title: Chemical characterization of the membrane-anchoring domain of human placental alkaline phosphatase.
abstract: Membrane and soluble forms of alkaline phosphatase (ALP) were selectively prepared from human placental microsomes by treatment with 1-butanol at pH 8.5 and 5.5, respectively. The purified membrane (mALP) and soluble (sALP) forms were analyzed for chemical compositions. mALP was found to contain 1 mol each of palmitate, stearate, and glycerol/subunit of ALP, which were absent in sALP. Both the forms contained 1 mol of inositol and 2 mol of ethanolamine/subunit. However, none of these compounds was detectable in another soluble form prepared by treatment with papain, which is known to cleave the carboxyl-terminal region. The results suggest that mALP contains diacylglycerol, the removal of which results in its conversion to sALP. We then prepared [3H]ethanolamine-labeled ALP by incubating choriocarcinoma cells (JEG-3) with the isotope. 3H-Labeled sALP was mixed with unlabeled sALP and treated with papain. A 3H-labeled single component was purified from the digests by sequential chromatography through anti-ALP-IgG-Sepharose, concanavalin A-Sepharose, Bio-Gel P-6, and TSK G-2000 columns. Chemical analyses revealed that the purified sample contains the tripeptide Thr-Thr-Asp, ethanolamine, glucosamine, mannose, inositol, and phosphate. Molar ratios of the latter five compounds were calculated to be 2, 1, 3, 1, and 2, respectively, by taking Asp as 1 mol. The tripeptide sequence was identified at positions 482-484 in the primary structure deduced from the cDNA sequence, which predicts a further extension to position 513, containing a hydrophobic amino acid sequence. Taken together, these results suggest that the mature ALP molecule lacks the predicted carboxyl-terminal peptide extension and is attached at Asp484 with a glycosylphospholipid, the components of which are characterized above. The glycosylphospholipid thus attached is considered to function as the membrane anchor of ALP.
authors.affiliation: Department of Biochemistry, Fukuoka University School of Medicine, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Ogata
authors.forename: Y
authors.initials: Y
authors.lastname: Hayashi
authors.forename: N
authors.initials: N
authors.lastname: Takami
authors.forename: Y
authors.initials: Y
authors.lastname: Ikehara
mesh_terms: D000469:Alkaline Phosphatase / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002460:Cell Line; D005260:Female; D006801:Humans; D007425:Intracellular Membranes / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology*; D010206:Papain; D010446:Peptide Fragments / Q000032:analysis; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011485:Protein Binding
chemical_list: D000596:Amino Acids; D010446:Peptide Fragments; D000469:Alkaline Phosphatase; D010206:Papain