title: Two rat intestinal alkaline phosphatase isoforms with different carboxyl-terminal peptides are both membrane-bound by a glycan phosphatidylinositol linkage.
abstract: Two cDNAs encode rat intestinal alkaline phosphatases having completely different carboxyl-terminal peptides; one is hydrophobic and fulfills the consensus requirements for glycan phosphatidylinositol linkage, and the other is neither hydrophobic nor hydrophilic, but contains a small amino acid domain (-NSASS-) just distal to a region of 17 threonine residues. Constructs were created using 80% of the amino-terminal portion of one alkaline phosphatase and the carboxyl-terminal portions of each of the isoforms. Both of the carboxyl-terminal peptides supported glycan phosphatidylinositol linkage as demonstrated by the following 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 glycoprotein cross-reacting 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 positions in the putative linkage site, -NSA-, and 6) evidence of surface membrane localization by immunofluorescence using antibody against rat intestinal alkaline phosphatase. These data demonstrate that a predicted hydrophobic carboxyl-terminal sequence is not essential for glycan phosphatidylinositol linkage. Moreover, because both isomers are membrane-bound, the origin of soluble enzyme in the serum is likely to arise from the action of serum phosphatidylinositol-specific phospholipase C.
authors.affiliation: Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Engle
authors.forename: A
authors.initials: A
authors.lastname: Mahmood
authors.forename: D H
authors.initials: DH
authors.lastname: Alpers
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D003110:Colonic Neoplasms / Q000473:pathology; D016384:Consensus Sequence; D018076:DNA, Complementary / Q000235:genetics; D004041:Dietary Fats / Q000494:pharmacology; D005796:Genes; D017261:Glycosylphosphatidylinositols / Q000737:chemistry*; D006801:Humans; D007422:Intestines / Q000201:enzymology* / Q000648:ultrastructure; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008871:Microvilli / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D051381:Rats / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D014407:Tumor Cells, Cultured
chemical_list: D018076:DNA, Complementary; D004041:Dietary Fats; D017261:Glycosylphosphatidylinositols; D007527:Isoenzymes; D008565:Membrane Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase