.     

A comparison of the inhibitory effects of new non-tricyclic amine uptake inhibitors on the uptake of norepinephrine and 5-hydroxytryptamine into synaptosomes of the rat brain

The effects of new non-tricyclic amine uptake inhibitors, FS32 and FS97, on the uptake of [³H]-norepinephrine (NE) into the hypothalamic synaptosomes and [³H]-5-hydroxytryptamine (5-HT) into whole brain synaptosomes were studied. Their effects were compared with those of tricyclic antidepressants. The uptake of [³H]-NE was inhibited competitively by FS32 and FS97 with a respective K_i value of 6.5 × 10^?7 M and 3.8 × 10^?7 M. The potency of FS32 and FS97 to inhibit this uptake was almost comparable to that of clomipramine and imipramine, respectively. In the case of [³H]-5-HT uptake, FS32 and FS97 also showed competitive inhibition with a respective K_i value of 2.9 × 10^?6 M and 5.9 × 10^?6M. The ability of FS32 to inhibit [³H]-5-HT uptake was almost equal to that of nortriptyline, while FS97 was two times more potent than iprindole in inhibiting this uptake.     

The interaction of bis-pyridinium oximes with mouse brain muscarinic receptor

The bispyridinium oximes toxogonin [N, N′-oxydimethylene bis (pyridinium 4-aldoxim) dichloride] and its structural analogs HS-3, HS-6, HI-6 and MMB-4, and the bispyridinium salt SAD-128, which serve as antidotes to certain types of organophosphorus poisoning, bind competitively to mouse brain muscarinic receptors. This was determined in vitro employing the potent and specific muscarinic antagonist ³H-4NMPB (³H-4-N-methyl piperidyl benzilate). All the bispyridinium compounds also exerted a mild anti-acetylcholine activity (K_d = 10^?4?10^?5M) measured physiologically in the guinea pig ileum, which correlated well with the dissociation constants obtained from binding studies with mouse brain homogenate. The most potent muscarinic blocker was SAD-128 (K′_d = (7.1 ± 1.2) × 10^?6M for whole mouse brain), whose remarkable therapeutic action against soman intoxication may be partly attributed to this antimuscarinic activity.The binding data are best fitted by a competitive model, and the deviation from the law of mass action observed here may be related either to the heterogeneity of muscarinic receptors in the mouse brain or to nonequivalency of the number of binding sites for bisquaternary pyridines and 4-NMPB.     

Studies on the cytostatic action, phosphorylation and deamination of 5-azacytidine and 5,6-dihydro-5-azacytidine in HeLa cells.

5-Azacytidine at 10 μM completely arrested the growth of HeLa cells, whereas 5,6-dihydro-5-azacytidine, a reduced stable analog of 5-azacytidine, at 100 and 200 μM did not inhibit their growth. 3,4,5,6-Tetrahydrouridine, a potent inhibitor of cytidine deaminase had no effect on cell growth at 100 μM, but in combination with 5,6-dihydro-5-azacytidine (100 μM) arrested cell growth completely. However, 3,4,5,6-tetrahydrouridine did not enhance the cytostatic action of 5-azacytidine at concentrations which were only slightly inhibitory to cell growth. Kinetic studies with HeLa cell preparations of cytidine kinase showed that 5-azacytidine is phosphorylated to a greater extent than 5,6-dihydro-5-azacytidine but neither analog is as good a substrate as cytidine. Both drugs are deaminated by cell free extracts but apparent K_m values indicate that the reduced 5-azacytidine compound has a 10-fold greater affinity for cytidine deaminase than the parent drug.     

C(2′)-substituted purine nucleoside analogs: Interactions with adenosine deaminase and purine nucleoside phosphorylase and formation of analog nucleotides

Four C(2′)-substituted 2′-deoxyadenosines were examined as substrates for human erythrocytic adenosine deaminase and for formation of intracellular nucleotide analogs in human erythrocytes, lymphocytes and murine Sarcoma 180 cells: 9-(2′-deoxy-2′-fluoro-β-D-ribofuranosyl)adenine, 9-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine, 9-(2′-azido-2′-deoxy-β-D-ribofuranosyl)adenine (2′-N₃-riboA) and 9-(2′-azido-2′-deoxy-β-D-arabinofuranosyl)adenine. All four adenosine analogs were substrates of human erythrocytic adenosine deaminase, but the corresponding inosine analogs (synthesized by the adenosine deaminase reaction) were highly resistant to cleavage by human erythrocytic purine nucleoside phosphorylase. Only 9-(2′-deoxy-2′-fluoro-β-D-ribofuranosyl)hypoxanthine underwent very slow phosphorolysis, and no inhibition of inosine phosphorolysis was detected when a 30 μM concentration of any studied inosine analog was added to a reaction mixture containing 30 μM inosine (the K_m concentration). Kinetic parameters were determined for the deamination of the adenosine analogs. The greatest affinity for adenosine deaminase was found with 2′-N₃-riboA (K_i=2μM), but the reaction velocity was highest with the F-substituted analogs. All four adenosine analogs formed triphosphate nucleotides after incubation with human erythrocytes, murine Sarcoma 180 cells, or human lymphocytes (tested only with the F analogs) in the presence of deoxycoformycin.     

Analysis of cytidine deaminase and tetrahydrouridine interaction by use of ligand techniques

Ligand techniques, as employed in radio-immunossay and radioreceptor assay, offer a sensitive and precise method for characterizing the interaction of enzymes and tight-binding inhibitors. Tetrahydrouridine (H₄U) inhibition of human liver cytidine deaminase (EC 3.5.4.5) has been examined by direct measurement of enzyme-inhibitor (EI) binding and release. With partially purified enzyme from human liver, the EI complex was found to have a dissociation constant (K_D) of 4.5 × 10^?8M at 37°, in close agreement with estimates based on inhibition of enzyme activity by H₄U. The presence of steady state conditions during competitive binding analysis was confirmed by direct measurement of the rate constants for EI binding at 25° and 37° (K^on 1.7 × 10⁴ and 5.6 x ×0⁴M^?1sec^?1 respectively). The rate constant for EI release at 37° was also determined experimentally (K^off = 4.0 × 10^?3sec^?1), and was in close agreement with the K^off value calculated from the experimentally determined K_D and K^on values (K_D = K^off/K^on). Scatchard analysis of H₄U-enzyme binding, both in the presence and in the absence of 10^?3M cytidine, showed no variation in total enzyme concentration (E_T) but a decrease in apparent inhibitor affinity for enzyme, suggesting that cytidine and H₄U compete for the same binding sites on cytidine deaminase, and confirming the competitive inhibition suggested by Lineweaver-Burk analysis. The turnover number for cytidine deaminase based on per H₄U binding sites was 3.9 × 10³ min^?1. Thermodynamic constants for cytidine deaminase-tetrahydrouridine binding were derived from data on the temperature dependence of binding and included an enthalpy change (ΔH) = ?12.7 kcal/mole, entropy change (ΔS) = ?8.68 cal/deg/mole and Gibbs free energy change (ΔG) = ?9.96 kcal/mole at 37.1°. This study indicates that ligand techniques can be applied to the difficult problem of characterizing the interaction of enzymes and tight-binding inhibitors.     

Inhibitory effects of cordycepin on cyclic nucleotide-dependent and cyclic nucleotide-independent protein kinases

The in vitro effect of cordydepin was tested using various protein kinase preparations. These included cyclic AMP-dependent protein kinase (A-PK) from bovine heart, cyclic GMP-dependent protein kinase (G-PK) from fetal guinea pig lung, and two cyclic nucleotide-independent nuclear protein kinases (PK-I and PK-II) prepared from rat hepatoma 3924A and rat liver. The 50 per cent inhibitory concentrations (id₅₀) of cordycepin for A-PK and G-PK ranged from 1.5–5.0 × 10^?4M and 2.5–8.0 × 10^?4 M, respectively, depending on the presence or absence of cyclic AMP and cyclic GMP in the assay. The id₅₀ of cordycepin with either hepatoma 3924A or rat liver PK-I and PK-II was 4.5 × 10^?5 M and 1.0 × 10^?3 M. respectively. The inhibitory effect of cordycepin was competitive with respect to ATP in all cases. The K_{m} for ATP was increased 3-fold and 5-fold by 5 × 10^?4 M cordycepin for G-PK and A-PK, respectively, while the K_m for ATP was increased 10-fold and 4-fold by 1 × 10^?3 M cordycepin for PK-I and PK-II, respectively.     

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.     

Studies on the biochemical actions of 6-selenoguanine and 6-selenoguanosine

Survival studies were performed in mice bearing Sarcoma 180 ascites tumor treated with 6-thio and 6-seleno analogs of guanine and guanosine. The selenium-containing analogs were somewhat superior to the sulfur-containing compounds in antitumor activity and therapeutic index. The formation of 6-SeGMP from 6-seleno-guanine (6-SeG) was demonstrated in Sarcoma 180 ascites cells. Guanine, 6-thioguanine (6-TG) and 6-SeG show comparable substrate activity whereas 8-azaguanine is a much poorer substrate for hypoxanthine-guanine phosphoribosyl transferase from Sarcoma 180 cells. Both 6-TG and 6-SeG are good substrates for purine nucleoside phosphorylase from Sarcoma 180 cells. Chemically and enzymatically synthesized 6-SeGMP behaved as a competitive inhibitor (K_i 1 × 10^?4 M) of erythrocytic and Sarcoma 180 guanylate kinases. Weak substrate activity was demonstrated in the presence of large amounts of erythrocytic guanylate kinase.