Release of alkaline phosphatase from human osteosarcoma cells by phosphatidylinositol phospholipase C: effect of tunicamycin.
Alkaline phosphatase (orthophosphoric-monoester phosphohydrolase [alkaline optimum], EC 3.1.3.1) expressed in two human osteosarcoma cell lines (Saos-2 and KTOO5) in culture was the tissue nonspecific type and was released from the plasma membrane by phosphatidylinositol (PI) phospholipase C. Despite a difference of 10-fold between the two cell lines in the amount of alkaline phosphatase expressed, the phospholipase solubilized nearly all of the phosphatase from resuspended cells of the two lines. Alkaline phosphatase released with Nonidet-P40 from Saos-2 cells had a Mr of 445,000 by gradient gel electrophoresis in the absence of detergent; that released by PI-phospholipase C was 200,000. The subunit Mr of both solubilized forms was 86,000. Thus, tetrameric alkaline phosphatase in the membrane is attached by a PI-glycan moiety and is converted to dimers when released by PI-phospholipase C. Tunicamycin treatment of Saos-2 cells in culture affected the release of alkaline phosphatase by a high concentration of PI-phospholipase C, but not by a low concentration; both the rate and extent of release were lower from treated cells. However, the enzyme released from the treated cells was in two forms with different molecular weights; it seems that both glycosylated and nonglycosylated dimers were transported to the cell surface and incorporated into the plasma membrane. Glycosylation does not appear to be necessary for alkaline phosphatase to be anchored in the membrane via PI.
Nakamura
T
T
Department of Pathology, University of Alberta, Edmonton, Canada.
Nakamura
K
K
Stinson
R A
RA
D000469:Alkaline Phosphatase / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D006801:Humans; D008970:Molecular Weight; D012516:Osteosarcoma / Q000201:enzymology*; D010716:Phosphatidylinositols / Q000502:physiology*; D014415:Tunicamycin / Q000494:pharmacology*; D010738:Type C Phospholipases / Q000502:physiology*
D010716:Phosphatidylinositols; D014415:Tunicamycin; D000469:Alkaline Phosphatase; D010738:Type C Phospholipases