The transport and utilization of purine and pyrimidine nucleosides and bases by germinating conidia of Neurospora crassa

Abstract

The relationship between intracellular enzymes involved in the utilization of uracil, uridine, and adenosine and the transport of these compounds by germinating conidia of Neurospora crassa was investigated. Crude extracts of the uracil transport-deficient strain uc-5, pyr-1 were shown to have uracil phosphoribosyltransferase (UPRTase) activity comparable to the wild-type strain, suggesting that uracil uptake in Neurospora does not occur by a group translocation mechanism involving phosphoribosylation. Specificity studies of UPRTase and uracil uptake indicated that phosphoribosylation was not an important determinant of the specificity of uracil uptake, as the specificity of uracil uptake was much broader than the specificity of UPRTase. This adds additional support for the independence of uracil uptake and phosphoribosylation in Neurospora. Similarly, crude extracts of the nucleoside transport deficient strain, ud-1, pyr-1 were shown to have both uridine and adenosine kinase activities which were comparable to these activities in crude extracts of wild-type conidia. Evidence that the ud-1 phenotype, expressed by concomitant loss of purine and pyrimidine nucleoside transport, is the result of a single mutational event is presented and provides additional support for the existence of a single nucleoside transport system in Neurospora. Resistance to the antibiotic, tubercidin (7-deazaadenosine), coupled with the inability to utilize uridine, proved to be an effective method for screening for nucleoside transport deficiency in pyrimidine auxotrophs. The separation of adenosine and uridine kinase activities by ammonium sulfate fractionation and the determination of the Km for adenosine of adenosine kinase (0.26 μM), which is significantly lower than the Km for adenosine of the nucleoside transport systems (8 μM), provides additional support for the independence of the phosphorylation of nucleosides and the transport of nucleosides in Neurospora...