title: Rat alkaline phosphatase. I. Purification and characterization of the enzyme from osteosarcoma: generation of monoclonal and polyclonal antibodies.
abstract: Alkaline phosphatase (AP) was purified to over 90% homogeneity from rat osteosarcoma by acetone precipitation followed by chromatography on DEAE-cellulose, Sephacryl S-200, and hydroxyapatite. The purified enzyme had a specific activity of 759 units/mg protein at its optimal pH (10.5), and a Km of 0.8 mM for p-nitrophenylphosphate. The enzyme's apparent subunit molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 82,000 Da. The heat-inactivation profile and homoarginine inhibition were characteristic of the bone-liver-kidney AP isoenzyme. Monoclonal and polyclonal anti-AP antibodies were prepared and characterized. Polyclonal rabbit antiserum quantitatively precipitated the activity from purified AP preparations and tissue extracts but did not inhibit AP catalytic activity. This antiserum was almost 10-fold less active against heat-inactivated enzyme when tested in a competition assay using 125I-AP. Two distinct monoclonal antibodies were each partly effective in immunoprecipitating AP when tested individually; however, together they precipitated over 90% of the AP activity.
authors.forename: B C
authors.initials: BC
authors.lastname: Nair
authors.forename: R J
authors.initials: RJ
authors.lastname: Majeska
authors.forename: G A
authors.initials: GA
authors.lastname: Rodan
mesh_terms: D000469:Alkaline Phosphatase / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D007120:Immunochemistry; D007700:Kinetics; D008970:Molecular Weight; D012516:Osteosarcoma / Q000201:enzymology*; D051381:Rats; D011912:Rats, Inbred ACI
chemical_list: D000911:Antibodies, Monoclonal; D000469:Alkaline Phosphatase