Purine and Pyrimidine Nucleotide Synthesis and Metabolism

Abstract

This chapter describes the purification procedure of enzyme adenosine deaminase from Escherichia Coli. Adenosine deaminase is an inducible enzyme that functions in a pathway that converts adenine, adenosine, and deoxyadenosine to guanine nucleotides. Addition of adenine, hypoxanthine, and their nucleosides to the cultures of E. coli induces the synthesis of adenosine deaminase. The continuous spectrophotometric assay is based on the decrease in optical density at 265 nm due to the removal of adenosine and the formation of inosine. In this assay, the adenosine deaminase activity is measured as the amount of [14C]inosine formed from [14C]adenosine with time. The assay is based on a chromatographic separation of adenosine from inosine. Because the chromatographic system also separates adenine and hypoxanthine from adenosine and inosine, a possible cleavage of adenosine and inosine by purine nucleoside phosphorylase can be determined. The adenosine deaminase preparation obtained by this procedure is about 90% pure as judged by analytical polyacrylamide gel electrophoresis.