Molecular forms of acetylcholinesterase in two sublines of human erythroleukemia K562 cells: Sensitivity or resistance to phosphatidylinositol-specific phospholipase C and biosynthesis

J. P. Toutant, M. K. Richards, J. A. Krall, T. L. Rosenberry

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Abstract

Acetylcholinesterase (AChE) in K562 cells exists in two molecular forms. The major form, an amphiphilic dimer (G 2a) which sediments at 5.3 S, and the minor form, an amphiphilic monomer (G 1a) which sediments at 3.5 S. Extraction in the presence of the sulfhydryl alkylating agent N-ethylmaleimide was required to preserve the G 2a form. In Triton X-100 extracts of the subline K562-243, phosphatidylinositol-specific phospholipase C (PtdIns-PLC) from Bacillus thuringiensis converted most of the G 2a AChE into a hydrophilic dimer (G 2h), indicating that the G 2a form possessed a hydrophobic glycoinositol phospholipid that mediated its attachment to the membrane. Treatment of intact K562-243 cells with PtdIns-PLC released approximately 60% of the total AChE activity and provided an estimate of the externally exposed AChE. The direct conversion from an amphiphilic to a hydrophilic dimeric form by PtdIns-PLC was not obtained in extracts or intact cells of the subline K562-48. Instead, pretreatment with alkaline hydroxylamine was necessary to render the amphiphilic G 2 form of this subline susceptible to digestion by the phospholipase. In this respect, the amphiphilic dimer of K562-48 AChE resembles the G 2a form of human erythrocyte AChE, which is resistant to PtdIns-PLC because of the direct palmitoylation of an inositol hydroxyl group in the anchor [Roberts et al. (1988) J. Biol. Chem. 263, 18766-18775]. Release of this acyl chain by hydroxylamine renders the enzyme susceptible to PtdIns-PLC [Toutant et al. (1989) Eur. J. Biochem. 180, 503-508]. In both K562 sublines, sialidase decreased the migration of the G 2a form but not of the G 1a form of AChE. G 1a forms thus appear to represent an intracellular pool of newly synthesized molecules residing in a compartment proximal to the trans-Golgi apparatus. This sialidase-resistant G 1a molecules were also resistant to PtdIns-PLC digestion; possible explanations for this resistance are presented.

Original languageEnglish (US)
Pages (from-to)31-38
Number of pages8
JournalEuropean Journal of Biochemistry
Volume187
Issue number1
DOIs
StatePublished - 1990
Externally publishedYes

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Phosphoinositide Phospholipase C
Leukemia, Erythroblastic, Acute
K562 Cells
Biosynthesis
Acetylcholinesterase
Dimers
Hydroxylamine
Neuraminidase
Digestion
Sediments
Lipoylation
Bacillus thuringiensis
Molecules
Ethylmaleimide
Phospholipases
Alkylating Agents
Octoxynol
Golgi Apparatus
Inositol
Bacilli

ASJC Scopus subject areas

  • Biochemistry

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Molecular forms of acetylcholinesterase in two sublines of human erythroleukemia K562 cells : Sensitivity or resistance to phosphatidylinositol-specific phospholipase C and biosynthesis. / Toutant, J. P.; Richards, M. K.; Krall, J. A.; Rosenberry, T. L.

In: European Journal of Biochemistry, Vol. 187, No. 1, 1990, p. 31-38.

Research output: Contribution to journalArticle

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abstract = "Acetylcholinesterase (AChE) in K562 cells exists in two molecular forms. The major form, an amphiphilic dimer (G 2a) which sediments at 5.3 S, and the minor form, an amphiphilic monomer (G 1a) which sediments at 3.5 S. Extraction in the presence of the sulfhydryl alkylating agent N-ethylmaleimide was required to preserve the G 2a form. In Triton X-100 extracts of the subline K562-243, phosphatidylinositol-specific phospholipase C (PtdIns-PLC) from Bacillus thuringiensis converted most of the G 2a AChE into a hydrophilic dimer (G 2h), indicating that the G 2a form possessed a hydrophobic glycoinositol phospholipid that mediated its attachment to the membrane. Treatment of intact K562-243 cells with PtdIns-PLC released approximately 60{\%} of the total AChE activity and provided an estimate of the externally exposed AChE. The direct conversion from an amphiphilic to a hydrophilic dimeric form by PtdIns-PLC was not obtained in extracts or intact cells of the subline K562-48. Instead, pretreatment with alkaline hydroxylamine was necessary to render the amphiphilic G 2 form of this subline susceptible to digestion by the phospholipase. In this respect, the amphiphilic dimer of K562-48 AChE resembles the G 2a form of human erythrocyte AChE, which is resistant to PtdIns-PLC because of the direct palmitoylation of an inositol hydroxyl group in the anchor [Roberts et al. (1988) J. Biol. Chem. 263, 18766-18775]. Release of this acyl chain by hydroxylamine renders the enzyme susceptible to PtdIns-PLC [Toutant et al. (1989) Eur. J. Biochem. 180, 503-508]. In both K562 sublines, sialidase decreased the migration of the G 2a form but not of the G 1a form of AChE. G 1a forms thus appear to represent an intracellular pool of newly synthesized molecules residing in a compartment proximal to the trans-Golgi apparatus. This sialidase-resistant G 1a molecules were also resistant to PtdIns-PLC digestion; possible explanations for this resistance are presented.",
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AU - Richards, M. K.

AU - Krall, J. A.

AU - Rosenberry, T. L.

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N2 - Acetylcholinesterase (AChE) in K562 cells exists in two molecular forms. The major form, an amphiphilic dimer (G 2a) which sediments at 5.3 S, and the minor form, an amphiphilic monomer (G 1a) which sediments at 3.5 S. Extraction in the presence of the sulfhydryl alkylating agent N-ethylmaleimide was required to preserve the G 2a form. In Triton X-100 extracts of the subline K562-243, phosphatidylinositol-specific phospholipase C (PtdIns-PLC) from Bacillus thuringiensis converted most of the G 2a AChE into a hydrophilic dimer (G 2h), indicating that the G 2a form possessed a hydrophobic glycoinositol phospholipid that mediated its attachment to the membrane. Treatment of intact K562-243 cells with PtdIns-PLC released approximately 60% of the total AChE activity and provided an estimate of the externally exposed AChE. The direct conversion from an amphiphilic to a hydrophilic dimeric form by PtdIns-PLC was not obtained in extracts or intact cells of the subline K562-48. Instead, pretreatment with alkaline hydroxylamine was necessary to render the amphiphilic G 2 form of this subline susceptible to digestion by the phospholipase. In this respect, the amphiphilic dimer of K562-48 AChE resembles the G 2a form of human erythrocyte AChE, which is resistant to PtdIns-PLC because of the direct palmitoylation of an inositol hydroxyl group in the anchor [Roberts et al. (1988) J. Biol. Chem. 263, 18766-18775]. Release of this acyl chain by hydroxylamine renders the enzyme susceptible to PtdIns-PLC [Toutant et al. (1989) Eur. J. Biochem. 180, 503-508]. In both K562 sublines, sialidase decreased the migration of the G 2a form but not of the G 1a form of AChE. G 1a forms thus appear to represent an intracellular pool of newly synthesized molecules residing in a compartment proximal to the trans-Golgi apparatus. This sialidase-resistant G 1a molecules were also resistant to PtdIns-PLC digestion; possible explanations for this resistance are presented.

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