Nicotinate Phosphoribosyltransferase of Yeast: PURIFICATION AND PROPERTIES

Abstract

Abstract Nicotinate phosphoribosyltransferase from bakers' yeast has been purified about 1,000-fold; the purified material showed only one band on cellulose acetate electrophoresis (pH 5.0 and 8.6). In addition to 5-phosphoribosyl 1-pyrophosphate, adenosine triphosphate (ATP) or an analogous nucleotide is required for enzyme activity and the stoichiometric hydrolysis of ATP to adenosine diphosphate (ADP) and orthophosphate with formation of products, nicotinic acid mononucleotide and pyrophosphate, was verified with this purified preparation. The nucleotide specificity, however, is broader than that of the liver form of this enzyme. The molecular weight of the yeast enzyme was estimated from Sephadex chromatography to be 43,000 (about one-half that of the liver enzyme) whether in the presence or absence of ATP. Phosphate ions greatly stimulate the enzyme activity, giving optimum activity in the range of 10 to 100 mm phosphate in the presence of 5 mm Mg++. At 1 and 5 mm Mg++, optimum enzyme activities are observed with equimolar concentration of ATP. The enzyme catalyzes isotope exchange between ATP and ADP at rates approximately 8 times its molecular activity with all substrates, although no net ATP hydrolysis occurs in the absence of other substrates. ATP, guanosine triphosphate, and the substrates, nicotinic acid and 5-phosphoribosyl 1-pyrophosphate, protect the enzyme from heat inactivation.