Incorporation of human liver and placental alkaline phosphatases into liposomes and membranes is via phosphatidylinositol.
As assessed by incorporation into liposomes and by adsorption to octyl-Sepharose, the integrity of the membrane anchor for the purified tetrameric forms of alkaline phosphatase from human liver and placenta was intact. Any treatment that resulted in a dimeric enzyme precluded incorporation and adsorption. An intact anchor also allowed incorporation into red cell ghosts. The addition of hydrophobic proteins inhibited incorporation into liposomes to varying degrees. Alkaline phosphatase was 100% releasable from liposomes and red cell ghosts by a phospholipase C specific for phosphatidylinositol. There was no appreciable difference in the rates of release of placental and liver alkaline phosphatases, although both were approximately 250 x slower in liposomes and 100 x slower in red cell ghosts than the enzyme's release from a suspension of cultured osteosarcoma cells. Both enzymes were released by phosphatidylinositol phospholipase C as dimers and would not reincorporate or adsorb to octyl-Sepharose. However, the enzyme incorporated, resolubilized by Triton X-100, and cleansed of the detergent by butanol treatment was tetrameric by gradient gel electrophoresis, was hydrophobic, and could reincorporate into fresh liposomes. A monoclonal antibody to liver alkaline phosphatase inhibited the enzyme's incorporation into liposomes, and abolished its release from liposomes and its conversion to dimers by phosphatidylinositol phospholipase C.
Kihn
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Department of Pathology, University of Alberta, Edmonton, Canada.
Rutkowski
D
D
Stinson
R A
RA
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000302:isolation & purification*; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane; D005260:Female; D006017:Glycolipids / Q000302:isolation & purification; D017261:Glycosylphosphatidylinositols; D006801:Humans; D008081:Liposomes; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D010713:Phosphatidylcholines; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000302:isolation & purification; D010727:Phosphoric Diester Hydrolases; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D012685:Sepharose / Q000031:analogs & derivatives
D000911:Antibodies, Monoclonal; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D008081:Liposomes; D046911:Macromolecular Substances; D010713:Phosphatidylcholines; D010716:Phosphatidylinositols; C027176:octyl-sepharose CL-4B; D012685:Sepharose; D000469:Alkaline Phosphatase; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase