Characterization of guanine and hypoxanthine phosphoribosyltransferases in Methanococcus voltae.
Phosphoribosyltransferase (PRTase) and nucleoside phosphorylase (NPase) activities were detected by radiometric methods in extracts of Methanococcus voltae. Guanine PRTase activity was present at 2.7 nmol min(-1) mg of protein(-1) and had an apparent Km for guanine of 0.2 mM and a pH optimum of 9. The activity was inhibited 50% by 0.3 mM GMP. IMP and AMP were not inhibitory at concentrations up to 0.6 mM. Hypoxanthine inhibited by 50% at 0.16 mM, and adenine and xanthine were not inhibitory at concentrations up to 0.5 mM. Guanosine NPase activity was present at 0.01 nmol min(-1) mg of protein(-1). Hypoxanthine PRTase activity was present at 0.85 nmol min(-1) mg of protein(-1) with an apparent Km for hypoxanthine of 0.015 mM and a pH optimum of 9. Activity was stimulated at least twofold by 0.05 mM GMP and 0.2 mM IMP but was unaffected by AMP. Guanine inhibited by 50% at 0.06 mM, but adenine and xanthine were not inhibitory. Inosine NPase activity was present at 0.04 nmol min(-1) mg of protein(-1). PRTase activities were not sensitive to any base analogs examined, with the exception of 8-azaguanine, 8-azahypoxanthine, and 2-thioxanthine. Fractionation of cell extracts by ion-exchange chromatography resolved three peaks of activity, each of which contained both guanine and hypoxanthine PRTase activities. The specific activities of the PRTases were not affected by growth in medium containing the nucleobases. Mutants of M. voltae resistant to base analogs lacked PRTase activity. Two mutants resistant to both 8-azaguanine and 8-azahypoxanthine lacked activity for both guanine and hypoxanthine PRTase. These results suggest that analog resistance was acquired by the loss of PRTase activity.
Bowen
T L
TL
Department of Microbiology, University of Georgia, Athens 30602-2605, USA.
Lin
W C
WC
Whitman
W B
WB
D001375:Azaguanine / Q000494:pharmacology; D006147:Guanine / Q000494:pharmacology; D006157:Guanosine Monophosphate / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D019271:Hypoxanthine; D007041:Hypoxanthine Phosphoribosyltransferase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D007042:Hypoxanthines / Q000494:pharmacology; D007291:Inosine Monophosphate / Q000494:pharmacology; D007700:Kinetics; D017017:Methanococcus / Q000201:enzymology* / Q000235:genetics; D009154:Mutation; D010430:Pentosyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D011683:Purine-Nucleoside Phosphorylase / Q000378:metabolism
D007042:Hypoxanthines; D007291:Inosine Monophosphate; C000149:8-azahypoxanthine; D019271:Hypoxanthine; D006147:Guanine; D006157:Guanosine Monophosphate; D010430:Pentosyltransferases; C028449:xanthosine phosphorylase; D011683:Purine-Nucleoside Phosphorylase; C024827:xanthine phosphoribosyltransferase; D007041:Hypoxanthine Phosphoribosyltransferase; D001375:Azaguanine