Chemical characterization of the membrane-anchoring domain of human placental alkaline phosphatase

• Published in: The Journal of biological chemistry Vol. 263; no. 21; pp. 10489 - 10494
• Main Authors: Ogata, S, Hayashi, Y, Takami, N, Ikehara, Y
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 25-07-1988; American Society for Biochemistry and Molecular Biology
• Subjects: Alkaline Phosphatase - isolation & purification; Alkaline Phosphatase - metabolism; alkaline phosphate; Amino Acid Sequence; Amino Acids - analysis; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Cell Line; Enzymes and enzyme inhibitors; Female; Fundamental and applied biological sciences. Psychology; glycolipids; Humans; Hydrolases; Intracellular Membranes - enzymology; man; Microsomes - enzymology; Papain; Peptide Fragments - analysis; placenta; Placenta - enzymology; Pregnancy; Protein Binding; Human; Alkaline phosphatase; Anchoring domaine; Composition; Molecular form; Placenta; Complementary DNA; Nucleotide sequence; Gel electrophoresis; Enzyme; Aminoacid; HPLC chromatography
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)81542-9

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.