Open AccessMolecular forms of acetylcholinesterase in two sublines of human erythroleukemia K562 cells
Author(s) -
TOUTANT JeanPierre,
RICHARDS Michael K.,
KRALL Jennifer A.,
ROSENBERRY Terrone L.
Publication year - 1990
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1990.tb15274.x
Subject(s) - phospholipase c , acetylcholinesterase , biochemistry , hydroxylamine , chemistry , myristoylation , phospholipase a2 , phosphatidylinositol , gtp' , dimer , hydrazide , phospholipid , enzyme , stereochemistry , membrane , signal transduction , organic chemistry
Acetylcholinesterase (AChE) in K562 cells exists in two molecular forms. The major form, an amphiphilic dimer (G 2 a) which sediments at 5.3 S, and the minor form, an amphiphilic monomer (G 1 a) which sediments at 3.5 S. Extraction in the presence of the sulfhydryl alkylating agent N ‐ethylmaleimide was required to preserve the G 2 a 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 2 a AChE into a hydrophilic dimer (G 2 h), indicating that the G 2 a 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 2 a 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 2 a form but not of the G 1 a form of AChE. G 1 a forms thus appear to represent an intracellular pool of newly synthesized molecules residing in a compartment proximal to the trans‐Golgi apparatus. The sialidase‐resistant G 1 a molecules were also resistant to PtdIns‐PLC digestion; possible explanations for this resistance are presented.