Radiometric measurement of phosphoribosylpyrophosphate and ribose 5-phosphate by enzymatic procedures.
Methods for the measurement of phosphoribosylpyrophosphate (PRPP) and ribose 5-phosphate (R-5-P) in tissues have been developed. The lability of these compounds during tissue extraction and the recovery of standards from tissue preparations have been examined. Enzymatic conversion of phosphoribosylpyrophosphate to [14C]AMP in the presence of labeled adenine or formation of [14C]GMP ([14C]IMP) in the presence of labeled guanine or hypoxanthine was accomplished in the first step. In the second step, the labeled product was separated from the substrate. For the measurement of R-5-P, the first step included phosphoribosylpyrophosphate synthetase, as well as the appropriate substrate and effector (ATP and Pi), in combination with adenine phosphoribosyl transferase. The product [14C]AMP was measured in three ways: (1) HPLC separation with an on-line radioisotope detector; (2) butanol extraction of the labeled base, and measurement of an aliquot of the aqueous phase in a scintillation counter; (3) filtration of the incubation mixture with chromatographic filter paper disks, which were then counted in a scintillation counter. When [14C]guanine was the substrate, HPLC separation was used because the butanol or paper separation was not adequate. Measurement of 5-125 pmol of PRPP or R-5-P gave a linear response.
National Institute on Alcohol Abuse and Alcoholism, Laboratory of Metabolism and Molecular Biology, Rockville, Maryland 20852.
M T
MT
King
J V
JV
Passonneau
R L
RL
Veech
D000228:Adenine Phosphoribosyltransferase / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002854:Chromatography, Paper; D004795:Enzyme Stability; D007041:Hypoxanthine Phosphoribosyltransferase / Q000378:metabolism; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D008297:Male; D009206:Myocardium / Q000378:metabolism; D010428:Pentosephosphates / Q000032:analysis*; D010754:Phosphoribosyl Pyrophosphate / Q000032:analysis*; D011874:Radiometry; D051381:Rats; D011919:Rats, Inbred Strains; D012267:Ribosemonophosphates / Q000032:analysis*; D012588:Scintillation Counting
D010428:Pentosephosphates; D012267:Ribosemonophosphates; C031626:ribose-5-phosphate; D010754:Phosphoribosyl Pyrophosphate; D000228:Adenine Phosphoribosyltransferase; D007041:Hypoxanthine Phosphoribosyltransferase