Direct assay method for guanosine 5'-monophosphate reductase activity.
A sensitive and simple micromethod for the accurate measurement of GMP reductase (EC 1.6.6.8) activity in crude extracts is described. The reaction product of [8-14C]IMP was separated from the substrate [8-14C]GMP by descending chromatography on Whatman DE81 ion-exchange paper. This separation method provides an analysis of the possible interfering reactions, such as the metabolic conversion of the substrate GMP to GDP, GTP, and/or guanosine, and guanine and the loss of the product IMP to inosine, hypoxanthine, and other metabolites. Low blank values (70-90 cpm) were obtained consistently with this assay because the IMP spot moves faster than the GMP spot. The major advantages of this method are direct measurement of GMP reductase activity in crude extracts, high sensitivity (with a limit of detection of < 10 pmol of IMP production), high reproducibility (< +/- 5%), and capability to measure activity in small samples (9 micrograms protein).
Nakamura
H
H
Laboratory for Experimental Oncology, Indiana University School of Medicine, Indianapolis 46202-5200.
Natsumeda
Y
Y
Nagai
M
M
Shiotani
T
T
Weber
G
G
D000818:Animals; D001921:Brain / Q000201:enzymology; D050899:GMP Reductase; D006147:Guanine / Q000302:isolation & purification / Q000378:metabolism; D006151:Guanosine / Q000302:isolation & purification / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000302:isolation & purification / Q000378:metabolism; D006157:Guanosine Monophosphate / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000302:isolation & purification / Q000378:metabolism; D007288:Inosine / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008832:Microchemistry / Q000379:methods; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D007042:Hypoxanthines; D006151:Guanosine; D006153:Guanosine Diphosphate; D019271:Hypoxanthine; D009249:NADP; D007288:Inosine; D006147:Guanine; D006157:Guanosine Monophosphate; D009247:NADH, NADPH Oxidoreductases; D050899:GMP Reductase