Nucleosides, exchange labeling

The assay is carried out both at 1 jaM and 1 mM substrate concentrations [162]. The enzyme preparation is incubated with tritium-labelled cyclic nucleotide in the presence of Mg, the reaction is stopped by brief heating in a water bath, the 5 -nucleotide formed is converted to the nucleoside by snake venom 5 -nucleotidase, the nucleoside is separated from the remaining cyclic nucleotide by anion exchange chromatography (Dowex 2), and the radioactivity of both compounds is counted. 

Photochemical cross-linking studies of ribosomal RNA and a tRNA derivative have been achieved using the 5 -[Y- P]-labelled 3 , 5 -bisphosphate of 2, 6-diazido-9-(P-D-ribofuranosyl)purine. The compound was prepared by reaction of the corresponding nucleoside with pyrophosphoryl chloride followed by exchange of the 5 -phosphate with the y-phosphate of [ y- 2p]j jp catalysed by T4 polynucleotide kinase. It was then enzymatically ligated to the 3 -terminus of a yeast tRNA he molecule. 

Exchange of tritium for hydrogen by tritiated biomolecules and s >sequent loss of THO into the environment was examined by incubating the labeled biomolecule and scintillator in a sealed film tube at ambient temperature. The sanples (in triplicate) were then counted at timed intervals. Three tritiated nucleosides were compared in this manner, adenosine (2,8-3h) from New England Nuclear (Boston, MA.) and guanosine (8-3h) and uridine (5-3h) both from ICN (Irvine, CA.). 

Adenine is inert in the nucleoside phosphorylase systems of both mammalian tissues and microorganisms, but isotopically labeled adenine is effectively incorporated into nucleic acid purines, both in rats " and in yeast.This poses a question as to the possible role of nucleoside phosphorylase in polynucleotide synthesis. It has been suggested that hypoxanthine or guanine nucleosides (or nucleotides) are synthesized first. Then an exchange reaction with free adenine (or a derivative) might occur, For example, adenine might react with inosine to form adenosine and hypoxanthine. Some known exchange reactions are discussed below. 

Rabbit livers, perfused free of blood, were then labeled by perfusion, in situ, via the portal vein, with a suitable purine nucleotide precursor tritium labeled adenine or hypoxanthine. The livers were then perfused, with an isotonic solution containing a carrier nucleoside, usually unlabeled adenosine Hepatic venous effluent was collected, during five minute periods, for twenty minutes. Cold dilute perchloric acid extracts were prepared and the purine components eluted by ion exchange chromatography on Dowex 50. The data obtained, following labeled adenine perfusion and subsequent perfusion with unlabeled adenosine are presented in Figure 1. 

Compound classes labeled by heterogeneous metal-catalyzed tritium exchange from water include amino acids, peptides and proteins, sugars, polycyclic aromatic hydrocarbons, purines, pyrimidines and nucleosides, alkyl-substituted aromatics, alicyclic compounds and steroids, A-heterocycles and anilines and some O- and 5-heterocycles. The method is firmly fixed in the repertoire of methods of the commercial custom labeling industry. 

---