Rat brain nucleoside diphosphatase: Purification and properties of type L isozyme

Type L nucleoside diphosphatase of rat brain was purified with procedures of solubilization by Triton X-100, ammonium sulfate fractionation and 4 steps of column chromatography using DE-52, Con A-Sepharose, Sephadex G-150, and Phenyl-Sepharose CL-4B. The purified enzyme had a molecular weight of 45 KDa and an isoelectric point of 4.6. The optimum pH for enzyme activity was 7.5≈8.0 and the K m value was 1.2 mM for GDP. The enzyme activity was markedly enhanced by ATP, the stimulatory effect being due to increase in the affinity of the enzyme for substrate. Inorganic pyrophosphate potently inhibited the activity. It was a competitive inhibitor at lower concentrations, while it also decreased the Vmax value at higher concentrations.