Hydrophobic interactions of brush border alkaline phosphatases: the role of phosphatidyl inositol.
Tissue-specific (intestinal) and tissue-nonspecific (kidney) rat alkaline phosphatases are released from their respective brush border membranes by different enzymes. To elucidate the mechanism underlying their membrane attachment, we tested the ability of these enzymes to partition into lipid or aqueous phases both before and after treatment with phospholipases and proteases. Interaction with Triton X-114 micelles was eliminated or decreased by treatment of intestinal enzyme with phospholipase A2 or papain, while only phosphatidylinositol (PI)-specific phospholipase C (PIPLC) and subtilisin were effective with the kidney enzyme. Binding to octyl Sepharose for the intestinal enzyme was decreased by phospholipase A2 more than by PIPLC, whereas the reverse was true for the kidney enzyme. Treatment with phospholipases decreased the apparent mass of the phosphatases by 50-80 kDa, presumably due to loss of bound lipid and detergent. PIPLC treatment of the kidney, but not the intestinal enzyme, prevented binding of the phosphatase to phospholipid vesicles. These results show that both enzymes are bound to respective membranes by hydrophobic anchor peptides to which phospholipids are bound. However, their sensitivity to phospholipases is different. The data are consistent with the hypothesis that, in the kidney enzyme, the PI is bound covalently, while with the intestinal enzyme, binding of PI appears to be tight but not covalent.
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D000440:Butanols; D002845:Chromatography; D007413:Intestinal Mucosa / Q000201:enzymology; D007668:Kidney / Q000201:enzymology; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008871:Microvilli / Q000201:enzymology*; D008970:Molecular Weight; D010716:Phosphatidylinositols / Q000502:physiology*; D010740:Phospholipases / Q000378:metabolism; D011092:Polyethylene Glycols; D011485:Protein Binding; D051381:Rats; D012995:Solubility
D000440:Butanols; D008081:Liposomes; D008565:Membrane Proteins; D010716:Phosphatidylinositols; D011092:Polyethylene Glycols; D010740:Phospholipases; D000469:Alkaline Phosphatase