Effect of nucleos(t)ide analogue on serum HBsAg level in chronic hepatitis B patients: A 3-years study.

AIM: We aim to explore the effects of nucleos(t)ide analogues (NUCs) on the changes of HBsAg in chronic hepatitis B (CHB) patients. METHODS: A total of 264 CHB patients were enrolled in our study. All of them were treated with NUCs for at least three years. Quantification of HBsAg levels were measured by Elecsys HBsAg II. RESULTS: Although HBsAg levels were significantly higher in HBeAg seropositive CHB patients at baseline than in HBeAg seronegative CHB patients (3.84 ±â€¯0.82 vs 3.21 ±â€¯0.59 IU/mL), HBsAg levels declined more rapidly in the HBeAg seropositive group (P < 0.001). In HBeAg-positive CHB patients, HBsAg level in the telbivudine (LDT)-treated group was 3.68 ±â€¯0.56 IU/mL after 52-week of treatment, which was significantly higher than that in lamivudine (LAM)-treated group (P = 0.009). Multivariable analyses showed that baseline HBV DNA viral load (OR = 0.75, P = 0.018), baseline ALT level (OR = 0.99, P = 0.015), and baseline HBsAg level (OR = 0.188, P < 0.001) were independent factors that affected HBsAg decline in HBeAg seropositive CHB patients. For HBeAg seronegative CHB patients, the average of serum HBsAg levels in LAM-, LdT-, adefovir (ADV)-, and entecavir (ETV)-treated groups at baseline, 52 weeks, 104 weeks, and 156 weeks were similar. Multivariable analyses showed that only baseline HBV DNA level (OR = 0.56, P = 0.020) and baseline HBsAg level (OR = 0.57, P = 0.012) were independent factors that affected HBsAg decline in HBeAg seronegative patients with CHB. Baseline HBV DNA level (OR = 0.72, P = 0.010) and baseline HBsAg level (OR = 0.19, P < 0.001) were independent factors that affected all CHB patients. CONCLUSIONS: CHB Patients who had received NUCs antiviral treatment showed a slow but significant decrease in serum HBsAg level. Long-term monitoring and continuous antiviral treatment are necessary, especially for those patients with risk factors associated with HBsAg decline.

Why myotoxin-containing snake venoms possess powerful nucleotidases?

The venom of the snake Bothrops asper causes muscle necrosis, pain and inflammation. This venom contains myotoxins which cause an increase in intracellular Ca(2+) concentration and release of K(+) and ATP from myotubes. ATP is a key danger molecule that triggers a variety of reactions, including activation of the innate immune response. Here, using ATP-luciferase bioluminescence imaging technique, we show for the first time in vivo, that the purified myotoxins induce rapid release of ATP, whilst the complete venom of B. asper does at a very small extent. This apparent contradiction is explained by the finding that the venom contains powerful nucleotidases that in vivo convert ATP into ADP, AMP and Adenosine. These findings indicate that high concentrations of adenosine are generated by the double action of the venom and provide the experimental basis to the suggestion that in situ generated adenosine plays an important role in envenomation via its hypotensive, paralyzing and anti-coagulant activities.

The pharmacological role of nucleotidases in snake venoms.

Several hydrolytic enzymes of snake venom have evolved to interfere in various physiological processes, which are well defined. However, hydrolytic enzymes such as nucleotidases (5'nucleotidase, ATPase, and ADPase) are less studied and their pharmacological role in venoms is not clearly defined. Very few studies have shown the pharmacological importance of these endogenous purine release related enzymes in venoms. The near-ubiquitous distribution of these enzymes in venoms, suggests a significant role for these enzymes in envenomation. It is suggested that their major function is in the generation of purines (mainly adenosine)-a multitoxin. Therefore, it appears that these enzymes play a central role in liberating adenosine and through the action of adenosine help in prey immobilization. However, apart from this, these enzymes could also possess other pharmacological activities. Further research is needed to biologically characterize these enzymes in snake venoms, such that their role in venom is clearly established.

The ecto-nucleotidase NTPDase1 differentially regulates P2Y1 and P2Y2 receptor-dependent vasorelaxation.

BACKGROUND AND PURPOSE: Extracellular nucleotides produce vasodilatation through endothelial P2 receptor activation. As these autacoids are actively metabolized by the ecto-nucleotidase nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), we studied the effects of this cell surface enzyme on nucleotide-dependent vasodilatation. EXPERIMENTAL APPROACH: Vascular NTPDase expression and activity were evaluated by immunohistochemistry and histochemistry. The vascular effects of nucleotides were tested in vivo by monitoring mean arterial pressure, and in vitro comparing reactivity of aortic rings using wild-type and Entpd1(-/-) (lacking NTPDase1) mice. KEY RESULTS: The absence of NTPDase1 in Entpd1(-/-) mice led to a dramatic drop in endothelial nucleotidase activity. This deficit was associated with an exacerbated decrease in blood pressure after nucleotide injection. Following ATP injection, mean arterial pressure was decreased in Entpd1(+/+) and Entpd1(-/-) mice by 5.0 and 17%, respectively, and by 0.1 and 19% after UTP injection (10 nmole.kg(-1) both). In vitro, the concentration-response curves of relaxation to ADP and ATP were shifted to the left, revealing a facilitation of endothelial P2Y1 and P2Y2 receptor activation in Entpd1(-/-) mice. EC(50) values in Entpd1(+/+) versus Entpd1(-/-) aortic rings were 14 microM versus 0.35 microM for ADP, and 29 microM versus 1 microM for ATP. In Entpd1(-/-) aortas, P2Y1 receptors were more extensively desensitized than P2Y2 receptors. Relaxations to the non-hydrolysable analogues ADPbetaS (P2Y1) and ATPgammaS (P2Y2) were equivalent in both genotypes confirming the normal functionality of these P2Y receptors in mutant mice. CONCLUSIONS AND IMPLICATIONS: NTPDase1 controls endothelial P2Y receptor-dependent relaxation, regulating both agonist level and P2 receptor reactivity.

Flavone as PARP-1 inhibitor: its effect on lipopolysaccharide induced gene-expression.

The nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) which was initially known for its role in the repair of oxidative stress-induced DNA damage, has also been reported to play a mediating role in the inflammatory response. Studies with PARP-1 knockout models have shown that PARP-1 is a co-activator of Nuclear Factor-kappa B (NF-kappaB), although this appears not to require its enzyme activity. In addition, drug-induced inhibition of the enzyme activity of PARP-1 was observed to reduce the production of pro-inflammatory mediators. In this study, the flavonoid compound flavone was demonstrated to significantly inhibit the enzyme activity of PARP-1. Further evaluation of flavone in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated human pulmonary epithelial and vascular endothelial cells revealed that both the decrease in NAD(+) levels, as well as the formation of PAR-polymers was dose-dependently attenuated by flavone. In addition, flavone was found to reduce the lipopolysaccharide (LPS)-induced interleukin (IL)-8 production in pulmonary epithelial cells, which was confirmed by transcription analysis. Furthermore, the transcription Inhibitor kappa B alpha (of IkappaBalpha) was significantly increased by flavone. The results of the present study indicate that the flavonoid flavone could be a potential candidate for application in treatment of chronic inflammatory diseases. PARP-1 inhibition could have beneficial effects in such diseases as Chronic Obstructive Pulmonary Disease (COPD) and diabetes, by preservation of cellular NAD(+) levels and attenuating inflammatory conditions.

Analysis of the influence of nucleotidases on the apparent activity of exogenous ATP and ADP at P2Y1 receptors.

1. ADP is a potent agonist of rat and human P2Y1 purinoceptors. ATP is a weak competitive antagonist. This study analyses the situation in which P2Y1 receptors are exposed to ATP in the presence of exogenous ecto-nucleotidases (apyrases) that have high or low ATPase/ADPase activity ratio. 2. Rat brain capillary endothelial cells of the B10 clone express P2Y1 receptors that couple to intracellular Ca2+ mobilization. They have low endogenous ecto-ATPase and ecto-ADPase activities. 3. ATP did not raise intracellular Ca2+ in B10 cells. Addition of apyrases III or VII (1 u ml(-1)) to ATP treated cells induced large intracellular Ca2+ transients. Apyrases had no action in the absence of ATP. 4. A 1 u ml(-1) apyrase III solution generated 20 microM ADP from 0.1 mM ATP within 15 s. This concentration of ADP was sufficient to produce maximal activation of P2Y1 receptors. 5. ATP was a full agonist of P2Y1 receptors in the presence of 1 u ml(-1) apyrase III. Dose response curves for the apparent actions of ATP were bell shaped in the presence of 0.1 u ml(-1) apyrase III. Apyrase III did not alter ADP dose response curves when coincubated with ADP for 15 s. 6. Apyrase VII (1 u ml(-1)) shifted dose response curves for the actions of ADP to larger concentrations. It induced a bell shaped ATP dose response curve. 7. Results suggest that ATPDases prevent P2Y1 receptor activation by degrading ADP but may contribute to P2Y1 receptor activation by generating ADP from ATP.

Effects of P2-purinoceptor antagonists on degradation of adenine nucleotides by ecto-nucleotidases in folliculated oocytes of Xenopus laevis.

The aim of the present study was to examine the effects of a number of P2-purinoceptor antagonists on degradation of adenine nucleotides by Xenopus laevis oocyte ecto-nucleotidase. Folliculated oocytes readily metabolize all three naturally-occurring nucleotides, the order of preferential substrates being ATP >ADP > AMP. The degradation of ATP and ADP was decreased significantly in the presence of several P2X- and P2Y-purinoceptor antagonists, including suramin, PPADS, Cibacron blue, Coomassie Brilliant blue, Evans blue, Trypan blue, Congo red, and PIT (each compound was used at 100 microM). All these compounds inhibited the degradation of ATP by up to 60%, whereas the hydrolysis of ADP was inhibited by Congo red and PIT by 75-80%. In addition, DIDS (100 microM) and TNP-ATP (100 microM) selectively inhibited the breakdown of ATP, and sodium azide (10 mM) selectively inhibited the breakdown of ADP. The enzymatic breakdown of either ATP or ADP was unaffected by 8-pSPT (100 microM), an antagonist of P1-purinoceptors, or by oxidized ATP (100 microM), an antagonist of P2Z-purinoceptors. The degradation of AMP was prevented completely by PIT (100 microM) and ingibited significantly by Congo red (100 microM). In conclusion, the present study shows that most of currently available antagonists of P2-purinoceptors inhibit the enzymatic breakdown of extracellular ATP and ADP. The inhibitory effect on ecto-nucleotidase activity should be taken into account when these antagonists are used in pharmacological experiments.

Kinetics of extracellular ATP hydrolysis by microvascular endothelial cells from rat heart.

We have characterized the ectonucleotidases that catalyse the reaction sequence ATP-->ADP-->AMP-->adenosine on microvascular endothelial cells cultured from the rat heart. Computer simulation and data fitting of progress of reaction curves showed that depletion of substrate at the cell surface dominates the regulation of the rate of hydrolysis of ATP when it is presented to the cells. Preferential delivery of AMP by ADPase to 5'-nucleotidase makes a significant contribution to the regulation of adenosine production from ATP or ADP. By contrast, we found no evidence for the preferential delivery of ADP from ATPase to ADPase. Feed-forward inhibition of AMP hydrolysis by ADP and/or ATP also modulated the rate of adenosine production. The properties of the ectonucleotidases on rat heart microvascular cells are such that adenosine is produced at a steady rate over a wide range of ATP concentrations.

Quantitation of the 32P-postlabeling reaction using cyclic N1,N2 and C8 modified deoxyguanosine 3'-monophosphates as substrates.

The 32P-postlabeling technique was used to investigate the efficiency of phosphorylation reaction by T4 polynucleotide kinase using seven synthetic adducted deoxyguanosine 3'-monophosphates. The adducts included cyclic N1,N2 derivatives and the C8 adduct of 4-aminobiphenyl. The adducted substrates were detected at a subfemtomole sensitivity except for one of the diastereomeric propanoguanine derivatives. In general, the recommended conditions were found to be proper for an efficient phosphorylation of the adducts studied. Sensitivity of the adducts to the 3'-dephosphorylation reaction of nuclease P1 was also tested. All the complex cyclic adducts were resistant towards P1. However, the ethenoguanine and 4-aminobiphenyl adducts were relatively sensitive towards P1. No differences were noted between diastereomers.

3-Deazaadenosine 5'-triphosphate: a novel metabolite of 3-deazaadenosine in mouse leukocytes.

Evidence has been obtained for the metabolic formation of small amounts (1-2% of the ATP pool) of 3-deazaadenosine 5'-triphosphate (c3ATP) from 3-deazaadenosine (c3Ado) in mouse cytolytic lymphocytes and mouse resident peritoneal macrophages. With intact leukocytes, pharmacological evidence was obtained that adenosine kinase was not the enzyme chiefly responsible for the phosphorylation of c3Ado. Moreover, in the presence of MgCl2, NaCl and IMP, purified rat liver 5'-nucleotidase catalyzed the phosphorylation of c3Ado to 3-deazaadenosine 5'-monophosphate (c3AMP). Two lines of evidence suggest that the metabolic formation of c3ATP is not involved in the inhibition of leukocyte function caused by c3Ado. First, the inhibitory action of c3Ado on antibody-dependent phagocytosis and lymphocyte-mediated cytolysis was reversed markedly upon removal of the drug from the medium. However, the intracellular content of c3ATP remained constant in lymphocytes and macrophages after removal of c3Ado. Second, in macrophages and in lymphocytes, similar intracellular amounts of c3ATP were formed from both c3Ado and 3-deazaadenine under conditions in which the former was biologically active and the latter was essentially inactive. Thus, it appears unlikely that the novel c3ATP metabolite is of relevance for the mechanism of action of c3Ado in mouse leukocytes.

Regulation of follicle-stimulating hormone binding to receptors on bovine calf testis membranes by cholera toxin-sensitive guanine nucleotide binding protein.

In a previous study we reported that FSH receptors in bovine testes membranes are physically and functionally associated with a guanine nucleotide-binding protein (N protein). In this study we examined the mechanism whereby GTP binding to N protein regulates FSH binding to its receptors. Binding of FSH to receptors decreased in the presence of GTP in a dose-dependent and noncompetitive manner. This effect did not require the presence of Mg+2 and is in contrast to the reported requirement for Mg+2 for GTP effects on human CG binding to ovarian receptors. Equilibrium binding experiments indicated that decreased hormone binding in the presence of GTP was not due to a decrease in the number of FSH receptors per se; rather, the altered binding isotherm was the result of a decrease in affinity of receptors for FSH. Moreover, the dissociation of [125I]human FSH from preformed FSH-receptor complex was rapid in onset and significantly accelerated in the presence of GTP. In a series of nucleotides, GTP was most effective in causing this effect. Evidently, occupancy of GTP binding sites on the N protein, including low affinity and high capacity sites, is necessary for GTP regulation of FSH binding to receptors. The fact that pretreatment of bovine testis membranes with cholera toxin plus NAD, but not pertussis toxin plus NAD, eliminates the GTP effect on FSH binding to its receptors suggests that the GTP regulatory binding protein mediating the GTP regulation of FSH binding is probably Ns and not Ni. Further characterization of FSH receptor sensitivity to GTP, however, indicated that the N protein involved does not exhibit all of the characteristics reported for Ns. For example, the affinity of GTP for N protein is relatively low even under conditions where GTP hydrolysis has a minimal effect in reducing the total concentration of GTP. Also, the absence of a requirement for Mg+2 in high affinity FSH receptor-N protein coupling is different from the requirement for Mg+2 seen with the beta-adrenergic receptor and Ns. Moreover, the N protein which mediates GTP regulation of FSH-receptor binding appears to be relatively insensitive to N-ethylmaleimide, unlike the N-ethylmaleimide sensitivity of the turkey erythrocyte Ns. These results suggest that differences may exist in the structure-function features of GTP regulatory binding protein associated with different types of hormone ligands and receptors.

On the role, inactivation and origin of endogenous adenosine at the frog neuromuscular junction.

1. The effects of adenosine deaminase, inosine, alkylxanthines (8-phenyltheophylline (8-PT), theophylline and isobutylmethylxanthine (IBMX], dipyridamole, alpha, beta-methylene ADP (AOPCP) and ATP analogues (alpha, beta-methylene ATP and beta, gamma-methylene ATP) on evoked end-plate potentials (e.p.p.s) were investigated in innervated sartorius muscles of the frog, in which twitches had been prevented with tubocurarine. The effects of 8-PT and IBMX on the amplitude and quantal content of e.p.p.s were also investigated in innervated sartorius muscles of the frog, in which twitches had been prevented with high-magnesium solutions. 2. Adenosine deaminase reversibly increased the amplitude of e.p.p.s and prevented the reduction caused by exogenously applied adenosine on e.p.p. amplitude. The increase caused by adenosine deaminase was equivalent to the decrease caused by 12 +/- 5.8 microM-adenosine on e.p.p. amplitude. 3. Inosine, the product of adenosine deamination, was virtually devoid of effect on e.p.p.s. 4. The adenosine receptor antagonists at the frog neuromuscular junction, 8-PT and theophylline, increased in a concentration-dependent manner the amplitude of e.p.p.s in the presence of tubocurarine. 8-PT increased the amplitude and quantal content of e.p.p.s in the presence of high magnesium. IBMX, which does not behave as an adenosine receptor antagonist at the frog neuromuscular junction, decreased the amplitude of e.p.p.s in the presence of tubocurarine or high-magnesium solutions. 5. Dipyridamole, an adenosine uptake blocker, decreased the amplitude of e.p.p.s, and in a concentration that did not affect neuromuscular transmission potentiated the depressing effect of adenosine, but not that of 2-chloroadenosine, on the amplitude of e.p.p.s. 6. AOPCP, an inhibitor of 5'-nucleotidase, increased the amplitude of e.p.p.s and markedly attenuated the depressing effect of ATP, but not that of adenosine, on e.p.p. amplitude. 7. The ATP analogue, alpha, beta-methylene ATP, which is not a substrate for 5'-nucleotidase, was virtually devoid of effect on e.p.p.s. beta, gamma-Methylene ATP, which can be a substrate for 5'-nucleotidase, mimicked the depressing effect of ATP on e.p.p. amplitude, an effect which was also reduced by AOPCP. 8. It is concluded that in conditions in which the initial quantal content is assumed to be normal (1) endogenous adenosine depresses neuromuscular transmission, (2) at the neuromuscular junction adenosine is inactivated through a dipyridamole-sensitive uptake process, and (3) released adenine nucleotides might contribute to the pool of endogenous adenosine which modulates neuromuscular transmission.

Nuclease P1-mediated enhancement of sensitivity of 32P-postlabeling test for structurally diverse DNA adducts.

Exceedingly sensitive procedures are required to detect the presence of covalent DNA adducts in humans exposed to environmental genotoxicants because of low levels of such derivatives (1 adduct in 10(8)-10(10) DNA nucleotides). A 32P-postlabeling assay for detection and quantitation of carcinogen--DNA adducts with a sensitivity limit of 1 adduct in 10(7)-10(8) nucleotides has been described previously. In the standard procedure, DNA is enzymatically digested to 3'-phosphorylated normal and adducted mononucleotides, which are quantitatively 32P-labeled at their 5'-hydroxyl groups by T4 polynucleotide kinase-catalyzed [32P]phosphate transfer from [gamma-32P]ATP. 32P-labeled derivatives are resolved by t.l.c., detected by autoradiography and quantitated by counting. We now report that a minor modification of this procedure, entailing the postincubation of DNA digests with Penicillium citrinum nuclease P1 before 32P-labeling, enhanced the technique's sensitivity to 1 adduct in approximately 10(10) nucleotides for a 10-micrograms DNA sample. Nuclease P1 cleaves deoxyribonucleoside 3'-monophosphates of normal nucleotides to deoxyribonucleosides which do not serve as substrates for polynucleotide kinase, while most adducted nucleotides were found to be totally or partially resistant to the 3'-dephosphorylating action of nuclease P1. The additional enzymatic step enabled specific labeling of adducts in up to 20 micrograms of DNA with excess carrier-free [gamma-32P]ATP. The enzymatic digestion conditions were standardized to afford optimal adduct recovery. The new procedure was found to be simple, highly reproducible, and applicable to the detection and measurement of aromatic or bulky non-aromatic DNA adducts formed with such structurally diverse carcinogens as benzo[a]pyrene, 7,12-dimethyl-benz[a]anthracene, dibenzo[c,g]carbazole, 4-aminobiphenyl, safrole and mitomycin C; most adducts were recovered quantitatively with a 500- to 1000-fold increase in 32P-count rates as compared with the standard procedure.

Reaction of DNA with chemically or enzymatically activated mitomycin C: isolation and structure of the major covalent adduct.

The antitumor antibiotic mitomycin C is shown to form a covalent complex with calf thymus DNA under anaerobic conditions in the presence of either NADPH cytochrome c reductase/NADPH, xanthine oxidase/NADH, or the chemical reducing system H2/PtO2. Digestion of the complex with DNase I/snake venom diesterase/alkaline phosphatase yields a single mitomycin deoxyguanosine adduct as the major DNA alkylation product, identified as N2-(2'' beta,7''-diaminomitosen-1'' alpha-yl) 2'-deoxyguanosine (Structure 2). Two minor adducts, 2-5% each of the total adduct pool, are isolated and identified as the 1'' beta stereoisomer of 2 (Structure 3), and 10''-decarbamoyl-2 (Structure 7). The same results were obtained with M13 DNA and poly(dG-dC).poly(dG-dC); however, in the latter case, a minor adduct apparently possessing two deoxyguanosine and one mitomycin unit is isolated. Digestion of the covalent mitomycin-calf thymus DNA complex with nuclease P1 yields four dinucleotide adducts, all of which consist of 2 linked at its 3' end to each of the four possible 5' nucleotides (A, T, G, and C). Upon treatment of each dinucleotide adduct with snake venom diesterase/alkaline phosphatase, 2 is released along with the corresponding free nucleoside. In apparent conflict with the present results, previous reports from another laboratory have indicated that modification of calf thymus DNA by mitomycin C under conditions identical to those described here result in the isolation of three mitomycin C mononucleotide adducts possessing linkages of the drug to N2 and O6 of guanine and N6 of adenine. Evidence is shown suggesting that the latter adducts are actually three of the above four dinucleotide derivatives of 2 obtained independently by us and, thus, all of them in fact possess an identical N2-mitosenylguanine adduct moiety. Model-building studies indicate an excellent fit of the guanine N2-linked drug molecule inside the minor groove of B-DNA with no appreciable distortion of the DNA structure.

Platelet aggregation inhibitors from Agkistrodon acutus snake venom.

Among all the purified components from A. acutus venom, including ADPase, 5'-nucleotidase, phospholipase A2 and fibrinogenases, only the venom ADPase (50-100 micrograms/ml) shows marked inhibitory action on ADP (10 microM)-, collagen (10 micrograms/ml)- and sodium arachidonate (100 microM)-induced platelet aggregations of rabbit platelet-rich plasma. The venom 5'-nucleotidase (100 micrograms/ml) inhibited ADP-induced platelet aggregation by 31 +/- 4% (n = 4, P less than 0.05). Fibrinogenolytic enzymes (fractions I and IX, 100 micrograms/ml) did not significantly inhibit platelet aggregation induced by ADP (10 microM), collagen (10 micrograms/ml) or sodium arachidonate (100 microM). However, when the fibrinogenase (fraction IX, 100 micrograms/ml) was preincubated with platelet-rich plasma for 30 min it inhibited collagen (20 micrograms/ml)- and ADP (10 microM)-induced platelet aggregations by 34 +/- 9% (n = 4, P less than 0.05) and 35 +/- 6% (n = 4, P less than 0.05), respectively. The phospholipase A2 (100 micrograms/ml) did not affect platelet aggregation. The venom ADPase is a single chain polypeptide with a molecular weight of 94,000. The specific ADPase activity is estimated to be 4.3 mu moles Pi/min/mg of protein. It also possesses phosphodiesterase and weak 5'-nucleotidase activities.

Cytoplasmic 5'-nucleotidase catalyzes acyclovir phosphorylation.

A cytoplasmic 5'-nucleotidase (EC 3.1.3.5) can catalyze the phosphorylation of inosine (Worku, Y., and Newby, A.C. (1982) Biochem. J. 205, 503-510). This enzyme was purified to determine whether it could catalyze the formation of trace levels of phosphorylated acyclovir (ACV), a nucleoside analog with antiherpes activity. Acyclovir phosphorylating activity from rat liver co-chromatographed with the enzyme throughout the 1200-fold purification and through size exclusion chromatography or polyacrylamide-gel electrophoresis. In addition, the pH optimum, ATP stimulation, and phosphate inhibition of the ACV phosphorylating activity paralleled those of the 5'-nucleotidase. Finally, ACV phosphorylation was competitively inhibited by inosine (Kis = 6.5 mM; K'm (inosine) = 5.0 mM). This was consistent with phosphorylation at a common catalytic site. In addition to inosine and ACV, the guanine derivatives Guo, dGuo, 9-beta-D-arabinofuranosylguanine, and 9-(1,3-dihydroxy-2-propoxymethyl)guanine were substrates for the enzyme. The relative phosphorylation rates were, respectively, 100, 0.7, 19, 4, 0.3, and 0.7, at 0.1 mM phosphate acceptor. Approximate K'm values were, respectively, 5, 90, 10, 10, greater than 100, and greater than 100 mM. Although the substrate activity of ACV with the 5'-nucleotidase was inefficient, it appeared to be sufficient to account for the small amounts of ACV phosphates formed in uninfected cells.

Ecto-5'-nucleotidase can provide the total purine requirements of mitogen-stimulated human T cells and rapidly dividing human B lymphoblastoid cells.

The ability of mitogen-stimulated human T cells or rapidly dividing human B lymphoblastoid cells to drive their total purine requirements from inosine 5'-monophosphate, inosine, or hypoxanthine was compared. Inosine 5'-monophosphate first must be converted to inosine by the action of the enzyme ecto-5'-nucleotidase before it can be transported into the cell; inosine and hypoxanthine, however, can be transported directly. Mitogen-stimulated human peripheral blood T cells were treated with aminopterin to inhibit purine synthesis de novo and to make the cells dependent on an exogenous purine source. Thymidine was added as a source of pyrimidines. Under these conditions, 30 microM inosine 5'-monophosphate, inosine, and hypoxanthine showed comparable abilities to support [3H]thymidine incorporation into DNA or [3H]leucine incorporation into protein at rates equal to that of untreated control cultures. Similar results were found when azaserine was used to inhibit purine synthesis de novo, and thus DNA synthesis. In parallel experiments with the rapidly dividing human B lymphoblastoid cell line WI-L2, treatment with aminopterin (plus thymidine) inhibited the growth rate by greater than 95%. The normal growth rate was restored by the addition of 30 microM inosine 5'-monophosphate, inosine, or hypoxanthine to the medium. However, in similar experiments with cell line 1254, a derivative of WI-L2 which lacks detectable ecto-5'-nucleotidase activity, inosine and hypoxanthine (plus thymidine), but not inosine 5'-monophosphate (and thymidine) were able to restore the growth inhibition due to aminopterin. These results show that the catalytic activity of ecto-5'-nucleotidase is sufficient to meet the total purine requirements of mitogen-stimulated human T cells or rapidly dividing human B lymphoblastoid cells, and suggest that this enzyme may be important for purine salvage when rates of purine synthesis de novo are limited and/or an extracellular source of purine nucleotides is available.

Oxidation of N-methylthiobenzamide and N-methylthiobenzamide S-oxide by liver and lung microsomes.

The in vitro oxidation of N-[14C]methylthiobenzamide (NMTB) and N-[14C]methylthiobenzamide S-oxide (NMTBSO) by rat lung and liver microsomes was studied. In the presence of an NADPH-generating system, NMTB was rapidly converted to NMTBSO and small amounts of N-methylbenzamide (NMBA) and covalently bound metabolites (CVB). Under similar conditions, NMTBSO was converted to NMBA and CVB. Studies with metabolic inhibitors indicate that both the S-oxidation of NMTB and its further conversion to NMBA and CVB, probably via enzymatic oxidation to the S,S-dioxide, are catalyzed by both cytochromes P-450 and the FAD-containing monooxygenase (MFMO). Based on differential effects of inhibitors with lung vs liver microsomes, it would appear that in lung microsomes the MFMO plays a significantly greater role than cytochrome P-450 in the oxidation of NMTB and NMTBSO, whereas in the liver the contribution of these two pathways is more nearly equal. 1-Methyl-1-phenyl-3-benzoylthiourea, which blocks the in vivo pneumotoxicity of both NMTB and NMTBSO, also inhibited their in vitro microsomal metabolism and CVB, suggesting that these oxidations are obligatory steps in the expression of toxicity by these compounds.