Nucleotides dephosphorylation

Although several enzymes can in theory convert purine ribonucleotides to ribonucleosides, in only a few cases is it known exactly which is acting. Certain strains of Bacillus svbtilis accumulate nucleotides extra-cellularly, but mutants lacking 5 -nucleotidase or alkaline phosphatase, or both, accumulate smaller quantities of nucleotides than cells which have both enzymes. 5 -Nucleotidase appeared to be quantitatively the more important for nucleotide dephosphorylation in these cells 23). Baer and Drummond 12) compared the rates of dephosphorylation of the 2 -, 3 -, and 5 -phosphates of adenosine by perfused rat heart and concluded that only a specific 5 -nucleotidase was actively in contact with the blood. More evidence for the action of specific, rather than nonspecific phosphatases, are the observations that in one system or another, adenylate, inosinate, xanthylate, or guanylate appears to be the nucleotide most rapidly de-phosphorylated. 

Introduction of the cobalt atom into the corrin ring is preceeded by conversion of hydrogenobyrinic acid to the diamide (34). The resultant cobalt(II) complex (35) is reduced to the cobalt(I) complex (36) prior to adenosylation to adenosylcobyrinic acid i7,i -diamide (37). Four of the six remaining carboxyhc acids are converted to primary amides (adenosylcobyric acid) (38) and the other amidated with (R)-l-amino-2-propanol to provide adenosylcobinamide (39). Completion of the nucleotide loop involves conversion to the monophosphate followed by reaction with guanosyl triphosphate to give diphosphate (40). Reaction with a-ribazole 5 -phosphate, derived biosyntheticaHy in several steps from riboflavin, and dephosphorylation completes the synthesis. 

Pyrimidine 5 -nucleotidase (P5N) is a unique enzyme that was recognized from studies of families with relatively common hemolytic disorders. The enzyme catalyzes the hydrolytic dephosphorylation of pyrimidine 5 -nucleotides but not purine nucleotides. The role of this enzyme is to eliminate RNA and DNA degradation products from the cytosol during erythroid maturation by conversion of nucleotide monophosphates to diffusible nucleosides. P5N is inhibited by lead, and its activity is considered to be a good indicator of lead exposure (PI). 

The answer is c. (Katzung, p 933.) Resistance to thioguanine occurs because of an increase in alkaline phosphatase and a decrease in hypoxanthine-guanine phosphoribosyl transferase. These enzymes are responsible, respectively, for the increase in dephosphorylation of thiopurine nucleotide and the conversion of thioguanine to its active form, 6-thioinosinic acid. 

MTX also has several effects on the purine synthetic pathway. MTXPGs inhibit the enzyme aminoimidazole carboxamide ribonucleotide (AlCAR) transformylase, which in turn causes intracellular accumulation of AICAR. AICAR and its metabolites can then inhibit two enzymes in the adenosine pathway adenosine deaminase and adenosine monophosphate (AMP) deaminase, which leads to intracellnlar accumulation of adenosine and adenine nucleotides. Subsequent dephosphorylation of these nucleotides results in increased extracellular concentrations of adenosine, which is a powerful anti-inflammatory agent (11). 

In addition to the enzymes that catalyse the formation of nucleotides and polynucleotides, a large number of catabolic systems exist which operate at all levels of the internucleotide pathways. The ribonucleases and deoxyribonucleases that degrade polynucleotides are probably not significantly involved in purine analogue metabolism, but the enzymes which dephosphorylate nucleoside 5 -monophosphates are known to attack analogue nucleotides and may be of some importance to their in vivo activity. Phosphatases of low specificity are abundant in many tissues [38], particularly the intestine [29]. Purified mammalian 5-nucleotidases hydrolyse only the nucleoside 5 monophosphates [28] and... 

The splitting of sugar to lactic acid is thus, briefly, the shifting of hydrogen by means of the nicotinamide moiety of diphosphopyridine nucleotide (also termed nicotinamide adenine dinuclcotide). Nicotinamide in DPN takes away two atoms of hydrogen from phosphorylated carbohydrate, and after dephosphorylation gives back two hydrogens fin DPNH ) to pyruvic acid. 

Derivatives bearing a 3 -monopbosphoryl group were originally classified as totally resistant to venom exonuclease. As the quality of the enzyme preparation improved, these compounds were found susceptible but required 1000-fold more enzyme than was needed to hydrolyze 5 -monophosphate-bearing compounds. This unusual resistance led to another erroneous conclusion, that the polarity of exonuclease changes (20). The basis for this belief were the experiments in which a mixture of tri-, tetra-, and pentanucleotides of the type d-N pNPpN pN p were used as substrates. The early products were nucleosides and nucleotides, whereas 3, 5 -mononucleoside diphosphates appeared considerably later. It is clear now that the mixture was contaminated with a small amount of dephosphorylated chains which were rapidly hydrolyzed to completion. 

The 32P-postlabeling technique allows to improve the sensitivity of the detection of DNA damage (Cadet et al. 1998). The damaged DNA is enzymatically degraded into nucleotide-3-phosphates [reaction (7)]. The resulting mixture of unchanged nucleoside-3-phosphates (dNp) and damaged ones (dXP) are separated by HPLC [reaction (8)]. They are then labeled at the 5 -position with 32P [reaction (9)] and subsequently dephosphorylated at the 3 -position [reaction (10)]. This allows to proceed with a second purification and their identification by, for example, two-dimensional TLC [reactions (11) and (12)]. 

Resistance to both 6-MP and 6-TG occurs most commonly by decrease in HGPRT activity an alternative mechanism in acute leukemia involves elevation of levels of alkaline phosphatase, which results in dephosphorylation of thiopurine nucleotide and cellular loss of the resulting ribonucleoside. 

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