Abstract
A rapid procedure for the large-scale purification of pig heart nucleoside diphosphate kinase is described. The purification procedure involves extraction of the enzyme, absorption on cibacron Blue 3G-A Sepharose, elution with ATP, ammonium sulfate precipitation, heat treatment, and rechromatography on Cibacron Blue 3G-A Sepharose. Typically, 10–12 mg of pure nucleoside diphosphate kinase is obtained from 1 kg of heart muscle (50% yield), with a purification factor of 1200 over the extract. The specific activity is 1500 units/mg at 25°C with 8-bromoinosine 5′-diphosphate as acceptor nucleotide. This method may be easily scaled up.
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