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Drosophila acetylcholinesterase: demonstration of a glycoinositol phospholipid anchor and an endogenous proteolytic cleavage

Journal Article · · Biochemistry; (United States)
DOI:https://doi.org/10.1021/bi00417a038· OSTI ID:6464902
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The presence of a glycoinositol phospholipid anchor Drosophila acetylcholinesterase (AChE) was shown by several criteria. Chemical analysis of highly purified Drosophila AChE demonstrated approximately one residue of inositol per enzyme subunit. Selective cleavage by Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) was tested with Drosophila AChE radiolabeled by the photoactivatable affinity probe 3-(trifluoromethyl)-3-(m-(/sup 125/I)iodophenyl)diazirine ((/sup 125/I)TID), a reagent that specifically labels the lipid moiety of glycoinositol phospholipid-anchored proteins. Digestion with PI-PLC released 75% of this radiolabel from the protein. Gel electrophoresis of Drosophila AChE in sodium dodecyl sulfate indicated prominent 55- and 16-kDa bands and a faint 70-kDa band. The (/sup 125/)I)TID label was localized on the 55-kDa fragment, suggesting that this fragment is the C-terminal portion of the protein. In support of this conclusion, a sensitive microsequencing procedure that involved manual Edman degradation combined with radiomethylation was used to determine residues 2-5 of the 16-kDa fragment. Comparison with the Drosophila AChE cDNA sequence confirmed that the 16-kDa fragment includes the N-terminus of AChE. Furthermore, the position of the N-terminal amino acid of the mature Drosophila AChE is closely homologous to that of Torpedo AChE. The presence of radiomethylatable ethanolamine in both 16- and 55-kDa fragments was also confirmed. Thus, Drosophila AChE may include a second posttranslational modification involving ethanolamine.

Research Organization:
Case Western Reserve Univ., Cleveland, OH (USA)
OSTI ID:
6464902
Journal Information:
Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 27:17; ISSN BICHA
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINES
ANIMALS
ARTHROPODS
BETA DECAY RADIOISOTOPES
BIOCHEMISTRY
CARBON 14 COMPOUNDS
CARBOXYLESTERASES
CHEMICAL REACTIONS
CHEMISTRY
CHOLINESTERASE
COLLAGEN
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
DIPTERA
DROSOPHILA
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
ENZYMATIC HYDROLYSIS
ENZYMES
ESTERASES
ESTERS
FLIES
FRUIT FLIES
HYDROLASES
HYDROLYSIS
INSECTS
INTERMEDIATE MASS NUCLEI
INVERTEBRATES
IODINE 125
IODINE ISOTOPES
ISOTOPES
LABELLED COMPOUNDS
LIPIDS
LYSIS
METHYLATION
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
PHOSPHOLIPIDS
POST-TRANSLATION MODIFICATION
PROTEINS
RADIOISOTOPES
SCLEROPROTEINS
SOLVOLYSIS