Bovine milk acid phosphatase. III. Purification and characterisation of the enzyme.
The acid phosphatase of bovine milk has been purified by ion-exchange chromatography on Amberlite CG-50 resin followed by Sephadex G-100 gel filtration and further purification steps using CM-Sephadex C-50, cellulose phosphate and affinity chromatography on alphas1-casein-Sepharose. These gave an apparently homogeneous enzyme with a specific activity of about 30 I.U./mg which possessed a molecular weight of about 42000 and E1%1cm = 9.4 at 280 nm. Electrophoresis under dissociating conditions, gel filtration in a 6.6 M urea buffer and ultracentrifugal analysis in the presence of 6 M guanidine HCl and 0.1 M 2-mercaptoethanol all gave molecular weight values close to 42000 indicating that the molecule was probably composed of a single polypeptide chain. The acid phosphatase was a glycoprotein with 2 mol/mol of galactose, 2 mol/mol of mannose and 4 mol/mol of glucosamine. Amino acid analysis showed that the enzyme was devoid of methionine.
D000135:Acid Phosphatase / Q000302:isolation & purification; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002846:Chromatography, Affinity; D005260:Female; D005944:Glucosamine / Q000032:analysis; D006146:Guanidines; D006601:Hexoses / Q000032:analysis; D046911:Macromolecular Substances; D008892:Milk / Q000201:enzymology*; D008970:Molecular Weight; D011485:Protein Binding; D011487:Protein Conformation; D014508:Urea
D000596:Amino Acids; D006146:Guanidines; D006601:Hexoses; D046911:Macromolecular Substances; D014508:Urea; D000135:Acid Phosphatase; D005944:Glucosamine