Inhibition of adenosine uptake in human erythrocytes by adenosine-5'-carboxamides,xylosyladenine, dipyridamole,hexobendine, and p-nitrobenzylthioguanosine

Adenosine uptake in human erythrocytes at 0° consists of a saturable and a concentration-proportional component, the latter seems to represent uptake into a pericellulai compartment inaccessible to inulin. Xylosyladenine and derivatives of adenosine-5'-carboxamide were found to be weak inhibitors of the saturable component of adenosine uptake with apparent K_i values at least one order of magnitude higher than the apparent K_m for adenosine (2.4 × 10^?6 M). The affinity of the adenine nucleosides to the saturable uptake process appears to depend not only on the 3'-hydroxy] group and its erythro-configuration but also on the 5'-substituent. Dipyridamole, hexobendine, and p-nitrobenzylthioguanosine, by contrast, had K_i values at least one order of magnitude lower than the K_m for adenosine. The steric requirements for binding of the adenine furanosides to the putative smooth muscle receptors mediating vasodilation. on the the one hand, and to the saturable cellular uptake mechanism, on the other hand, were found to be different.     

Reassessment of the interactions of guanylate kinase and 6-thioguanosine 5'-phosphate.

Contrary to published studies, 6-thioguanosine 5'-phosphate (6-thioGMP) was found to be a relatively weak inhibitor of the phosphorylation of GMP catalyzed by guanylate kinase. The inhibition constant for 6-thioGMP was 2.3 mM for the enzyme from human erythrocytes. This K_i value is 30-fold larger than previously reported values. The weak inhibition by 6-thioGMP is related to the alternate substrate activity of this compound. It has an apparent K_m value of 2.1 mM and a maximal velocity of 3 per cent of that attainable with GMP as the saturating substrate. The discrepancy between these findings and those previously reported was found to be due to a spectrophotometric artifact that was associated with the high absorbance of 6-thioGMP at the wavelength previously used for the velocity measurements. Guanylate kinase from rat liver, hog brain, Sarcoma 180 tumor cells and Ehrlich ascites tumor cells was also inhibited by 6-thioGMP with a K_i in the mM range.     

5'-Methylthioadenosine phosphorylase-L. Substrate activity of 5'-deoxyadenosine with the enzyme from Sarcoma 180 cells

5′-Deoxy-5′-methylthioadenosine phosphorylase (MTA phosphorylase), an enzyme involved in the salvage of adenine moieties from 5′-deoxy-5′-methylthioadenosine (MTA) produced primarily during polyamine biosynthesis, is present in Sarcoma 180 cells (0.0026 ± 0.0002 μM units/mg cytosol protein). 5′-Deoxyadenosine (5′-dAdo), an adenosine analog previously thought not to be metabolizable, has been shown [D. Hunting and J.F. Henderson, Biochem. Pharmac. 27, 2163 (1978)] to have a number of biochemical effects on Ehrlich ascites cells. We have now found that 5′-dAdo is a substrate for the MTA phosphorylase from Sarcoma 180 cells, yielding free adenine and 5-deoxyribose-1-phosphate. The reaction was reversible and totally dependent upon phosphate. Evidence that MTA phosphorylase is responsible for 5′-dAdo phosphorylase activity includes the following: (1) Sarcoma 180 MTA phosphorylase preparations did not show additive rates of adenine production in the presence of saturating concentrations of both 5′-dAdo and MTA; (2) double-reciprocal plots of the rates of adenine formation from 5′-dAdo by Sarcoma 180 enzyme preparations in the presence of MTA displayed a pattern characteristic of alternative, competing substrates; (3) the rate of depletion of 5′-dAdo by Sarcoma 180 preparations was inhibited by the presence of MTA; (4) the K_i value of a competitive inhibitor of Sarcoma 180 MTA phosphorylase, 5′-deoxy-5′-chloroformycin, was the same when either MTA or 5′-dAdo was employed as substrate; and (5) the apparent K_m values of phosphate for both MTA and 5′-dAdo phosphorylase activities were identical (3.5mM). The K_m of Sarcoma 180 MTA phosphorylase for MTA is 4 μM; the K_m for 5′-dAdo is 23 μM (V_max relative to MTA = 180 per cent). Incubation of Sarcoma 180 cells with either 5′-dAdo or MTA caused profound elevations of adenine nucleotides, as well as an inhibition of 5-phosphoribosyl-l-pyrophosphate (PRPP) accumulation. The reaction of 5′-dAdo with MTA phosphorylase to yield free adenine, which is then salvaged to adenine nucleotides, can account for many of the previously reported biochemical effects of 5′-dAdo, such as inhibitions of PRPP accumulation, purine de novo synthesis, and glycolysis that have previously been attributed to the unmetabolized nucleoside. The other product of this reaction, 5-deoxyribose-l-phosphate, may also contribute to these effects.     

Phosphodiesterase in the liver fluke, Fasciola hepatica.

Phosphodiesterase was found in homogenates of the liver fluke, Fasciola hepatica, and was distributed between a supernatant and particulate fraction after centrifugation at 2000 g. Mg²⁺ was necessary for enzyme activity; Ca²⁺ in the presence of Mg²⁺ did not affect enzyme activity. Enzyme kinetics followed the Michaelis-Menten model with a K_m of 8 μM for cAMP and 300 μM for cGMP as the substrate. The most potent inhibitor tested was 1-ethyl-4-(isopropylidenehydrazino)-1 H-pyrazolo- (3,4-b)-pyridine-5-carboxylic acid, ethyl ester, HC1 (SQ 20009) which had a K_i of 26 μM. The K_i for isobutyl methyl xanthine (IBMX) was 45 μM; for 6,7 dimethyl-4 ethylquinazoline (Quazodine) 75 μM; papaverine. 100 μM; theophylline, 550 μM; and for caffeine or D-lysergic acid diethylamide (LSD), 800 μM. The effects on fluke motility of these phosphodiesterase inhibitors were tested. All phosphodiesterase inhibitors except caffeine stimulated the rhythmical movement of the flukes. None of the inhibitors tested significantly increased the endogenous cAMP concentrations of fluke heads. IBMX potentiated the rise in endogenous cAMP caused by 5-hydroxytryptamine (5-HT) but SQ 20009, LSD, and papaverine prevented it. The latter results could not be explained on the basis of phosphodies-terase inhibition, but might be attributed to interference with the stimulation of adenylate cyclase by 5-HT.     

Inhibition of cyclic AMP phosphodiesterase by 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid 3-[2-(N-benzyl-N-methylamino)] ethyl ester 5-methyl ester hydrochloride (YC-93), a potent vasodilator.

A new, potent vasodilator (YC-93), 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid 3-[2-(N-benzyl-N-methyl amino)] ethyl ester 5-methyl ester hydrochloride, competitively inhibited cyclic adenosine 3′,5′-monophosphate (cyclic AMP) phosphodiesterase in the 105,000 g supernatant solutions from canine basilar, carotid, coronary and femoral arteries. The inhibition constant (K_i) of YC-93 for these enzyme preparations was in the range of 2.0–4.3 μM at substrate concentrations near the low K_m (about 1 μM for each enzyme preparation), and was 4.0–12 μM at substrate concentrations near the high K_m (50–70 μM). The potency of YC-93 for inhibition of coronary phosphodiesterase at 1 μM cyclic AMP and 50 μM cyclic AMP was much greater than that of papaverine and 3-isobutyl-l-methyl xanthine (IBMX). Commercially available cyclic AMP phosphodiesterase purified from beef heart was also strongly inhibited by YC-93 in a competitive manner and its K_i value was 2.0 μM in the wide range of substrate concentrations tested. Studies on the structure-activity relationship using low K_m phosphodiesterase from canine coronary artery and high K_m phosphodiesterase from beef heart, demonstrated that 3,5-diethoxycarbonyl-1,4-dihydro-2, 4,6-collidine, the simplest 1,4-dihydropyridine derivative (tested in the present studies) resulted in slightly less inhibition than papaverine, and the inhibitory potency of the former compound was greatly increased mainly by two structural modifications. Firstly, addition of a nitrophenyl group at position 4 of the 1,4-dihydropyridine ring, secondly, the replacement of ethylester at position 3 of the 1,4-dihydro-pyridine ring by N-benzyl-N-methylaminoethyl ester. A few dihydropyridine derivatives together with YC-93 were the most potent inhibitors of cyclic AMP phosphodiesterase among the compounds tested. The finding that the level of cyclic AMP in canine arterial strips was increased by 64 per cent (P < 0.01) even after 1 min exposure to 1 μM YC-93 supports the possibility of at least a partial involvement of phosphodiesterase inhibition in vasodilation by the drug.     

Adenosine deaminase from human erythrocytes: Purification and effects of adenosine analogs

Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) has been purified about 3000-fold from human erythrocytes. The molecular weight of the enzyme was estimated to be 33,000. With the partially purified erythrocytic adenosine deaminase, K_m and V_max values relative to adenosine were: adenosine, 25 μM, 100 per cent; formycin A, 1000 μM, 753–850 per cent; 8-aza-adenosine, 130 μM, 310 per cent; 6-chloropurine ribonuclcoside, 1000 μM, 91 per cent; 2,6-diaminopurine ribonucleoside, 74 μM, 91 per cent; 2'-deoxyadenosine. 7 μm, 60 per cent; xylosyladenine, 33 μm, 62 per cent; arabinosyi adenine, 100 μM, 47 per cent; 3'-deoxyadenosine (cordycepin), 41 μM, 100 per cent; 3'-amino3'-deoxyadenosine. 133 μM, 89 per cent: 4'-thioadenosine, 13 μM, 43 per cent; and 6-methylselenopurine ribonucleoside, 27 μM, 88 per cent. Apparent K_ti values of reaction products and some adenosine analogs using adenosine as a substrate were as follows; inosine. 116 μM; 2'-deoxyinosine, 60 μM; guanosine, 140 μM; 2-fluoroadenosine, 60 μM; 2-fluorodeoxyadenosine. 19 μM; N⁶-methyladenosine, 17 μM; N₁-methyladenosine, 275 μM; 6-thioguanosine, 92 μM; 6-thioinosine, 330 μM; 6-methylthioinosine, 270 μM; arabinosyl 6-thiopurine, 360 μM; and coformycin, 0.01 μM. Tubercidin (7-deaza-adenosine) and toyocamycin were devoid both of substrate and inhibitor activity. Also. N⁷-methylinosine, N⁷-methylguanosine and dipyridamole (Persantin®) did not inhibit the enzymic activity.     

Adenosine 3',5'-monophosphate phosphodiesterase activity in experimental animal tumours which are either sensitive or resistant to bifunctional alkylating agents.

The adenosine 3′,5′-monophosphate phosphodiesterase of Walker rat carcinoma 256, ADJ/ PC6 plasma cell tumour, NK lymphoma. Sarcoma 180 and TLX5 lymphoma behaves kinetically as if two separate activities exist, one with a low affinity for the substrate and the other with a high affinity. The high K_m values are 82·5, 566, 590, 1975 and 1075 μM for the enzyme from each tumour respectively, and the low K_m values are 1·1, 17·7, 5·75, 7·1 and 4·4 μM. In the Walker carcinoma and PC6 plasma cytoma, tumours which are sensitive to alkylating agents, the apparent V_max of the low K_m forms are respectively 38 and 25 per cent of the total activity. In those tumours which are naturally resistant to the growth inhibitory properties of the alkylating agents, the apparent V_max of the low K_m form is less than 10 per cent of the total activity. Walker carcinoma showing a 20-fold resistance to chlorambucil[p-(di-2-chloroethylamino) phenylbutyric acid] has the V_max of the high affinity form decreased to 15 per cent of the total, while a 70-fold resistant line has the V_max of this form of the enzyme decreased to 9 per cent of the total. This decrease in the activity of the high affinity form of the enzyme in the resistant tumours does not appear to be due to the presence of an endogenous inhibitor. While the high K_m form of the enzyme in the Walker carcinoma is mainly confined to the cytosol, about half of the activity of the low K_m form appears to be associated with particulate fractions. This subcellular distribution does not differ appreciably in the sensitive and resistant tumours. The possible reasons for such an isoenzyme shift are discussed.     

Stimulatory effect of ionophores on adenosine 3',5'-monophosphate content in human mononuclear leukocytes

Ionophores A23187 and bromo-lasalocid ethanolate enhanced the cyclic AMP content in human mononuclear leukocytes. The maximum effect of A23187 with a 10-min incubation was found with 0.3–1.0μM concentrations with or without l-isoproterenol (1 μM) or prostaglandin E ₁ (pge ₁) (0.3 μM). The maximum effect after 5 min of incubation at 37° was observed with 0.05, 0.2 and 1 μm A23187. The effect of ionophore A23187 was enhanced by both aminophylline (1 mM) and isobutyl-methylxanthine (1 mM). Calcium (1 mM). aspirin (1 mM) and indomethacin (100 μM) decreased the stimulatory action of A23187. Bromo-lasalocid ethanolate increased cyclic AMP content in cells maximally at a 3 μM concentration with or without 0.3 μM pge ₁.     

Serotonin synthesis with rat brain synaptosomes. Effects of L-dopa, L-3-methoxytyrosine and catecholamines

Experiments were done with a fraction of rat brain containing mitochondria and synaptosomes. L-DOPA∥ (K_t 0·3 mM) and L-3-methoxytyrosine (K_t 0·5 mM) were found to be competitive inhibitors of tryptophan accumulation, while dopamine and l-noradrenaline had no effect on the accumulation of tryptophan. Furthermore, L-DOPA (K_t 5·6 μM) was about 100 times more potent than L-3-methoxytyrosine in inhibiting the synthesis of 5HT from tryptophan. The inhibition of 5HT synthesis by L-DOPA appeared to be competitive to tryptophan and was not linear at concentrations of L-DOPA higher than 1 μM. L-DOPA also interfered with the rate of deamination of 5HT synthesized in vitro. Dopamine and l-noradrenaline also inhibited the synthesis of 5HT.     

Selegiline Metabolism and Cytochrome P450 Enzymes:In vitro Study in Human Liver Microsomes*

Although being a drug therapeutically used for a long time, the enzymatic metabolism of selegiline has not been adequately studied. In the current work we have studied the cytochrome P450 (CYP)‐catalyzed oxidative metabolism of selegiline to desmethylselegiline and l‐methamphetamine and the effects of selegiline, desmethylselegiline and l‐methamphetamine on hepatic CYP enzymes in human liver microsomes in vitro. The apparent K_m values for desmethylselegiline and l‐methamphetamine formation were on an average 149 μM and 293 μM, and the apparent V_max values, 243 pmol/min./mg and 1351 pmol/min./mg, respectively. Furafylline and ketoconazole, the known reference inhibitors for CYP1A2 and CYP3A4, respectively, inhibited the formation of desmethylselegiline with K_i value of 1.7 μM and 15 μM. Ketoconazole inhibited also the formation of l‐methamphetamine with K_i of 18 μM. Fluvoxamine, an inhibitor of CYP1A2, CYP2C19 and CYP3A4, inhibited the formation of desmethylselegiline and l‐methamphetamine with K_i values of 9 and 25 μM, respectively. On the basis of these results we suggest that CYP1A2 and CYP3A4 contribute to the formation of desmethylselegiline and that CYP3A4 participates in the formation of l‐methamphetamine. In studies with CYP‐specific model activities, both selegiline and desmethylselegiline inhibited the CYP2C19‐mediated S‐mephenytoin 4′‐hydroxylation with average IC₅₀ values of 21 μM and 26 μM, respectively. The K_i for selegiline was determined to be around 7 μM. Selegiline inhibited CYP1A2‐mediated ethoxyresorufin O‐deethylation with a K_i value of 76 μM. Inhibitory potencies of selegiline, desmethylselegiline and l‐methamphetamine towards other CYP‐model activities were much lower. On this basis, selegiline and desmethylselegiline were shown to have a relatively high affinity for CYP2C19, but no evidence about selegiline metabolism by CYP2C19 was obtained.     

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.     

Cytidine and deoxycytidylate deaminase inhibition by uridine analogs

Cytidine deaminase, an enzyme found in the supernatant fluid of hepatocytes, granulocytes and tumor cells, and in plasma, degrades the antitumor agents cytosine arabinoside and 5-azacytidine. Uridine and its analogs, 3-deazauridine, 5-bromodeoxyuridine, 5-fluorodeoxyuridine and 6-azauridine, were found to competitively inhibit cytidine deaminase; the most potent inhibitor was 3-deazauridine (K_i = 1.9 × 10^?5 M). In addition, deoxycytidylate deaminase, which degrades cytosine arabinoside monophosphate to the inactive uracil arabinoside monophosphate (K_m = 9 × 10^?4 M), was competitively inhibited by 3-deazauridine monophosphate, as well as by the nucleotides of other uridine analogs. These results suggest that uridine analogs such as 3-deazauridine may have value in protecting cytosine arabinoside, 5-azacytidine and their monophosphate nucleotides from degration by neucleoside and nucleotide deaminases.     

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).     

Biotransformation of 4-dimethylaminophenol in the isolated perfused rat liver and in the rat

Isolated rat livers were perfused with various concentrations of 4-dimethylaminophenol-[¹⁴C] (DMAP). During single pass perfusion with modified protein-free Krebs-Henseleit solution up to 70μM prehepatic 4-dimethylaminophenol (DMAP) were metabolized by the liver. The main route of biotransformation was conjugation. At steady state conditions glucuronide formation showed an apparent V_max of 8.5 μmoles × min^?1 × g protein^?1, and K_m of 562 μM, whereas sulfate formation had an apparent V_max of 1.2 and a K_m of 35. Thus, at low substrate concentration the sulfate conjugation outweighed glucuronidation whereas at high substrate concentration the ratio of conjugates was reversed. In contrast to DMAP-sulfate, some DMAP-glucuronide was stored by the liver and was released with a half life of about 15 min which showed positive correlation with the dose of DMAP during the washout period. Perfusion with human or rat erythrocytes demonstrated the other important path of biotransformation of DMAP within erythrocytes, namely thioether formation with glutathione and SH-groups of hemoglobin. The pattern of DMAP-conjugation was affected depending on the time of prehepatic exposure to erythrocytes, and the species of red cells. The results obtained from the isolated metabolic system resemble the hepatic part of the overall metabolism under in vivo conditions.     

Mestranol-induced hypertension: characterization of cytochrome P-450 dependent catechol estrogen formation in brain microsomes

An animal model of estrogen-induced hypertension was used to study the effects of chronic administration of the synthetic estrogen mestranol on cytochrome P-450 content and catechol estrogen formation in brain microsomes. Cytochrome P-450 content of brain microsomes from untreated female rats in estrus was 0.034 nmole/mg protein and the dithionite-reduced carbon monoxide absorbance peak (γ_max) was 452 nm. Catechol estrogen formation in brain microsomes was optimal in the presence of both NADPH and NADH cofactors with an apparent K_m value of 71 μM for 17β-estradiol substrate. Brain microsomes from animals in estrus and diestrus were compared, and no significant differences were observed in cytochrome P-450 content, or in the apparent K_m and V_max values of catechol estrogen formation. Administration of mestranol, 15 μg biweekly, for 3–4 weeks resulted in a significant increase in systolic blood pressure in unanesthetized female rats. Mestranol treatment was not associated with a change in microsomal cytochrome P-450 content or the spectral γ_max. At 10 μM substrate concentration, catechol estrogen formation in microsomes from mestranol-treated animals was increased 2- to 3-fold, with enzyme activity being expressed either per mg protein or per nmole cytochrome P-450. In contrast, no difference was observed between groups when enzyme activity was measured at 100 μM substrate concentration. These data suggest that chronic administration of a synthetic estrogen can regulate the enzyme system involved in formation of brain catechol estrogen metabolites, a mechanism which may alter the biological impact of the parent steroid.     

Affinity of Drugs for Cytochrome P-450 Determined by Inhibition of p-Nitrophenetole O-Deethylation by Rat Liver Microsomes

Abstract: The rate of conversion of p-nitrophenetole to p-nitrophenol by rat liver microsomes was studied. Inhibition of the reaction by CO and by SKF 525A and the absolute dependence on NADPH and oxygen indicate that cytochrome P-450 catalyzes the reaction. The apparent K_m for oxygen was 0.07 μM. Furthermore, cytochrome b₅ seemed to be involved in the formation of p-nitrophenol. The effect on p-nitrophenol formation of drugs known to be involved in drug interaction in clinical practice was studied. There was a competitive inhibition by phenytoin (inhibitor constant, K_i, 30 μM), disulfiram (K_i, 2 μM) and chloramphenicol (K_i, 20 μM), whereas a mixed-type inhibition by isoniazid was observed (K_is, 1,3 mM and K_ii, 10,6 mM).     

Inosine 5′-monophosphate dehydrogenase from Sarcoma 180 cells—Substrate and inhibitor specificity

The substrate and inhibitor specificity of IMP dehydrogenase from Sarcoma 180 ascites tumor cells has been studied with twenty purine nucleotide analogs. Several were found to be substrates with the following efficiencies (V_max/K_m):IMP (4000), 8-azaIMP (1360), 6-thioIMP (250), araIMP (250) and dIMP (240). While substrate activity was not detected with the 5′-phosphates of 6-methylmercaptopurine riboside or 1-ribosylallopurinol (rates less than 1/10,000th that of IMP), they were competitive inhibitors with respect to IMP (both with K_i, values of 0.43 mM). Seven XMP analogs and six GMP analogs were also found to be competitive inhibitors with respect to IMP. Methods for the synthesis of the 5′-phosphates of arabinosylhypoxanthine. arabinosylxanthine. arabinosylguanine and 2′-deoxyxanthosine are described.     

Pyridine nucleotide involvement in rat hepatic microsomal drug metabolism--I. Factors that influence NADPH kinetic estimations during mixed function oxidase reactions.

The localisation of significant amounts of nucleotide pyrophosphatase activity in the rat hepatic microsomal fraction results in erroneous values of apparent K_m (NADPH) for aminopyrine-Ndemethylase. This was demonstrated by the inclusion of 20 mM pyrophosphate, which inhibits nucleotide pyrophosphatase activity and reduces the apparent K_m (NADPH) value from 27.9 μM to 7.92 μM. The apparent K_m (NADPH) values determined in the presence of three Type I substrates were not statistically different from each other, but the value in the presence of aniline was lower. The kinetic constants of NADPH for NADPH cytochrome P450-reductase in the presence of either aminopyrine, ethylmorphine or aniline, are also reported.     

A novel metabolic effect of the adenosine deaminase inhibitor coformycin,a potentiator of antiviral adenosine analogues

Coformycin [$\text{(R)-3-(β-}\text{d}\text{-erythropentofuranosyl}\text{)-3,6,7,8-}\text{tetrahydroimidazo}\text{[4,5}\text{d}\text{]-[1,3]}\text{diazepin}\text{-8-(R)-}\text{ol}$] is an inhibitor of adenosine deaminase and thus increases the antiviral potency of a series of adenosine analogues. The influence of chemically synthesized coformycin triphosphate on RNA-polymerizing enzymes (RNA polymerases from E. coli and from calf thymus, poly(A) polymerase) and on DNA-synthesizing enzymes (DNA polymerases and terminal deoxynucleotidyltransferase from calf thymus) was studied. Only the poly(A) polymerase was found to be sensitively inhibited by coformycin triphosphate. The inhibition is non-competitive with respect to ATP (K_i = 2.9 μM). Inhibition was reversed by simple dilution. The inhibitor does not act as chain terminator.     

On the substrate specificity of the digitoxigenin monodigitoxoside conjugating UDP-glucuronyltransferase in rat liver

The aim of the present study was to investigate the specificity of the UDP-glucuronosyltransferase (EC 2.4.1.17) involved in the conjugation of digitoxigenin monodigitoxoside. By in vitro assays with detergent activated liver musomes it was found that (1) digitoxigenin monodigitoxoside is by far the best substrate of all cardenolides and cardenolide digitoxosides tested. (2) In the presence of saturating UDP-glucuronate concentrations an apparent K_m of 5.8 μM was obtained from linear Lineweaver-Burk plots together with a V_max of about 150 pmoles/mg musomal protein/min. (3) Neither phenobarbital nor polycyclic aromatic hydrocarbons caused a considerable induction of the enzyme without change of the apparent K_m, but spironolactone did. (4) The conjugation of the substrate (4 μM) could only be inhibited by the 3'-epi-digitoxoside of digitoxigenin. (5) 25–50 μM substrate inhibited only the conjugation of the 3'-epimer and that of digoxigenin monodigitoxoside. It is suggested that there is a form of glucuronyltransferase which specifically conjugates digitoxigenin monodigitoxoside.