title: Alkaline phosphatase is an ectoenzyme that acts on micromolar concentrations of natural substrates at physiologic pH in human osteosarcoma (SAOS-2) cells.
abstract: Alkaline phosphatase (ALP) was examined in cultured human osteosarcoma cells (SAOS-2) with respect to isoenzyme form, kinetic properties toward two natural substrates, and topography and nature of attachment to the plasma membrane. ALP in SAOS-2 homogenates is the tissue-nonspecific (TNS) isoenzyme and a phosphoethanolamine (PEA) and pyridoxal 5'-phosphate (PLP) phosphatase, as demonstrated by heat and inhibition profiles and electrophoretic mobility. Kinetic studies indicate that TNSALP in SAOS-2 cells has both a low- and a high-affinity activity. The high-affinity activity (showing the greater catalytic efficiency) is active at physiologic pH toward physiologic concentrations (microM) of PEA and PLP. TNSALP was shown to be an ectoenzyme in SAOS-2 cells by our findings in intact cell suspensions, where (i) PEA and PLP degradation in the medium nearly equaled that of whole cell homogenates, (ii) greater than 85% of ALP activity was inactivated by acid treatment, and (iii) ALP activity was quantitatively released by phosphatidylinositol-specific phospholipase C. Our findings indicate that, in SAOS-2 cells, TNS (bone) ALP functions as an ectoenzyme to degrade physiologic concentrations of extracellular natural substrates at physiologic pH.
authors.affiliation: Division of Bone and Mineral Diseases, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: K N
authors.initials: KN
authors.lastname: Fedde
authors.forename: C C
authors.initials: CC
authors.lastname: Lane
authors.forename: M P
authors.initials: MP
authors.lastname: Whyte
mesh_terms: D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001842:Bone and Bones / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004983:Ethanolamines / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007422:Intestines / Q000201:enzymology; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D012516:Osteosarcoma / Q000201:enzymology*; D010920:Placenta / Q000201:enzymology; D011732:Pyridoxal Phosphate / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D004983:Ethanolamines; D007527:Isoenzymes; D011732:Pyridoxal Phosphate; C005448:phosphorylethanolamine; D000469:Alkaline Phosphatase