Ribose radioactive

Partial phosphodiesterase digestion reduced both bound and nonbound poly(ADP-ribose) radioactivity to 50% (Fig. 3). A newly generated radioactive peak was comigrating with phosphodiesterase itself. 

C-labeled carbon dioxide is administered to a green plant, and shortly thereafter the following compounds are isolated from the plant 3-phosphoglycerate, glucose, erythrose-4-phosphate, sedoheptulose-l,7-bisphosphate, ribose-5-phosphate. In which carbon atoms will radioactivity be found ... 

Scheme 27.—A degradation of D-ribose, which allows the localization of radioactivity in each carbon atom.

The experiments with (U-l3C)AIRs showed that this nucleoside supplied all of the carbon atoms of pyramine. Because out of 6 carbon atoms of pyramine, only three may come from the imidazole part of AIRs, it can be concluded that the three other carbon atoms come from the ribose part of this nucleoside. In complete agreement with these results, radioactivity from AIRs, labeled mainly with, 4C in its ribose part, was found to incorporate into the three carbon atoms of pyramine, the origin of which was, at the time, unknown. Owing to the minute amount of AIRs supplied (as compared with that of glucose) in both experiments, the incorporation of label from AIRs after metabolic degradation is ruled out. 

Further experiments with labeled precursors were necessary to shed a little more light on this puzzling observation. Pyramine, biosynthesized from AIRs labeled with, 4C on C-l on the ribose part, exhibited only marginal radioactivity. This result rules out C-l of ribose in AIRs as a precursor of pyramine. This conclusion was confirmed with a precursor labeled at the C-l position with the stable l3C isotope. The mass spectrum of the ethylthio derivative of pyramine was identical with that of an unlabeled sample (Scheme 9). 

On the other hand, the fragmentation of pyramine obtained from (2 -l3C)AIRs indicated clearly that C-2, in the ribose part, was the precursor of carbon C-7 of the methyl on C-2 of the pyrimidine ring (Scheme 29). This result was confirmed by an experiment with a sample of AIRs labeled with l4C on C-l, C-2, C-3, on the ribose, and C-5 on the imidazole, with an approximate distribution of 1, 1, 3, 3. This precursor produced pyramine with the methyl group almost as radioactive as C-l or C-2, and much less than C-3 of AIRs. Because of the incorporation of C-5 of imidazole into C-4 of pyramine, and the comparable activities of C-3 and C-5 in the precursor AIRs, the specific activity of pyramine... 

The above transketolase and transaldolase reactions were found inadequate to explain the metabolism of D-ribose 5-phosphate, because of the non-accumulation of tetrose phosphate, the 75 % yield of hexose phosphate, and the results of experiments with C14 (the distribution of which differed markedly from the values predicted for such a sequence). 24(b) Thus, with D-ribose-l-C14, using rat-liver enzymes, any hexose formed should have equal radioactivity at Cl and C3, whereas, actually, 74% appeared at Cl. Furthermore, D-ribose-2,3-Cl42 should have given material having equal labels at C2 and C4 in the resultant hexose, whereas, in fact, it had 50% of the activity at C4, C3 was nearly as active as C2, and Cl had little activity. Similar results were obtained with pea-leaf and -root preparations.24 The following reactions, for which there is enzymic evidence,170(b) were proposed, in addition to those involving D-aftro-heptulose, to account for these results.24(b) (o) 200... 

From the reaction of D-ribose-J-14C with secondary amine salts in aqueous acid, Peer and van den Ouweland215 isolated 4-hydroxy-5-methyl-3(2H)-furanone (120) (11.4%), and found it to be labeled entirely at the methyl carbon atom. Thus, in the presence of amines, the formation of 120 must proceed through the 1-deoxydiulose by the mechanism described in Section II (see p. 168). In contrast, the reaction of D-ribose-J-14C 5-phosphate218 gave 120 having no radioactivity in the methyl carbon atom, from which it was concluded that the methyl carbon atom originates from C-5 of the D-ribose. The... 

Recently, Gleason and Barker studied the behavior of D-ribose in aqueous potassium hydroxide under aerobic and anaerobic conditions (15). They found that the D-arabinose formed from D-ribose-2-f contains a substantial amount of radioactivity at C-l and concluded that rearrangement of D-ribose occurs principally by the hydride-transfer mechanism. 

Carbon shuffling. Ribose 5-phosphate labeled with Cat C-1 is added to a solution containing transketolase, transal dolase, phosphopenlose epimerase, phosphopentosc isu-merase, and glyceraldehyde 3-phosphate. What is the distribution of the radioactive label in the erythrose 4-phosphate and fructose 6-phosphate that are formed in this reaction mixture ... 

Studies on the biogenesis of cordycepin with labeled precursors demonstrated (375,376) that glucose-7- C and glucose-6- C, as well as adenine-8- C, adenosine-and formate- C, were incorporated into cordycepin, whereas ribose-/- C was essentially unutilized. Determining the radioactivity distribution led to the conclusion that cordycepin is generated from adenosine, with retention of the glucoside bond intact. 

In this case, we shall have the transfer of a radioactive atom (represented by bold letters) from substrate A to product P. If the nucleotide in the adenine, or the ribose portion of ATP, is labeled with we shall have the transfer of a radioactive atom from A to product Q. On the other hand, if the C label is in the fmctose part of fmctose-6-P or label in the 6-phosphate of fmctose-6-P, we shall have the transfer of a radioactive atom from B to product P. Most isotope exchange studies are conducted between A and Q, or B and P. 

Fig. 1. Size exclusion chromatography of ADP-ribose made in nucleotide permeable SVT2 cells [1] and purified as described somewhere else [4], using one Bio Sil TSK-125 column. The running buffer used was 0.1 A/ sodium phosphate, pH 6.8 at a flow rate of 1.0 ml min". 0.5 ml fractions were collected. The arrows indicate void volume and included volume, respectively. — Radioactivity profiles -absorbance profile...

Fig. 4A-D. Digestion of poly(ADP-ribose) synthetase with chymotrypsin or papain after modification of 3-(bromoacetyl)pyridine in the presence and absence of NAD. The enzyme sample (25 jug), chemically modified in the presence ( ) and absence (O) of NAD and labeled with NaB H4, was digested with 0.5 Mg of a-chymotrypsin (left) or 0.5 jug of papain (right) and subjected to SDS gel electrophoresis. A, C Quantification of the radioactivity in SDS-gel B, D the difference between the values denoted by and O in A and C was replotted...

Fig. 5. Digestion of automodified poly(ADP-ribose) synthetase with a-chymotrypsin and with papain. The enzyme (40 Mg) was automodified with 1.4 idM [adenine- ]- C]NAD (423 dpm/ pmol) and then digested with oi-chymotrypsin or further with papain. The radioactivity was detected by fluorography. a The automodified enzyme b the enzyme after digestion with a-chymotrypsin c the fragment obtained by digestion with papain after chymotryptic digestion...

The hen liver nuclei contain both ADP-ribosyltransferase and poly(ADP-ribose) synthetase. To separate the ADP-ribosyltransferase from poly(ADP-ribose) synthetase, the 0.6 M potassium chloride extract from the nuclei was applied to a Sephadex G-200 column and eluted with the 0.1 Af Tris-buffer, pH 8.0. Each fraction was incubated with 1 vaM [adenine- H]NAD and 100 jug of whole histones, in a total volume of 0.2 ml containing 50 mAf Tris-Cl" buffer, pH 9.0, and the radioactivity in the acid-insoluble fraction was determined. The result shows the two fractions containing the enzyme activities which catalyze the incorporation of the ADP-ribose moiety from NAD to the whole histones. From the hydroxyapatite column chromatographic analysis of the products formed by the respective fraction, we found that the former fraction contains poly(ADP-ribose) synthetase and the latter fraction contains poly-... 

ADP-ribose) synthetase and the latter fraction involves ADP-ribosyltransferase. To clarify that the radioactive compound formed by the latter fraction was indeed the mono(ADP-ribose) molecule, the acid-insoluble reaction product was treated with alkali at 37°C for 2 h. The radioactive material solubilized was adjusted to pH 7.0 and subjected to high performance liquid chromatography with reverse phase column. The eluate was monitored by UV and fractionated and radioactivity of the fraction was measured. The retention time of the radioactive product coincided with that of authentic mono(ADP-ribose). Furthermore, by treatment with snake venom phosphodiesterase this radioactive peak, tentatively considered to be ADP-ribose, migrated to the position corresponding to the 5 -AMP. These results indicate that hen liver nuclei contain ADP-ribosyltransferase. We purified this enzyme to a homogeneous state through salt extraction, gel filtration, hydroxyapatite, phenyl-Sepharose, Cm-cellulose, and DNA-Sepharose [3]. 

With prolonged incubation, we detected that 1 mol of ADP-ribose can be incorporated per mole of histone HI. [ PJADP-ribosylated histone HI thus prepared was treated with N-bromosuccinimide and then subjected to gel filtration. The radioactivity was recovered in the fraction corresponding to the N2 fragment reported by Bustin and... 

Cole [6]. The N2 fragment is known to contain the cAMP-dependent phosphorylation site, serine residue 38 [7]. We confirmed this with cAMP dependency phosphoiylated and subsequently N-bromosuccinimide-treated histone HI. The [ P]ADP-ribosylated N2 fragment was further treated with three peptidases in the order of cathepsin D, aminopeptidase M, and carboxypeptidase B, and the product was analyzed by high performance liquid chromatography. The radioactive product was identified as ADP-ribose-arginine adduct [8]. 

The measurement of ADP-ribosyltransferase activity in situ, either in nuclei or in permeabilised cells is a procedure simple in execution but fraught with difficulties of interpretation. The incorporation of radioactivity from labelled NAD into TCA insoluble products represents at best the net rate of ADP-ribosylation [ADPRT activity less poly(ADP-ribose) gjycohydrolase and ADP-ribosylprotein lyase activities]. At zero time we may hope that the degradative enzymes, having no radiolabelled substrate, do not contribute to the rate. 

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