2 RAT INTESTINAL ALKALINE-PHOSPHATASE ISOFORMS WITH DIFFERENT CARBOXYL-TERMINAL PEPTIDES ARE BOTH MEMBRANE-BOUND BY A GLYCAN PHOSPHATIDYLINOSITOL LINKAGE

Two cDNAs encode rat intestinal alkaline phosphatases having completel y different carboxyl-terminal peptides; one is hydrophobic and fulfill s the consensus requirements for glycan phosphatidylinositol linkage, and the other is neither hydrophobic nor hydrophilic, but contains a s mall amino acid domain (-NSASS-) just distal to a region of 17 threoni ne residues, Constructs were created using 80% of the amino terminal p ortion of one alkaline phosphatase and the carboxyl terminal portions of each of the isoforms. Both of the carboxyl-terminal peptides suppor ted glycan phosphatidylinositol linkage as demonstrated by the followi ng criteria: 1) plasma membrane targeting in transfected COS-1 cells, 2) release of transfected alkaline phosphatase by phosphatidylinositol -specific phospholipase C, 3) appearance of the trypanosome variable g lycoprotein crossreacting determinant after phospholipase C treatment, 4) ethanolamine incorporation into newly synthesized enzyme, 5) loss of phospholipase C release after mutation of the omega and omega + 2 p ositions in the putative linkage site, -NSA-, and 6) evidence of surfa ce membrane localization by immunofluorescence using antibody against rat intestinal alkaline phosphatase. These data demonstrate that a pre dicted hydrophobic carboxyl-terminal sequence is not essential for gly can phosphatidylinositol linkage, Moreover, because both isomers are m embrane bound, the origin of soluble enzyme in the serum is likely to arise from the action of serum phosphatidylinositol-specific phospholi pase D.