Isolation of Nucleoside Metabolizing Enzymes from Alaska Pea Seeds (Pisum sativum L. cultivar Alaska)

Abstract

The activities of number of metabolizing enzymes were determined from dry Alaska Pea Seeds (Pisum sativum L. cultivar Alaska). Then tested for presence of three groups of enzymes; nucleosidase, deaminases and phosphorylases. The highest activity of the nucleosidases was cytidine nucleosidase with an activity of 4.82 μmole/min and the lowest activity was 0.97 μmol/min inosine nucleosidase. In addition, deaminase activities in dry Alaska pea seeds were tested using adenine, adenosine, and cytidine as substrates. Deaminase activities of 0.70, 4.37, 4.82 μmole/min respectively were found for those substrates. However, no cytosine deaminase was detected in dry Alaska pea seeds. Phosphorylase activities for adenosine, guanosine, and cytidine were observed in ungerminated seeds with activities 0.71, 0.51, and 0.39 μmole/min respectively. In all cases tested the phosphorylase activities were lower than the corresponding nucleosidases.

Adenosine nucleosidase, an enzyme of the purine metabolic pathway, hydrolyzes adenosine to adenine and pentose sugar. It is involved in controlling the level of cytokinins, cell differentiation, division, and development in plants. Adenosine deaminase, also an enzyme of the purine metabolic pathway, catalyzes adenosine or to inosine.

Adenosine nucleosidase was purified from Alaska pea seeds 5 days after germination. The specific activity of the initial extract was 5810-4 to 560-3 μmole/min/mg. Dialysis of the initial extract increased the specific activity to 6810-4 μmole/min/mg. The first step of purification was ammonium sulfate precipitation. The purity of the enzyme was increased 2.4 fold to a specific activity of 1410-3 μmole/min/mg. The second step, ion-exchange chromatography (DEAE), increased purity 4-fold with a specific activity to 24 10-3 μmole/min/mg. The third purification step was ω-aminohexyl agarose chromatography, with a 4.2-fold increase to a specific activity 2510-3 μmole/min/mg. The final step, Sephacryl S100 chromatography, increased purification-fold to 44 with a specific activity to 2610-2 μmole/min/mg.

Finally, adenosine nucleosidase from Alaska pea has a molecular weight of 36 kDa determined by SDS-PAGE.