ADDITIONAL CHARACTERIZATION OF ADENOSINE NUCLEOSIDASE FROM ALASKA PEA SEEDS

Abstract

Adenosine nucleosidase was purified from Alaska pea seeds six days after germination. A 3-fold purification has been reached with a 0.56 % recovery. The purification scheme involved ammonium sulfate precipitation between 30% and 60% saturation, followed by ion exchange chromatography on a DEAE column. A final chromatography step used a hydroxyapatite column. The subunit molecular weight of adenosine nucleosidase was determined by SDS-PAGE to be 26.7 kD. The Michaelis constant, Km, and the maximum velocity, V max, for inosine were determined to be 377.7 141.7 M and 0.00078 0.000106 M/min respectively.The substrate specificity of the enzyme was investigated using 2’-deoxyinosine, allopurinol riboside, 3-deazauridine, and adenine- β-arabinofuranoside. Based on the observed substrate specificity, adenosine nucleosidase from Alaska pea seeds hydrolyzes both purines and pyrimidines. While these results place adenosine nucleosidase in the non-specific inosine-uridine nucleoside hydrolases (IU-NH), significant differences in its substrate specificity compared to the lead enzyme of the class, (IU-NH) from Crithidia fasciculata were found.