Purification and characterization of nucleoside diphosphatase from rat-liver microsomes. Evidence for metalloenzyme and glycoprotein.
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 units/mg protein and appeared homogeneous by gel electrophoresis. The enzyme had a sedimentation coefficient of 6.5 S by sucrose-density gradient centrifugation. 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 molecular weight is 130,000, but only one single band of Mr 65,000 was detected after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The native enzyme seems thus to be composed of two identical subunits. The purified enzyme was confirmed to be a glycoprotein containing approximately 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 found to be a metalloenzyme containing 0.9 mol zinc and 0.1 mol manganese/mol 65,000-Mr subunit. Metal-free nucleoside diphosphatase has been prepared. The activity of the metal-free enzyme was restored by the addition of several divalent cations, zinc being the most effective.
D017766:Acid Anhydride Hydrolases; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D006023:Glycoproteins / Q000032:analysis; D007526:Isoelectric Point; D007700:Kinetics; D008345:Manganese / Q000032:analysis; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D015032:Zinc / Q000032:analysis
D000596:Amino Acids; D002241:Carbohydrates; D006023:Glycoproteins; D008345:Manganese; D010744:Phosphoric Monoester Hydrolases; D017766:Acid Anhydride Hydrolases; C023837:nucleoside-diphosphatase; D015032:Zinc