A possible role of purine nucleotide pyrophosphorylases in the regulation of purine uptake by Bacillus subtilis.

The uptake of radioactive purines in resting cell suspensions of Bacillus subtilis has been studied. Initial rates of uptake obeyed saturation kinetics (e.g. apparent Km adenine, 0.6 PM). Subsequent uptake proceeded in steplike increments. Further studies were aimed at delineating the control mechanism for this uptake. Purines were highly concentrated with respect to the medium, but the intracellular concentration was not significantly different whether uptake had reached a plateau or was rising steeply. Over 95% of the label was present as nucleotides. A dual isotope method developed for determination of specific activities in small amounts of material suggested that the intracellular adenine pool was compartmentalized with respect to the formation of nucleotide. Uptake varied independently of nucleic acid synthesis suggesting that the magnitude of the labeled pool was not controlled directly by polymer synthesis. Evidence that the enzymes purine nucleotide pyrophosphorylase (EC 2.4.2.7, S), which catalyze the initial reaction leading from free purine to nucleotide, may control uptake has been obtained. The relative uptake of different purines was proportional to the relative activity of this enzyme with respect to the same purines. The reaction was measured with approximately physiological substrate concentrations. A variety of specific nucleotide feedback inhibitors were shown to exist. By feedback, the level of such nucleotides would modify pyrophosphorylase activity and hence uptake. Under certain conditions, such a mechanism could lead, for example, to the steplike increments observed. The effects of a variety of inhibitors including actinomycin D and chloramphenlcol on uptake were also consistent with this mechanism. The relation of this enzyme control system to other aspects of the regulation of purine nucleotide biosynthesis is discussed.