8-Aminoinosine and 8-Aminohypoxanthine Inhibit Purine Nucleoside Phosphorylase and Exert Diuretic and Natriuretic Activity.

8-Aminoguanine and 8-aminoguanosine (via metabolism to 8-aminoguanine) are endogenous 8-aminopurines that induce diuresis, natriuresis, and glucosuria by inhibiting purine nucleoside phosphorylase (PNPase); moreover, both 8-aminopurines cause antikaliuresis by other mechanisms. Because 8-aminoinosine and 8-aminohypoxanthine are structurally similar to 8-aminoguanosine and 8-aminoguanine, respectively, we sought to define their renal excretory effects. First, we compared the ability of 8-aminoguanine, 8-aminohypoxanthine, and 8-aminoinosine to inhibit recombinant PNPase. These compounds inhibited PNPase with a potency order of 8-aminoguanine > 8-aminohypoxanthine = 8-aminoinosine. Additional studies showed that 8-aminoinosine is a competitive substrate that is metabolized to a competitive PNPase inhibitor, namely 8-aminohypoxanthine. Administration of each 8-aminopurine (33.5 µmol/kg) reduced the guanine-to-guanosine and hypoxanthine-to-inosine ratios in urine, a finding confirming their ability to inhibit PNPase in vivo. All three 8-aminopurines induced diuresis, natriuresis, and glucosuria; however, the glucosuric effects of 8-aminohypoxanthine and 8-aminoinosine were less pronounced than those of 8-aminoguanine. Neither 8-aminohypoxanthine nor 8-aminoinosine altered potassium excretion, whereas 8-aminoguanine caused antikaliuresis. In vivo administration of 8-aminoinosine increased 8-aminohypoxanthine excretion, indicating that 8-aminohypoxanthine mediates, in part, the effects of 8-aminoinosine. Finally, 8-aminohypoxanthine was metabolized to 8-aminoxanthine by xanthine oxidase. Using ultraperformance liquid chromatography-tandem mass spectrometry, we identified 8-aminoinosine as an endogenous 8-aminopurine. In conclusion, 8-aminopurines have useful pharmacological profiles. To induce diuresis, natriuresis, glucosuria, and antikaliuresis, 8-aminoguanine (or its prodrug 8-aminoguanosine) would be preferred. If only diuresis and natriuresis, without marked glucosuria or antikaliuresis, is desired, 8-aminohypoxanthine or 8-aminoinosine might be useful. Finally, here we report the in vivo existence of another pharmacologically active 8-aminopurine, namely 8-aminoinosine. SIGNIFICANCE STATEMENT: Here, we report that a family of 8-aminopurines affects renal excretory function: effects that may be useful for treating multiple diseases including hypertension, heart failure, and chronic kidney disease. For diuresis and natriuresis accompanied by glucosuria and antikaliuresis, 8-aminoguanine (or its prodrug 8-aminoguanosine) would be useful; if only diuresis and natriuresis is called for, 8-aminohypoxanthine or 8-aminoinosine would be useful. Previously, we identified 8-aminoguanine and 8-aminoguanosine as endogenous 8-aminopurines; here, we extend the family of endogenous 8-aminopurines to include 8-aminoinosine.

PRE-CLINICAL AND CLINICAL VALIDATION OF AN ANTI-CANCER MODALITY THAT ABLATES REFRACTORY, LOW GROWTH FRACTION TUMORS.

Intratumoral expression of the E. coli purine nucleoside phosphorylase (PNP) gene was originally described by our laboratories as a means to inhibit growth of solid tumors in vivo. The approach generates purine bases that disrupt DNA, RNA, and protein synthesis, a unique mechanism when compared with all approved or experimental cancer therapeutics. Use of PNP has been validated by numerous laboratories worldwide against human tumor xenografts (lung, liver, pancreas, bladder, glioma, and prostate, among others). Data from a recently completed phase 1 clinical trial has indicated substantial anti-cancer activity in human subjects with no serious toxicities.

Targeted enzyme prodrug therapy for metastatic prostate cancer - a comparative study of L-methioninase, purine nucleoside phosphorylase, and cytosine deaminase.

BACKGROUND: Enzyme prodrug therapy shows promise for the treatment of solid tumors, but current approaches lack effective/safe delivery strategies. To address this, we previously developed three enzyme-containing fusion proteins targeted via annexin V to phosphatidylserine exposed on the tumor vasculature and tumor cells, using the enzymes L-methioninase, purine nucleoside phosphorylase, or cytosine deaminase. In enzyme prodrug therapy, the fusion protein is allowed to bind to the tumor before a nontoxic drug precursor, a prodrug, is introduced. Upon interaction of the prodrug with the bound enzyme, an anticancer compound is formed, but only in the direct vicinity of the tumor, thereby mitigating the risk of side effects while creating high intratumoral drug concentrations. The applicability of these enzyme prodrug systems to treating prostate cancer has remained unexplored. Additionally, target availability may increase with the addition of low dose docetaxel treatment to the enzyme prodrug treatment, but this effect has not been previously investigated. To this end, we examined the binding strength and the cytotoxic efficacy (with and without docetaxel treatment) of these enzyme prodrug systems on the human prostate cancer cell line PC-3. RESULTS: All three fusion proteins exhibited strong binding; dissociation constants were 0.572 nM for L-methioninase-annexin V (MT-AV), 0.406 nM for purine nucleoside phosphorylase-annexin V (PNP-AV), and 0.061 nM for cytosine deaminase-annexin V (CD-AV). MT-AV produced up to 99% cell death (p < 0.001) with limited cytotoxicity of the prodrug alone. PNP-AV with docetaxel created up to 78% cell death (p < 0.001) with no cytotoxicity of the prodrug alone. CD-AV with docetaxel displayed up to 60% cell death (p < 0.001) with no cytotoxicity of the prodrug alone. Docetaxel treatment created significant increases in cytotoxicity for PNP-AV and CD-AV. CONCLUSIONS: Strong binding of fusion proteins to the prostate cancer cells and effective cell killing suggest that the enzyme prodrug systems with MT-AV and PNP-AV may be effective treatment options. Additionally, low-dose docetaxel treatment was found to increase the cytotoxic effect of the annexin V-targeted therapeutics for the PNP-AV and CD-AV systems.

Effects of S-adenosylmethionine and methylthioadenosine on inflammation-induced colon cancer in mice.

Chronic inflammation is an underlying risk factor for colon cancer. Tumor necrosis factor alpha (TNF-α) plays a critical role in the development of inflammation-induced colon cancer in a mouse model. S-adenosylmethionine (SAMe) and its metabolite methylthioadenosine (MTA) can inhibit lipopolysaccharide-induced TNF-α expression in macrophages. The aim of this work was to examine whether SAMe and MTA are effective in preventing inflammation-induced colon cancer and if so identify signaling pathways affected. Balb/c mice were treated with azoxymethane (AOM) and dextran sulfate sodium to induce colon cancer. Two days after AOM treatment, mice were divided into three groups: vehicle control, SAMe or MTA. Tumor load, histology, immunohistochemistry, gene and protein expression were determined. SAMe and MTA treatment reduced tumor load by ∼40%. Both treatments raised SAMe and MTA levels but MTA also raised S-adenosylhomocysteine levels. MTA treatment prevented the induction of many genes known to play pathogenetic roles in this model except for TNF-α and inducible nitric oxide synthase (iNOS). SAMe also had no effect on TNF-α or iNOS and was less inhibitory than MTA on the other genes. In vivo, both treatments induced apoptosis but inhibited proliferation, ß-catenin, nuclear factor kappa B activation and interleukin (IL) 6 signaling. Effect of SAMe and MTA on IL-6 signaling was examined using Colo 205 colon cancer cells. In these cells, SAMe and MTA inhibited IL-6-induced IL-10 expression. MTA also inhibited IL-10 transcription and signal transducer and activator of transcription 3 activation. In conclusion, SAMe and MTA reduced inflammation-induced colon cancer and inhibited several pathways important in colon carcinogenesis.

Enzymatic dissolution of calcium and struvite crystals: in vitro evaluation of biochemical requirements.

OBJECTIVE: To evaluate the factors that affect the enzymatic dissolution rate of calcium oxalate monohydrate (COM), calcium phosphate (brushite), and magnesium ammonium phosphate (struvite) crystals as enzymatic digestion of kidney stones could enhance lithotripsy or provide alternatives to surgical removal. METHODS: At pH 4.2, pelleted COM crystals were combined with oxalate decarboxylase (ODC from Bacillus subtilis), oxalate oxidase (from Hordeum vulgare), or control. Crystal dissolution was followed by measuring increases in solution calcium ion concentration. For phosphate-based crystals, the rates of phosphorolysis by the enzyme purine nucleoside phosphorylase (PNP, assay form) were compared to the control solution using spectrophotometry. RESULTS: The addition of ODC to COM crystals resulted in production of highly soluble calcium formate and a 15-fold increase in COM solubility. By adding a formate-catabolizing enzyme (formate dehydrogenase), dissolution increased 47-fold compared with controls with nearly one half of the mineral dissolved. Oxalate oxidase showed much lower activity than ODC in COM dissolution. Using inorganic phosphate as a substrate, PNP was able to dissolve both brushite and struvite minerals in water at concentrations near saturation. Measuring dissolution by adding more PNP was not possible because of equilibrium and assay detection restraints. CONCLUSION: Stone dissolution using enzymes appears to be viable, particularly for oxalate-based minerals. In a closed system, product inhibition by calcium formate appeared to limit the extent of COM crystal dissolution using ODC. Although phosphate-containing minerals appear to be suitable phosphate sources for PNP, the reversibility of the reaction limits the use of this enzyme.

A transition state analogue of 5'-methylthioadenosine phosphorylase induces apoptosis in head and neck cancers.

Methylthio-DADMe-immucillin-A (MT-DADMe-ImmA) is an 86-pm inhibitor of human 5'-methylthioadenosine phosphorylase (MTAP). The sole function of MTAP is to recycle 5'-methylthioadenosine (MTA) to S-adenosylmethionine. Treatment of cultured cells with MT-DADMe-ImmA and MTA inhibited MTAP, increased cellular MTA concentrations, decreased polyamines, and induced apoptosis in FaDu and Cal27, two head and neck squamous cell carcinoma cell lines. The same treatment did not induce apoptosis in normal human fibroblast cell lines (CRL2522 and GM02037) or in MCF7, a breast cancer cell line with an MTAP gene deletion. MT-DADMe-ImmA alone did not induce apoptosis in any cell line, implicating MTA as the active agent. Treatment of sensitive cells caused loss of mitochondrial inner membrane potential, G(2)/M arrest, activation of mitochondria-dependent caspases, and apoptosis. Changes in cellular polyamines and MTA levels occurred in both responsive and nonresponsive cells, suggesting cell-specific epigenetic effects. A survey of aberrant DNA methylation in genomic DNA using a microarray of 12,288 CpG island clones revealed decreased CpG island methylation in treated FaDu cells compared with untreated cells. FaDu tumors in a mouse xenograft model were treated with MT-DADMe-ImmA, resulting in tumor remission. The selective action of MT-DADMe-ImmA on head and neck squamous cell carcinoma cells suggests potential as an agent for treatment of cancers sensitive to reduced CpG island methylation.

Relationship between activating and editing functions of the adenylation domain of apo-tyrocidin synthetase 1 (apo-TY1).

Tyrocidine synthetase 1 (TY1), the initial monomodular constituent of the tyrocidine biosynthetic system, exhibits relaxed substrate specificity, however an efficient editing of the mis-activated amino acid provides for fidelity of product formation. We chose to analyse the consequence of single amino acid substitutions, in the amino acid activation site of apo-TY1, on the editing functions of the enzyme. Discrimination between L-Phe and D-Phe by apo-TY1 depends primarily on the editing reaction. Distraction of unnatural amino acid substrates, such as L-PheSer, implies that editing is not designated to select a specific mis-activated amino acid, but instead to discriminate all mis-activated amino acid analogues. It was shown that active site residues which interact with the adenylate are essential for both activation and editing. Substitution of Lys186 with arginine substantially reduces the editing capacity of the protein. Loss of amino acid discrimination ability by the apo-K186T and apo-R416T mutant proteins suggests a role of active site residues in maintaining the structural determinants for substrate selection. Inadequate conformational changes, induced by non-cognate amino acid substrates, promote ATP breakdown yielding P(i) and ADP. Replacement of residue Lys186 or Arg416 enhances ATP hydrolysis implying a role in binding or adjusting of the triphosphate chain for adenylate formation and pyrophosphate cleavage.

Neuroblastoma-specific cytotoxicity mediated by the Mash1-promoter and E. coli purine nucleoside phosphorylase.

BACKGROUND: Neuroblastoma is derived from cells of neural crest origin and often expresses the transcription factor human achaete-scute homolog 1 (HASH1). The aim of this study was to selectively kill neuroblastoma cells by expressing the suicide gene E. coli purine nucleoside phosphorylase (PNP) under the control of the Mash1 promoter, the murine homolog of HASH1. PROCEDURE: The E. coli PNP gene regulated by the Mash1 promoter was cloned into an expression vector and transfected into neuroblastoma and non-neuroblastoma cell lines. After addition of the prodrug M2-fluoroadenine 9-beta-D-arabinofuranoside (F-araA) the cell-specific toxicity was examined. To optimize the cell specific activity, different sizes of the Mash1 promoter were analyzed in neuroblastoma cell lines and compared with the activity in non-neuroblastoma cells. RESULTS: Estimated as the percentages of CMV enhancer-promoter, the activity was significantly higher in the neuroblastoma cells, ranging from 17 to 58% when the shortest and the most active promoter was measured. The non-neuroblastoma cells yielded only 1-6% of the CMV promoter activity. When the shortest Mash1 promoter was combined with the E. coli PNP gene the cytotoxicity was 65% in the neuroblastoma cells with low cell death in the non-neuroblastoma cell lines, relative to the cytotoxicity where the E.coli PNP gene was regulated by the strong but non-specific CMV enhancer-promoter. CONCLUSIONS: We show here that the Mash1 promoter regulating the PNP gene confers a cell-type selective toxicity in neuroblastoma cell lines. These results indicate the feasibility to use the Mash1 promoter for regulating E.coli PNP expression in gene-directed enzyme prodrug therapy (GDEPT) of neuroblastoma.

Purine nucleoside phosphorylase and fludarabine phosphate gene-directed enzyme prodrug therapy suppresses primary tumour growth and pseudo-metastases in a mouse model of prostate cancer.

Gene-directed enzyme prodrug therapy based on the E. coli purine nucleoside phosphorylase (PNP) gene produces efficient tumour cell killing. PNP converts adenosine analogs into toxic metabolites that diffuse across cell membranes to kill neighbouring untransduced cells (PNP-GDEPT). Interference with DNA, RNA and protein synthesis kills dividing and non-dividing cells, an important consideration for slow-growing prostate tumours. This study examined the impact of administering PNP-GDEPT into orthotopically grown RM1 prostate cancers (PCas) on the growth of lung pseudo-metastases of immunocompetent mice. C57BL/6 mice bearing orthotopic RM1 PCas received a single intraprostatic injection of OAdV220 (10(10) particles), a recombinant ovine atadenovirus containing the PNP gene controlled by the Rous Sarcoma virus promoter, followed by fludarabine phosphate (approximately 600 mg/m(2)/day) administered intraperitoneally (ip) once daily for 5 days. Pseudo-metastases were induced 2 days after intraprostatic vector administration by tail-vein injection of untransduced RM1 cells. Mice given PNP-GDEPT showed a significant reduction both in prostate volume (approximately 50%) and in lung colony counts (approximately 60%). Apoptosis was increased two-fold in GDEPT-treated prostates compared with controls (P < 0.01), but was absent in the lungs. Staining for proliferating cell nuclear antigen (PCNA) indicated that proliferation of both RM1 prostate tumours (P < 0.01) and lung colonies (P < 0.01) was significantly suppressed after GDEPT. Although prostate tumour immune cell infiltration did not differ significantly between treatments, immunostaining for Thy-1.2 (CD90) showed that GDEPT promoted Thy-1.2(+) cell infiltration into the prostate tumour site. This study showed that a single course of PNP-GDEPT significantly suppressed local PCa growth and reduced lung colony formation in the aggressive RM1 tumour model.

Status of methylthioadenosine phosphorylase and its impact on cellular response to L-alanosine and methylmercaptopurine riboside in human soft tissue sarcoma cells.

The aim of this study was to examine the expression of methylthioadenosine phosphorylase (MTAP) in 21 fresh tumor samples from patients with soft tissue sarcomas (STS) and 11 human soft tissue sarcoma cell lines, and to determine if loss of expression of this enzyme was correlated with increased sensitivity to L-alanosine and/or 6-methylmercaptopurine. We used a polyclonal antibody to measure the expression of MTAP in soft tissue sarcoma cell lines and in fresh tumor samples. Transfection of the HT-1080 cell line with a plasmid containing the cDNA for the MTAP gene was also performed to generate cell lines for in vitro and in vivo comparative sensitivity studies. MTAP was not expressed in 8 of 21 fresh STS tumors. The expression of MTAP was also not detectable in 3 of the 11 soft tissue sarcoma cell lines (HT-1080, HS42, and M-9 110). These three cell lines were more sensitive to L-alanosine, a potent inhibitor of de novo AMP synthesis, and to an inhibitor of de novo purine nucleotide synthesis, 6-methylmercaptopurine riboside (MMPR). The IC50 values for L-alanosine and MMPR were >20-fold lower in MTAP-deficient cells than in MTAP-positive cells. Restoration of MTAP into HT-1080 MTAP-deficient cells also led to decreased sensitivity to L-alanosine and MMPR. An in vivo study using HT-1080 cell tumors with and without MTAP expression confirmed that tumors lacking MTAP were more sensitive to L-alanosine than tumors expressing MTAP. These results provide the basis for selective therapy using inhibitors of de novo purine nucleotide synthesis such as L-alanosine or MMPR to treat patients with STS lacking this enzyme.

Enzyme activities controlling adenosine levels in normal and neoplastic tissues.

Adenosine is known to be associated with effects such as inhibition of immune response, coronary vasodilation, stimulation of angiogenesis, and inhibition of inflammatory reactions. Some authors suggest that adenosine may also have similar functions in tumor tissues. Tissue levels of adenosine are under close regulation by different enzymes acting at different levels. Adenosine is produced from AMP by the action of 5'-nucleotidase (5'-NT) and is converted back into AMP by adenosine kinase (AK) or into inosine by adenosine deaminase (ADA). Inosine is converted into purine catabolites by purine nucleoside phosphorylase (PNP), whereas AMP is converted into ADP and ATP by adenylate kinase (MK). The aim of this study was to analyze the activities of the above enzymes in fragments of neoplastic and apparently normal mucosa, obtained less than 5 cm and at least 10 cm from tumors, in 40 patients with colorectal cancer. The results showed much higher activities of ADA, AK, 5'-NT, and PNP in tumor tissue than in neighboring mucosa (p > 0.01 for ADA, AK, and PNP; p > 0.05 for 5'-NT), suggesting that the activities of purine metabolizing enzymes increase to cope with accelerated purine metabolism in cancerous tissue. The simultaneous increase in ADA and 5'-NT activities might be a physiological attempt by cancer cells to provide more substrate to accelerate salvage pathway activity.

Gene-directed enzyme prodrug therapy for prostate cancer in a mouse model that imitates the development of human disease.

BACKGROUND: Gene-directed enzyme prodrug therapy (GDEPT) based on the E. coli enzyme purine nucleoside phosphorylase (PNP) represents a new approach for treating slow growing tumours like prostate cancer (PCa). Expressed enzyme converts a systemically administered prodrug, fludarabine phosphate, to a toxic metabolite, 2-fluoroadenine. Infected and neighbouring cells are killed by a bystander effect that results from the inhibition of DNA and RNA synthesis. METHODS: These studies were carried out using the transgenic adenocarcinoma of the prostate (TRAMP) model that mimics human PCa development and progression. Control TRAMP mice were injected intraprostatically with vector vehicle and thereafter intraperitoneally with saline or fludarabine phosphate ( approximately 600 mg/m(2)/day) once daily for 5 consecutive days. Treated mice received a single intraprostatic injection containing 10(10) particles of OAdV220, an ovine atadenovirus which expresses the E. coli PNP gene under the control of the Rous sarcoma virus promoter, followed by systemic fludarabine treatment. The weight of the genitourinary tract, seminal vesicles and the prostate as well as animal survival were monitored. Tumours were also analysed histologically. RESULTS: Preliminary studies showed that fludarabine alone caused no significant change in genitourinary (GU) tract weight in TRAMP mice. Animals injected with vector and prodrug showed a significant reduction (36-47%) in GU tract weight (ANOVA p = 0.0002) and a 35-50% reduction in seminal vesicle weight (ANOVA p = 0.0007). In particular, the target organ showed a significant 57% reduction in prostate weight (ANOVA p = 0.0007). PNP-GDEPT mice also showed a survival advantage over control mice. Histological analysis suggested that the cancer progression was slowed in GDEPT-treated animals. CONCLUSION: A single course of GDEPT based on OAdV-delivered PNP and fludarabine produced highly significant suppression of PCa progression in immune-competent TRAMP mice.

Deficiencia proteica: impacto sobre el timo de ratas en crecimiento / Protein deficiency: effect of thymus of growing rats

Se analiza las consecuencias provocadas por desequilibrios nutricionales sobre el contenido de DNA y la actividad de las enzimas ADA y PNP en timo de ratas en crecimientos. Ratas Wistar al destete fueron alimentadas con: 1- dieta libre de proteínas hasta asemejar cuadros de malnutrición proteica leve, moderada y severa; 2- dieta conteniendo harina de maíz en baja concentración (6,5 por ciento). La subnutrición durante la lactancia se obtuvo duplicando la camada (12-14 crías por madre). Como controles se utilizaron ratas bien nutridas de igual edad, que desde el destete recibieron dieta stock. Al finalizar la experiencia, se les extrajo el timo (Pt)(mg). Se determinó DNA (mg/órgano), el número de núcleos, el tamaño celular- Pt(mg)/No. de Núcleos- y la actividad de las enzimas ADA y PNP (umol de ácido úrico x 10 - 1/P)(P=Pt(mg) /P corporal 0.75). Los resultados muestran que tanto la subnutrición durante la lactancia, como la malnutrición proteica al destete y la administración de dieta de baja calidad, afectan la proliferación celular en el timo, Sólo la carencia de proteína o su baja calidad, aumenta la actividad de ADA y PNP

Cell to cell contact is not required for bystander cell killing by Escherichia coli purine nucleoside phosphorylase.

Expression of Escherichia coli purine nucleoside phosphorylase (PNP) activates prodrugs and kills entire populations of mammalian cells, even when as few as 1% of the cells express this gene. This phenomenon of bystander killing has been previously investigated for herpes simplex virus-thymidine kinase (HSV-TK) and has been shown to require cell to cell contact. Using silicon rings to separate E. coli PNP expressing cells from non-expressing cells sharing the same medium, we demonstrate that bystander cell killing by E. coli PNP does not require cell-cell contact. Initially, cells expressing E. coli PNP convert the non-toxic prodrug, 6-methylpurine-2'-deoxyriboside (MeP-dR) to the highly toxic membrane permeable toxin, 6-methylpurine (MeP). As the expressing cells die, E. coli PNP is released into the culture medium, retains activity, and continues precursor conversion extracellularly (as determined by reverse phase high performance liquid chromatography of both prodrug and toxin). Bystander killing can also be observed in the absence of extracellular E. coli PNP by removing the MeP-dR prior to death of the expressing cells. In this case, 100% of cultured cells die when as few as 3% of the cells of a population express E. coli PNP. Blocking nucleoside transport with nitrobenzylthioinosine reduces MeP-dR mediated cell killing but not MeP cell killing. These mechanisms differ fundamentally from those previously reported for the HSV-TK gene.

Effect of purine nucleoside phosphorylase substrates on the mitogen-induced stimulation of murine T and B spleen cells.

The effect of culture with exogenous purine nucleoside phosphorylase substrates (especially deoxyguanosine) on the proliferation of mitogen-stimulated murine spleen cells was investigated. Con A-stimulated 3H-thymidine incorporation in unpurified and purified T cells was appreciably inhibited by culture in the presence of 100 microM deoxyguanosine. LPS-stimulated incorporation in unpurified and purified B cells was affected in a similar manner. Culture with guanosine inhibited incorporation in both mitogen-stimulated T and B cells to almost the same extent as deoxyguanosine. Inhibition of 3H-thymidine incorporation in T cells by deoxyguanosine was not modified by concomitant inclusion of deoxycytidine in the culture medium. In addition, deoxyguanosine had effects on T cell proliferative responses during the early phases of stimulation and even prior to stimulation with the mitogen. These results contrast with those reported for human lymphoid cells, where deoxyguanosine was much more potent that guanosine, and where only T cells were affected. They suggest that mechanisms other than the one involving inhibition of ribonucleotide reductase may also be important in the effects of deoxyguanosine on certain lymphoid cells.

Kinetic properties of spermine synthase from bovine brain.

A kinetic analysis including initial-velocity and product-inhibition studies were performed with spermine synthase purified from bovine brain. The enzyme activity was assayed in the presence of 5'-methylthioadenosine phosphorylase as an auxiliary enzyme to prevent the accumulation of the inhibitory product, 5'-methylthioadenosine, and thus to obtain linearity of the reaction with time. Initial-velocity studies gave intersecting or converging linear double-reciprocal plots. No substrate inhibition by decarboxylated S-adenosylmethionine was observed at concentrations up to 0.4 mM. Apparent Michaelis constants were 60 microM for spermidine and 0.1 microM for decarboxylated S-adenosylmethionine. Spermine was a competitive product inhibitor with respect to decarboxylated S-adenosylmethionine, but a mixed one with respect to the other substrate, spermidine. 5'-Methylthioadenosine showed a mixed inhibition with both substrates, predominantly competitive with respect to decarboxylated S-adenosylmethionine and predominantly uncompetitive with respect to spermidine. The observed kinetic and inhibition patterns are consistent with a compulsory-order mechanism, where both substrates add to the enzyme before products can be released.

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-beta-D-ribofuranosyl)adenine, 9-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)adenine, 9-(2'-azido-2'-deoxy-beta-D-ribofuranosyl)adenine (2'-N3-riboA) and 9-(2-azido-2'-deoxy-beta-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-beta-D-ribofuranosyl)hypoxanthine underwent very slow phosphorolysis, and no inhibition of inosine phosphorolysis was detected when a 30 microM concentration of any studied inosine analog was added to a reaction mixture containing 30 microM inosine (the Km concentration). Kinetic parameters were determined for the deamination of the adenosine analogs. The greatest affinity for adenosine deaminase was found with 2'-N3-ribo A (Ki = 2 microM), 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.