Purification and Characterization of Nucleoside Diphosphatase from Rat‐Liver Microsomes

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Abstract
Nucleoside diphosphatase was purified from rat liver microsomes more than 3000-fold with a 16% yield using a procedure including concanavalin-A-Sepharose and phenyl-Sepharose column chromatography. The purified enzyme had a specific activity of 2500 U/mg protein and appeared homogeneous by gel electrophoresis. The enzyme had a sedimentation coefficient of 6.5 S by sucrose-density gradient centrifugation, and a Stokes'' radius of 4.8 nm was estimated by the gel filtration technique. Its MW is 130,000, but only 1 single band of MW 65,000 was detected after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The native enzyme seems thus to be composed of 2 identical subunits. The purified enzyme was confirmed to be a glycoprotein containing .apprx. 9% carbohydrates. The enzyme had a pH optimum of 7.5, an isoelectric point of 4.85 and a Km of 2.5 mM for UDP. On the basis of direct measurement of metal content in the native enzyme, the rat liver nucleoside diphosphatase was a metalloenzyme containing 0.9 mol Zn and 0.1 mol Mg/mol 65,000-MW subunit. Metal-free nucleoside diphosphatase was prepared. The activity of the metal-free enzyme was restored by the addition of several divalent cations, Zn being the most effective.