trans-N-Deoxyribosylase (EC 2.4.2.6) is usually considered as a single protein catalyzing indifferently the transfer of the deoxyribosyl moiety to and from a purine or a pyrimidine base. Affinity chromatography of an extract from Lactobacillus helveticus with two types of ligands allowed the separation and purification of two distinct trans-N-deoxyribosylases. One catalyzes specifically the deoxyribosyl transfer to and from purine bases exclusively : trans-N-deoxyribosylase-I, the other catalyzes the transfer to and from pyrimidine and purine bases : trans-N-deoxyribosylase-11. A Tris inhibition study showed a markedly different susceptibility of the two enzymes. Preliminary results indicate that the purine-specific enzyme is a polymeric enzyme of molecular weight 86000 (f. 4000).In some Lactobacilli a trans-N-deoxyribosylase catalyses the transfer of the deoxyribosyl moiety from one purine or pyrimidine base to another [I]. dRib-Pur + Pur'gdRib-Pur' + Pur (PurePur) dRib-Pur + PyredRib-Pyr + Pur (Pur=Pyr) dRib-Pyr + Pyr'edRib-Pyr' + Pyr (PyrePyr) For short these three transfer reactions will be referred to as (PurePur), (Pur+Pyr) and (PyrePyr) transfers respectively.Early studies of the enzyme from Lactobacillus helveticus [l] have shown that the transfer reaction does not require the presence of phosphate ions. On the other hand Tris was found to have a partial inhibitory action [2]. For a Tris concentration of 0.1 M, (PurGPyr) and (PyrePyr) transfers could be completely inhibited whereas higher concentrations of Tris resulted in a maximum inhibition of 30 % for the (PurePur) transfer. These observations led Roush and Betz to suggest the presence of two distinct proteins : one responsible for the (Pur+Pur) transfer not inhibited by Tris and the other (inhibited by Tris) catalyzing the ( P u r e Pyr) and (PyrePyr) transfers and possibly also the (PurePur) transfer.Abbreviations. Abbreviations follow CBN recommandations, see Eur. J . Biochem. 15, 203-208 (1970): Pur, a purine base; Pyr, a pyrimidine base; (dIno + Ade) denotes a transfer in which deoxyinosine and adenine are the substrates. SE-, sulfoethyl; CM-, carboxymethyl.Enzymes. trans-N-Deoxyribosylase or purine(pyrimidine) nucleoside : purine(pyrimidine) deoxyribosyl transferase (EC 2.4.2.6); xanthine oxidase or xanthine : oxygen oxidoreductase (EC 1.2.3.2).

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A series of purine bases and analogues were tested as substrates for trans-N-deoxyribosylase (EC 2.4.2.6). It was observed that the pyrimidine ring and its substituents on positions 1 , 2 and 6, are of minor importance. On the other hand only a few modifications are tolerated on the imidazole moiety, as follows.1. A tautomeric proton must be present on the imidazole ring. The "usual" shift is between position 9 and 7.2. The position of the tautomeric proton governs the site of substitution. 3 . For steric reasons no substituent is allowed on position 8.The direct transfer of the deoxyribosyl moiety from a purine (or a pyrimidine) deoxyribonucleoside to a purine (or pyrimidine) base is catalyzed by trans-N-deoxyribosylase [l]. The presence of this enzyme seems to be limited to a few bacteria species in the Lactobacillus genus. In a previous report it was shown that trans-N-deoxyribosylase from Lactobacillus helveticus is made up of at least two different enzymes [2]. trans-N-Deoxyribosylase-I specifically catalyzes the transfer between purine bases. trans-N-Deoxyribosylase-I1 catalyzes the (Pur $ Pyr) transfer. Since this transfer proceeds via a ping-pong bi-bi mechanism it is expected that the (Pur z $ Pur) and (Pyr + Pyr) transfers will also be observed. Information concerning the specificity of trans-N-deoxyribosylase appears in a number of reports, some of which are relevant to the present study [1,. In spite of a few conflicting results the broad specificity of transAbbreviutions. Pur, a purine base; Pyr, a pyrimidine base; (Pur + Pyr) is a transfer reaction where the deoxyribosyl moiety is transferred from a purine to a pyrimidine ; deoxyribonucleosides are abbreviated as recommended by IUPAC-IUB; (dIno + Ade) denotes a transfer in which deoxy inosine and adenine are the substrates.Enzyme. trans-N-Deoxyribosylase or nucleoside : purine (pyrimidine) deoxyribosyl transferase (EC 2.4.2.6).N-deoxyribosylase for the acceptor base is unanimously accepted. Using a pyrimidine deoxyribonucleoside as donor and a purine base as acceptor we were able to study the specificity of the acceptor base for trans-N-deoxyribosylase-11. In the present report we make a first attempt to give the structural requirements necessary for a purine base to be a competent acceptor in the (dRib-Pyr --+ Pur) transfer. MATERIALS AND METHODS ReagentsSome of the purine bases used in this study were commercial products from Aldrich, Sigma, Fluka, Cyclo, Schwarz-Mann or Calbiochem. The purity was checked by : (a) thin-layer chromatography on cellulose MN 300 (Macherey & Nagel) using six different solvents [12]; (b) absorption spectroscopy in the ultraviolet or visible range when the spectrum could be found in the literature for comparison [13].When a base gave several spots by thin-layer chromatography a purification was attempted by Eur.

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purity than those obtained by other means. Preliminary experiments with other plant ribonucleases suggest that the media described will be of general application and that, when used in combination with other nucleotide-containing media, they should simplify the search for base-specific nucleases.