Ribonucleoside separation

A wide variety of bases, nucleosides and nucleotides have been separated using porous layer bead ion exchangers. A representative chromatogram of the separation of ribonucleoside mono-phosphoric acids from the work of Smukler ( ) is shown in Figure 4. Recently, ion exchangers chemically bonded to small particle diameter (> 10 ym) silica have been successfully applied to the separation of nucleic acid constitutents (37). The rapid separations using such supports undoubtedly mean that they will find increasing use in the future. 

Figure 4. Separation of ribonucleoside monophosphofic acids. Conditions 250-cm anion exchange column gradient, 0.01M KHgPO containing HsPOi, (pH 2.6) to 0.15M KHiFO in 30 min column tempera-ture, 70 C detector, UV at 254 nm. 1, cyti-dine-S -monophosphoric acid 2, uridine-5 -monophosphoric acid 3, adenosine-5 -mon-ophosphofic acid 4, inosine-5 -monophosphoric acid 5, 3, 5 -cyclic adenosine mono-phosphoric add 6, guanosine-5 -monophosphoric acid (36).

Breter, H.-J., Seibert, G., and Zahn, R. K., Single-step separation of major and rare ribonucleosides and deoxyribonucleosides by high-performance liquid cation-exchange chromatography for the determination of the purity of nucleic acid preparations, ]. Chromatogr., 140, 251, 1977. 

In view of the difficulty of hydrolyzing the pyrimidine nucleosidic linkages, ribonucleic acids have been hydrolyzed to a mixture of purine bases and pyrimidine nucleotides which is then separated by paper chromatography.132, 163 164 This method has been employed extensively for the analysis of ribonucleic acids, and gives reproducible results,166 but it has not been used to any great extent for deoxyribonucleic acids, probably because, under these conditions of hydrolysis, they yield some pyrimidine deoxy-ribonucleoside diphosphates.166... 

Transfer RNA precursors may undergo further posttranscriptional processing. The 3 -terminal trinucleotide CCA(3 ) to which an amino acid will be attached during protein synthesis (Chapter 27) is absent from some bacterial and all eukaryotic tRNA precursors and is added during processing (Fig. 26-23). This addition is carried out by tRNA nucleotidyltransferase, an unusual enzyme that binds the three ribonucleoside triphosphate precursors in separate active sites and catalyzes formation of the phosphodiester bonds to produce the CCA(3 ) sequence. The creation of this defined sequence of nucleotides is therefore not dependent on a DNA or RNA template—the template is the binding site of the enzyme. 

These were differently affected by different procedures. For example, when the enzyme was activated at 55°, the increment in ki was slight, but k2 increased 3.5-fold. Similarly, in the presence of EDTA, fc, and k2 values decreased independently, suggesting that the sites for both activities were different. Center and Behai (5) found that with the P. mirabilis enzyme, cyclic 2, 3 -UMP competitively inhibited the hydrolysis of bis(p-nitrophenyl) phosphate. The Ki was 40 pAf very close to the Km for the cyclic nucleotide (Km, 75 yM) which indicated that the two compounds could serve as alternate substrates being hydrolyzed at the same active site. In contrast, 3 -AMP was a mixed inhibitor of cyclic 2, 3 -UMP and bis(p-nitrophenyl) phosphate hydrolysis. Adenosine was a mixed inhibitor of bis(p-nitrophenyl) phosphate hydrolysis but a competitive inhibitor of 3 -AMP hydrolysis. From such kinetic studies Center and Behai (5) suggested that two separate and adjacent sites A and B are involved in the hydrolysis of the diester and phos-phomonoester substrates. Site A serves as a binding site for hydrolysis of ribonucleoside 2, 3 -cyclic phosphates and together with site B catalyzes the hydrolysis of the diester bond. During this reaction 3 -... 

Solms and Deuel95 initially prepared a wholly synthetic, borylated polymer by using m-phenylenediamine, p-aminophenylboron dichloride, and formaldehyde, and they investigated carbohydrate separations on it, but addition polymers have usually been favored. Thus, ribonucleosides and deoxyribonucleosides have been efficiently separated on a column of a mixed copolymer of the methacroyl derivatives96 51 and 52, and the method has been extended to... 

Walseth et al. (W3) separated 5 -ribonucleoside monophosphates using ion-pair chromatography. The chromatognq>hic system has been used to analyze tissue nucleotides, to purify nucleoside monophosphates and cyclic nucleotides, and to monitor enzymatic reactions involving cyclic nucleotide phosphodiesterase, 5 -nucleotidase, and adenylate cyclase. 

Davis and co-workers (Dl) also analyzed enzyme hydrolysates of tRN A by RPLC. They report excellent separation and resolution of both the major and modified ribonucleosides however, enzyme hydrolysis may be less than desirable for quantitative release of ribonucleosides from tRN A. Figure 19 illustrates the tRN A hydrolysate separation of ribonucleosides in Hodgkin s tumor tRNA. 

Mundry (1965) has also described the use of DEAE-Sephadex for the separation of ribonucleotides and nucleosides. Using a 1.1 x85 cm column and a 1.1-1 linear gradient from 0.04 M Tris-HCl pH 8.8 to 0.20 M Tris-HCl, 0.25 M NaCl pH 9.5 or a linear gradient from 0.04 M triethylamine carbonate pH 8.8 to 0.35 M triethylamine carbonate, 0.15 M NaCl pH 9.5 he has separated the 8 main ribonucleosides and nucleotides. The nucleoside separation is very sensitive to traces of salt and some difficulty with reproducibility was experienced. 

Some ribonucleoside and nucleotide separations on 1.5x34 cm columns of DEAE-cellulose eluted with 0.09 M sodium acetate, 0.2 M acetic acid pH 4.4 have been described (Whatman Data Sheet DS/13). 

Stereodefined oligo(ribonucleoside phosphorothioate)s were available only by the enzymatic method [51 -54] or by partially stereocontrolled H-phosphonate procedure [55,56]. However, both procedures could provide only products with RP configuration. Alternatively, compounds containing the single phosphorothioate modification were synthesized by nonstereospecific phosphoramidite or H-phosphonate methods and then separated into diastereomers by tedious HPLC procedure with various degree of success [57-59]. Such constructs became important tools for investigation of the mechanism of action of ri-bozymes. 

CPMAS solid-state NMR has been used to investigate the dependence of sugar chemical shifts on specific conformational parameters of a variety of ribonucleotides and ribonucleosides. The distinct effects of sugar puckering on the Cl, C4, and C5 resonances of Cj endo (S type) and C3 endo (N type) furanoid conformations allow us to separate them into two groups. 

There are three distinct PRTases in L. donovani (30). One has its major activity with hypoxanthine and guanine a second with adenine and a third with xanthine. Pyrazolo (3,4-d)pyrimidines, such as allopurinol, are efficient substrates for the HGPRtase. Promastigotes accumulate large quantities of allopurinol ribonucleoside-5 -phosphate when exposed to allopurinol (31). A separate PRTase for xanthine is unusual in a eukaryotic cell. XPRTase is also present in L. mexicana and L. amazonensis as well as in four non-pathogenic trypanosomatids (32,33). 

Chain initiation requires formation of the open complex. Recent studies show that a portion of the P and the a-subunits initiate strand separation, melting about 14 base pairs surrounding the transcription start site. A purine ribonucleoside triphosphate is the first base in RNA, and it binds to its complementary DNA base at position +1. Of the purines, A tends to occur more often than G. This first residue retains its 5 -triphosphate group (indicated by ppp in Figure 11.3). 

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