FIG. 4.

E-NPP activity depends on NPP1 and NPP2. (A) E-NPP activity was measured in strains lacking NPP1 and/or NPP2. Nucleotide phosphate hydrolysis was measured by a coupled enzyme assay where ATP hydrolysis was monitored as a function of oxidation of NADH. Cultures were grown over a 24-h time course in regular or phosphate-depleted medium. Lack of both genes caused a significant deficiency in E-NPP activity under low-phosphate conditions. (B) Protein expression levels were monitored over a 24-h time course. Cellular lysates were prepared at each time point, and TAP fusion proteins were detected from whole-cell lysates through immunoblotting. Protein expression of Npp1p and Npp2p increased during log-phase growth, and this expression was enhanced for cells grown under low-phosphate conditions during the same phase of growth. Detection of the constitutively expressed Nap62p was used a protein-loading control.