Adenine incorporation into

In this paper we have examined [ H]-MVA incorporation into cytokinins in crown gall tissues. In addition, results are described which provide evidence of [ C]-adenine incorporation into cytokinins by germinating seeds and young leaves under normal physiological conditions. 

Unfortunately, this experiment could not be repeated due to the loss of the tobacco crown gall line by contamination of stock cultures. Further experiments were therefore carried out with D. innoxia crown gall tissue. The details of incubation conditions were exactly as described in a previous study of [U- Cj-adenine incorporation into cytokinins by tissues of different ages [22]. The tissues (2 g) were preincubated with compactin (0,5,20,100 juM) for 4 h after which 38 /xCi of [ H]-MVA lactone was added to each dish. The tissues were extracted and analyzed following further incubation for 8 h. Two additional treatments were included in which compactin (20 /xM) was present only during preincubation or during incubation with [ H]-MVA (Table 1). The extracts were first chemically treated with sodium periodate [19] to convert cytokinin ribosides and nucleotides... 

Table 2. C]-Adenine incorporation into putative cytokinins by intact lupin seed and isolated embryos...

Sulphonamides are structural analogues of PABA. They competitively inhibit the incorporation of PABA into dihydropteroic acid and there is some evidence for their incorporation into false folate analogues which inhibit subsequent metabolism. The presence of excess PABA will reverse the inhibitory action of sulphonamides, as will thymine, adenine, guanine and methionine. However, these nutrients are not normally available at the site of infections for which the sulphonamides are used. 

Fludarabine is an analog of the purine adenine. It interferes with DNA polymerase to cause chain termination and inhibits transcription by its incorporation into RNA. Fludarabine is dephosphorylated rapidly and converted to 2-fluoro-Ara-AMP (2-FLAA), which enters the cells and is phosphorylated to 2-fluoro-Ara-ATP, which is cytotoxic. Fludarabine is converted rapidly to 2-FLAA. The pharmacokinetics of 2-FLAA... 

Seasonal variations in the metabolic fate of adenine nucleotides prelabelled with [8—1-4C] adenine were examined in leaf disks prepared at 1-month intervals, over the course of 1 year, from the shoots of tea plants (Camellia sinensis L. cv. Yabukita) which were growing under natural field conditions by Fujimori et al.33 Incorporation of radioactivity into nucleic acids and catabolites of purine nucleotides was found throughout the experimental period, but incorporation into theobromine and caffeine was found only in the young leaves harvested from April to June. Methy-lation of xanthosine, 7-methylxanthine, and theobromine was catalyzed by gel-filtered leaf extracts from young shoots (April to June), but the reactions could not be detected in extracts from leaves in which no synthesis of caffeine was observed in vivo. By contrast, the activity of 5-phosphoribosyl-1-pyrophosphate synthetase was still found in leaves harvested in July and August. 

Biotin-dUTP derivatives are formed by modification of the C-5 position of uridine. This location is not involved in hydrogen bonding activity with complementary DNA strands, thus hybridization efficiency is not immediately compromised. By contrast, biotin-dCTP or biotin-dATP derivatives involve modification of the bases at the N-4 position of cytosine and the N-6 position of adenine, locations directly involved in hydrogen bond formation with complementary bases. Thus, DNA biotinylation through the use of modified deoxynucleoside triphosphates to be incorporated into existing DNA strands may result in better activity of the probe if dUTP is used over dATP or dCTP. 

Three years later, Lajtha, Oliver, and Ellis performed similar studies with human bone-marrow cultures exposed to 32P, or 14C-adenine. Control smears were treated with M HC1 at 60 °C for 6.5 min to remove 32P not incorporated into DNA. Grain counts were made over individual nuclei so that the rate of uptake into DNA could be estimated. The cycle time for the dividing cells in the culture was 40-48 h. DNA synthesis took 12-15 h in the second half of the cycle and was divided from mitosis by a 3-4 h non-synthesizing period (G2). 

The effect of 6-mercaptopurine on the incorporation of a number of C-labelled compounds into soluble purine nucleotides and into RNA and DNA has been studied in leukemia L1210, Ehrlich ascites carcinoma, and solid sarcoma 180. At a level of 6-mercaptopurine that markedly inhibited the incorporation of formate and glycine, the utilization of adenine or 2-aminoadenine was not affected. There was no inhibition of the incorporation of 5(or 4)-aminoimidazole-4(5)-carboxamide (AIC) into adenine derivatives and no marked or consistent inhibition of its incorporation into guanine derivatives. The conversion of AIC to purines in ascites cells was not inhibited at levels of 6-mercaptopurine 8-20 times those that produced 50 per cent or greater inhibition of de novo synthesis [292]. Furthermore, AIC reverses the inhibition of growth of S180 cells (AH/5) in culture by 6-mercaptopurine [293]. These results suggest that in all these systems, in vitro and in vivo, the principal site at which 6-mercaptopurine inhibits nucleic acid biosynthesis is prior to the formation of AIC, and that the interconversion of purine ribonucleotides (see below) is not the primary site of action [292]. Presumably, this early step is the conversion of PRPP to 5-phosphoribosylamine inhibited allosterically by 6-mercaptopurine ribonucleotide (feedback inhibition is not observed in cells that cannot convert 6-mercaptopurine to its ribonucleotide [244]. 

Azaguanine was the first purine analogue shown to be incorporated into polynucleotides [337] and, since its primary metabolic effect is on protein synthesis, the incorporation into RNA is considered the basis for its biologic activity [338]. In microbial systems 8-aza-adenine, 8-azahypoxanthine, 8-azaxanthine, and 5(4)-amino-l/f-l, 2, 3-triazole-4(5)-carboxamide are all incorporated into RNA as 8-azaguanylic acid [336]. 

Several analogues of adenine or adenosine are reported to be incorporated into nucleic acids 2-fluoroadenosine [342], tubercidin [190, 192, 342a], toyacamycin [193,342a], sangivatnycin [342a, b], cordycepin [168,343,344], 4-aminopyrazolo[3, 4-d] pyrimidine [119], formycin [344a], and 9- -D-arabinofuranosyladenine [152, 154], The evidence for the incorporation of 9-(3-D-arabinofuranosyladenine has been questioned [345]. 

These substances are analogues of thymine (azidothymidine, stavudine), adenine (didanosine), cytosine (lami-vudine, zaldtabine), and guanine (car-bovir, a metabolite of abacavir). They have in common an abnormal sugar moiety. Like the natural nucleosides, they undergo triphosphorylation, giving rise to nucleotides that both inhibit reverse transcriptase and cause strand breakage following incorporation into viral DNA. 

The cytostatic drugs administered (indicated by a syringe in the illustration) are often not active themselves but are only converted into the actual active agent in the metabolism. This also applies to the adenine analogue 6-mercaptopurine, which is initially converted to the mononucleotide tIMP (thioinosine monophosphate). Via several intermediate steps, tIMP gives rise to tdGTP, which is incorporated into the DNA and leads to crosslinks and other anomalies in it. The second effective metabolite of 6-mercaptopurine is S-methylated tIMP, an inhibitor of amidophos-phoribosyl transferase (see p. 188). 

The alkaloids are also relevant to drug design. Alkaloids are complex heterocyclic compounds that contain nitrogen and thus have base-like (hence the term alkaloid ) properties they are extremely structurally diverse. Nicotine is one of the simplest alkaloids. Oxidation of nicotine produces nicotinic acid, a vitamin that is incorporated into the important coenzyme nicotinamide adenine dinucleotide, commonly referred to as NAD" (oxidized form). The neurotransmitter serotonin is an alkaloid containing the aromatic indole ring system. 

Niacin (vitamin B3) is converted in the body to the amide, which is incorporated into niacinamide adenine dinucleotide (NAD). It is excreted in the urine unmodified and as several metabolites. 

Although initially and abortively developed for treatment of HIV infection, adefovir dipivoxil gained approval, at lower and less toxic doses, for treatment of HBV infection. Adefovir dipivoxil is the diester prodrug of adefovir, an acyclic phosphonated adenine nucleotide analog (Rgure 49-2). It is phosphorylated by cellular kinases to the active diphosphate metabolite and then competitively inhibits HBV DNA polymerase to result in chain termination after incorporation into the viral DNA. Adefovir is active in vitro... 

Perhydropyrimidine-2,4,6-trione (barbituric acid) has been incorporated into a polymer framework by modification of poly(diethyl methylenemalonate) (245) with urea (Scheme 119) (72NKKil79). The modified polymers (246) were useful for the adsorption of adenine. 

One way in which chemical compounds can induce base substitution mutation is through their incorporation into the structure of DNA itself. Thus, 5-bromodeoxyuridine (or bromouracil) can replace thymidine in DNA, where it serves as an efficient mutagenic agent.767 2-Aminopurine, an analog of adenine, pairs with thymine, just as does adenine when incorporated into DNA. 

As can be seen, the codons for glutamic acid (GAA and GAG) are very similar to two of the codons (GUA and GUG) for valine. Replacement of adenine in the glutamic acid codons by uracil causes valine to be incorporated into hemoglobin instead of glutamic acid and is responsible for the sickle cell trait. 

Fig. 4.2. Covalent incorporation of [3H]3-(3H-diazirino)pyridine adenine dinucleotide into lactate dehydrogenase as a function of time. Lactate dehydrogenase (I. I mM in subunits) in 100 mM Tris-HCl at pH 8.0 was mixed with 3H]3-(3-H-diazirino) pyridine adenine dinu-...

Purine derivatives were also synthesized starting from HCN derivatives. Yamada described in 1972 the one-pot synthesis of purine 12 obtained simply by heating neat formamide, a product of HCN hydrolysis [70,71] at 160 °C [72]. Adenine 1 was obtained as the main reaction product along with a low amount of 12 upon repetition of the reaction under similar experimental conditions in the presence of HCN (Scheme 7) [73,74], On the basis of 13C-NMR studies of the synthesis performed with the enriched substrate, three molecules of HCN and two molecules of formamide were found to be incorporated into the adenine scaffold by a C - N bond fission process. 

Pharmacokinetics Niacin is administered orally. It is converted in the body to nicotinamide, which is incorporated into the cofactor nicotinamide adenine dinucleotide (NAD+). Niacin, its nicotinamide derivative and other metabolites are excreted in the urine. [Note Nicotinamide alone does not decrease plasma lipid levels.]... 

Thus, it uses the four ribonucleoside triphosphates (ATP, GTP, UTP, and CTP) to assemble an RNA chain, the sequence of which is determined by the template strand of DNA. Nucleotide addition occurs sequentially, the phosphodiester bond being formed through the same mechanism as described for DNA polymerase (see Chap. 16, Fig. 16-9). RNA chain growth is in the 5 — 3 direction. An important distinction between RNA polymerase and DNA polymerase, however, is the ability of the former to start a new chain de novo i.e., it does not have an obligatory requirement for a primer. The first nucleotide to be incorporated into the chain of RNA contains either adenine or guanine and retains its 5 triphosphate. 

D-[l-HC]ribose — no incorporation [U-l4C]adenosine — incorporation into cordycepin without change in the adenine/ribose activity ratio856 Enzymic mechanism by which reduction of the 3 -hydroxyl group occurs is not known... 

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