Identification of amine components in a glycolipid membrane-binding domain at the C-terminus of human erythrocyte acetylcholinesterase

Robert Haas, Patricia T. Brandt, Jonathan Knight, Terrone L. Rosenberry

Research output: Contribution to journalArticle

56 Citations (Scopus)

Abstract

Purified human erythrocyte acetylcholinesterase was labeled by reductive radiomethylation with saturating amounts of [14C]formaldehyde and sodium cyanoborohydride. Acid hydrolysis and automated amino acid analysis permitted both identification of radiomethylated components by their coelution with radiomethylated standards and quantitation of these components. The methylated N-terminal amino acids glutamate and arginine were observed at levels of 0.66 and 0.34 residues, respectively, per 70-kilodalton subunit, and lysine residues were methylated on their ε-amino groups to a level of 7.40 residues per subunit [Haas, R., & Rosenberry, T. L. (1985) Anal. Biochem. 148, 154-162]. In addition, each subunit contained 1.35 residues of methylated ethanolamine and 0.98 residue of methylated glucosamine. Papain digestion cleaved the intact enzyme into two fragments, an enzymatically active hydrophilic fragment and a small hydrophobic fragment that represented the membrane-binding domain. The radiomethylated amino acids were quantitatively retained in the hydrophilic fragment, while the methylated ethanolamine and glucosamine were confined exclusively to the hydrophobic domain fragment. This fragment included the C-terminal dipeptide of the subunit. Peptide sequencing by manual Edman methods was combined with radiomethylation to demonstrate the sequence His-Gly-ethanolamine-Z for the hydrophobic domain fragment. The ethanolamine residue in this sequence is in amide linkage to the C-terminal Gly and is clearly distinct from the ethanolamine residues in Z which are susceptible to radiomethylation in the intact enzyme. Since Z also includes glucosamine and 2 mol of fatty acids [Roberts, W. L., & Rosenberry, T. L. (1985) Biochem. Biophys. Res. Commun. 133, 621-627], we conclude that the membrane-binding domain of human erythrocyte acetylcholinesterase is a covalently linked glycolipid at the C-termini of the subunits. Analogies to the membrane-binding domains of murine Thy-1 glycoprotein and trypanosome variant surface glycoproteins are discussed.

Original languageEnglish (US)
Pages (from-to)3098-3105
Number of pages8
JournalBiochemistry
Volume25
Issue number11
StatePublished - 1986
Externally publishedYes

Fingerprint

Ethanolamine
Glycolipids
Acetylcholinesterase
Amines
Erythrocytes
Glucosamine
Membranes
arginine glutamate
Amino Acids
histidylglycine
Papain
Trypanosomiasis
Dipeptides
Membrane Glycoproteins
Enzymes
Amides
Formaldehyde
Lysine
Digestion
Hydrolysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification of amine components in a glycolipid membrane-binding domain at the C-terminus of human erythrocyte acetylcholinesterase. / Haas, Robert; Brandt, Patricia T.; Knight, Jonathan; Rosenberry, Terrone L.

In: Biochemistry, Vol. 25, No. 11, 1986, p. 3098-3105.

Research output: Contribution to journalArticle

Haas, Robert ; Brandt, Patricia T. ; Knight, Jonathan ; Rosenberry, Terrone L. / Identification of amine components in a glycolipid membrane-binding domain at the C-terminus of human erythrocyte acetylcholinesterase. In: Biochemistry. 1986 ; Vol. 25, No. 11. pp. 3098-3105.
@article{9ce1b6d58860405faebc61fd740edf73,
title = "Identification of amine components in a glycolipid membrane-binding domain at the C-terminus of human erythrocyte acetylcholinesterase",
abstract = "Purified human erythrocyte acetylcholinesterase was labeled by reductive radiomethylation with saturating amounts of [14C]formaldehyde and sodium cyanoborohydride. Acid hydrolysis and automated amino acid analysis permitted both identification of radiomethylated components by their coelution with radiomethylated standards and quantitation of these components. The methylated N-terminal amino acids glutamate and arginine were observed at levels of 0.66 and 0.34 residues, respectively, per 70-kilodalton subunit, and lysine residues were methylated on their ε-amino groups to a level of 7.40 residues per subunit [Haas, R., & Rosenberry, T. L. (1985) Anal. Biochem. 148, 154-162]. In addition, each subunit contained 1.35 residues of methylated ethanolamine and 0.98 residue of methylated glucosamine. Papain digestion cleaved the intact enzyme into two fragments, an enzymatically active hydrophilic fragment and a small hydrophobic fragment that represented the membrane-binding domain. The radiomethylated amino acids were quantitatively retained in the hydrophilic fragment, while the methylated ethanolamine and glucosamine were confined exclusively to the hydrophobic domain fragment. This fragment included the C-terminal dipeptide of the subunit. Peptide sequencing by manual Edman methods was combined with radiomethylation to demonstrate the sequence His-Gly-ethanolamine-Z for the hydrophobic domain fragment. The ethanolamine residue in this sequence is in amide linkage to the C-terminal Gly and is clearly distinct from the ethanolamine residues in Z which are susceptible to radiomethylation in the intact enzyme. Since Z also includes glucosamine and 2 mol of fatty acids [Roberts, W. L., & Rosenberry, T. L. (1985) Biochem. Biophys. Res. Commun. 133, 621-627], we conclude that the membrane-binding domain of human erythrocyte acetylcholinesterase is a covalently linked glycolipid at the C-termini of the subunits. Analogies to the membrane-binding domains of murine Thy-1 glycoprotein and trypanosome variant surface glycoproteins are discussed.",
author = "Robert Haas and Brandt, {Patricia T.} and Jonathan Knight and Rosenberry, {Terrone L.}",
year = "1986",
language = "English (US)",
volume = "25",
pages = "3098--3105",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "11",

}

TY - JOUR

T1 - Identification of amine components in a glycolipid membrane-binding domain at the C-terminus of human erythrocyte acetylcholinesterase

AU - Haas, Robert

AU - Brandt, Patricia T.

AU - Knight, Jonathan

AU - Rosenberry, Terrone L.

PY - 1986

Y1 - 1986

N2 - Purified human erythrocyte acetylcholinesterase was labeled by reductive radiomethylation with saturating amounts of [14C]formaldehyde and sodium cyanoborohydride. Acid hydrolysis and automated amino acid analysis permitted both identification of radiomethylated components by their coelution with radiomethylated standards and quantitation of these components. The methylated N-terminal amino acids glutamate and arginine were observed at levels of 0.66 and 0.34 residues, respectively, per 70-kilodalton subunit, and lysine residues were methylated on their ε-amino groups to a level of 7.40 residues per subunit [Haas, R., & Rosenberry, T. L. (1985) Anal. Biochem. 148, 154-162]. In addition, each subunit contained 1.35 residues of methylated ethanolamine and 0.98 residue of methylated glucosamine. Papain digestion cleaved the intact enzyme into two fragments, an enzymatically active hydrophilic fragment and a small hydrophobic fragment that represented the membrane-binding domain. The radiomethylated amino acids were quantitatively retained in the hydrophilic fragment, while the methylated ethanolamine and glucosamine were confined exclusively to the hydrophobic domain fragment. This fragment included the C-terminal dipeptide of the subunit. Peptide sequencing by manual Edman methods was combined with radiomethylation to demonstrate the sequence His-Gly-ethanolamine-Z for the hydrophobic domain fragment. The ethanolamine residue in this sequence is in amide linkage to the C-terminal Gly and is clearly distinct from the ethanolamine residues in Z which are susceptible to radiomethylation in the intact enzyme. Since Z also includes glucosamine and 2 mol of fatty acids [Roberts, W. L., & Rosenberry, T. L. (1985) Biochem. Biophys. Res. Commun. 133, 621-627], we conclude that the membrane-binding domain of human erythrocyte acetylcholinesterase is a covalently linked glycolipid at the C-termini of the subunits. Analogies to the membrane-binding domains of murine Thy-1 glycoprotein and trypanosome variant surface glycoproteins are discussed.

AB - Purified human erythrocyte acetylcholinesterase was labeled by reductive radiomethylation with saturating amounts of [14C]formaldehyde and sodium cyanoborohydride. Acid hydrolysis and automated amino acid analysis permitted both identification of radiomethylated components by their coelution with radiomethylated standards and quantitation of these components. The methylated N-terminal amino acids glutamate and arginine were observed at levels of 0.66 and 0.34 residues, respectively, per 70-kilodalton subunit, and lysine residues were methylated on their ε-amino groups to a level of 7.40 residues per subunit [Haas, R., & Rosenberry, T. L. (1985) Anal. Biochem. 148, 154-162]. In addition, each subunit contained 1.35 residues of methylated ethanolamine and 0.98 residue of methylated glucosamine. Papain digestion cleaved the intact enzyme into two fragments, an enzymatically active hydrophilic fragment and a small hydrophobic fragment that represented the membrane-binding domain. The radiomethylated amino acids were quantitatively retained in the hydrophilic fragment, while the methylated ethanolamine and glucosamine were confined exclusively to the hydrophobic domain fragment. This fragment included the C-terminal dipeptide of the subunit. Peptide sequencing by manual Edman methods was combined with radiomethylation to demonstrate the sequence His-Gly-ethanolamine-Z for the hydrophobic domain fragment. The ethanolamine residue in this sequence is in amide linkage to the C-terminal Gly and is clearly distinct from the ethanolamine residues in Z which are susceptible to radiomethylation in the intact enzyme. Since Z also includes glucosamine and 2 mol of fatty acids [Roberts, W. L., & Rosenberry, T. L. (1985) Biochem. Biophys. Res. Commun. 133, 621-627], we conclude that the membrane-binding domain of human erythrocyte acetylcholinesterase is a covalently linked glycolipid at the C-termini of the subunits. Analogies to the membrane-binding domains of murine Thy-1 glycoprotein and trypanosome variant surface glycoproteins are discussed.

UR - http://www.scopus.com/inward/record.url?scp=0022485383&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0022485383&partnerID=8YFLogxK

M3 - Article

VL - 25

SP - 3098

EP - 3105

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 11

ER -