Synthesis of (14C)-Ribose-5-Phosphate and (14C)-Phosphoribosyl-Pyrophosphate and Their use in New Enzyme Assays

Biochemical characterization of the Mycobacterium tuberculosis phosphoribosyl-1-pyrophosphate synthetase

“…A coomassie-stained 12% SDS–PAGE analysis of Mt-PrsA revealed a band that migrated to a position of 35 kDa, which is in accordance with the expected molecular weight of Mt-PrsA, and was estimated to be at least 98% pure. To determine if recombinant Mt-PrsA was active, we performed a radiochemical assay to measure the pyrophosphate transfer from ATP to the C1–OH group of [ 14 C]-uniformly labeled R5P (Boss et al 1984). Starting with [ 14 C]-U- d -glucose and replicating the first three enzymic reactions of the pentose phosphate pathway in vitro using commercially available enzymes (Roche and Sigma), we prepared a source of purified [ 14 C]-U-α- d -R5P as a substrate for subsequent enzyme assays.…”

“…We prepared a source of [ 14 C]-R5P using a modified process as described in Boss et al (1984), for the subsequent use in Mt-PrsA assays. One hundred microcuries of [ 14 C]-U- d - glucose (100 µL in 70% ethanol; ARC Radiochemicals) was dried in a 14 mL glass tube under compressed nitrogen and then resuspended in 500 µL of 50 mM KH 2 PO 4 (pH 7.9), 150 mM NaCl, 5 mM MgCl 2 and 0.5 mM MnCl 2 .…”