5'-Nucleotidase from bull seminal plasma. Biochemical and biophysical aspects

Abstract

In bull seminal plasma 5'-nucleotidase is present in heterogeneous forms. The heterogeneity is abolished by treatment of bull seminal plasma with the detergent sodium cholate. The purified enzyme, which is a glycoprotein, shows an apparent molecular mass of 160 kDa on gel filtration in the presence of 50 mmol sodium cholate and an apparent molecular mass of 72 kDa upon SDS/polyacrylamide-gel electrophoresis. The 5'-nucleotidase of bull seminal plasma is a metalloprotein containing 2 zinc ions per molecule of dimeric protein. The removal of the two zinc ions from the protein results in a completely inactive apoenzyme. The substitution of the endogenous zinc with Co(II) Cu(II) produces a holoenzyme which is slightly activated in the case of Co(II), whereas, in the case of Cu(II) only 65% of the initial activity is recovered. The enzyme has a covalently attached glycosyl-phosphatidylinositol moiety which can be removed by treatment with phosphatidylinositol-specific phospholipase C. ESR studies have indicated a radius of 35 A for the protein and that Cu(II) binds to the metal-free enzyme to a site in which sulphur donors can be excluded.