Purification of 5'-nucleotidase from human placenta after release from plasma membranes by phosphatidylinositol-specific phospholipase C.
5'-Nucleotidase was purified greater than 1000-fold from human placenta by treatment of plasma membranes with S. aureus phosphatidylinositol-specific phospholipase C and affinity chromatography on Con A Sepharose and AMP-Sepharose. The resulting enzyme had a specific activity of greater than 5000 mumol/hr/mg protein and a subunit molecular weight of 73,000. Goat antibodies against 5'-nucleotidase inhibited enzyme activity and detected 5'-nucleotidase after Western blotting. These antibodies also recognized a soluble form of 5'-nucleotidase and residual membrane-bound 5'-nucleotidase which could not be released by phosphatidylinositol-specific phospholipase C treatment, suggesting that the three forms of the enzyme are structurally related. The soluble 5'-nucleotidase may be derived from the membrane-bound form by the action of an endogenous phospholipase C. The structural basis for the inability of some of the membrane-bound 5'-nucleotidase to be released by phosphatidylinositol-specific phospholipase C is unknown.
D015720:5'-Nucleotidase; D001402:B-Lymphocytes / Q000201:enzymology; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity / Q000379:methods; D005260:Female; D006801:Humans; D008970:Molecular Weight; D009708:Nucleotidases / Q000302:isolation & purification* / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D010920:Placenta / Q000201:enzymology*; D013601:T-Lymphocytes / Q000201:enzymology; D010738:Type C Phospholipases / Q000378:metabolism*
D010716:Phosphatidylinositols; D009708:Nucleotidases; D015720:5'-Nucleotidase; D010738:Type C Phospholipases