Superiority of HPLC to Assay for Enzymes Regulating Adenine Nucleotide Pool Intermediates Metabolism: 5'-Nucleotidase, Adenylate Deaminase, Adenosine Deaminase, and Adenylosuccinate Lyase-A Simple and Rapid Determination of Adenosine

Abstract

A new application of HPLC analysis to assay for all the enzymes involved in regulation of adenine nucleotide pool metabolism is described. In vitro, enzymatic reactions were carried out in buffered reaction media containing appropriate concentrations of metal ions, specific substrate, inhibitors and enzymes. Following an incubation period, the enzymatic reactions were terminated and extracted with cold trichloroacetic acid. The soluble acid extracts were neutralized and injected in a HPLC system. Using a C18-Nova Pak reverse phase column, we were able to separate, identify and quantify the substrate, products and their possible catabolites and UV-detectable inhibitors. A complete separation and quantitation of metabolites was accomplished within 16-18 minutes. However, rapid and simple HPLC runs were also developed which can be routinely used to determine adenosine levels within 3-4 minutes, using a single solvent HPLC system. This procedure is extremely reproducible and very reliable as demonstrated in assaying for 5'-nucleotidase, adenylate deaminase, adenosine deaminase and adenylosuccinate lyase activities.