Ribose-4-phosphate, chromatography

TALDO deficiency can be confirmed in lymphoblasts, fibroblasts and in erythrocytes. These cells are incubated with ribose-5-phosphate, after which formation of transketolase and TALDO products are analysed by gas chromatography with nitrogen phosphorous detection by liquid chromatography tandem mass spectrometry [8, 11]. A similar enzyme assay is available for RPI [2]. Confirmation of the gene defect can be performed by sequence analysis. Disease-causing mutations have been detected in all TALDO-deficient patients and in the RPI-deficient patient. 

The third enzyme in the pathway, KD0-8-phosphate phosphatase, has been purified to homogeneity (26). Because of its abosolute specificity, it should be a focal point for chemotherapeutic studies. jThe apparent for KD0-8-phosp te was+ etermined to be 5.8 x 10 M in the presence of 1.0 mM Co or Mg. This specific KD0-8-phosphate phosphatase was separated from enzymes, present in crude extracts, having phosphatase activity on other phosphorylated compounds by column chromatography on DGAE-Sephadex (26). Three distinct peaks of activity were detected. Fractions from each peak were pooled and the rates for the hydrolysis of five compounds were measured. Peak A possessed phosphatase activity for D-glucose-6-phosphate, D-arabinose-5-phosphate, D-ribose-5-phosphate and j-nitrophenylphosphate Peak B dephosphorylated D-arabinose-5-phosphate, D-ribose-5-phosphate and D-glucose-6-phos-phate. Peak C, which was well separated from the other two peaks, could only utilize KD0-8-phosphate as a substrate. KD0-8-phos-phate was not hydrolyzed by the phosphatases present in peaks A and B. 

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...

Amino acid analysis of a strong acid hydrolysate (2 N HCl at 100° C for 3 h) identified only glutamic acid and this was confirmed by gas chromatography-mass spectroscopy (Fig. 4). Mild acid hydrolysis (0.5 N HCl at 80°C for 1 h) followed by direct insertion mass spectroscopy gave a spectrum identical to authentic ribose-5-phosphate (Fig. 5). Borate electrophoresis of the alditol derived from the stored material and of its acid... 

Fig. 1A-C. Chromatographic analysis of (ADP-ribose)n residues associated with HMG proteins and histone HI isolated from control and 3-ABm treated cells. Radioactive material (fraction V, control) was treated with 0.3 N NaOH for 1 h at 0°C (A). Fraction V [control (B) and 3-ABm (C)] was digested with snake venome phosphodiesterase for 3 h at 37°C. The hydrolytic product was analyzed by cellulose thin layer chromatography, a solvent system containing 0.1 A/ sodium phosphate buffer (pH 6.8) ammonium sulfate, and -propanol (100 60 2, v/w/v)...

N.a. are purified by column chromatography (e.g. methylalbumin-silicic acid columns), by electrophoresis (e.g. polyacrylamide gels) and by density gradient centrifugation (e.g. sucrose or CsCl). A N.a. may be determined quantitatively from the UV absorption of its bases, by determination of the phosphate content, or by specific color reactions for ribose... 

Bull seminal plasma also contains 5-nucleotidase activity. The enzyme has been purified from this source and found to split adenosine-5 -phosphate, uridine-5 -phosphate, cytidine-5 -phosphate, guanosine-5 -phosphate, and nicotinamide-ribose-5 -phosphate. Of several dozen other esters, only the desoxyribonucleotides have been found active as substrates. Snake venom contains a highly specific 5-nucleotidase which can be separated from phosphodiesterase by means of cellulose column chromatography. ... 

A mixture of pentose phosphates has been found among the reaction products of phosphogluconate degradation. The first analysis of these products by Scott and Cohen following the use of the enzyme preparation of Dickens revealed a small proportion of ribose-5-phosphate, the pentose of which was identified by paper chromatography and adaptive enzymic analysis. However, most of the pentose phosphate which was isolated on paper and subsequently as the free sugar was a previously uncharacterized substance. Horecker and Smyrniotis used a considerably purified enzyme preparation, and pentose phosphate was produced under conditions in which the following stoichiometry prevailed ... 

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