Selective radiolabeling and isolation of the hydrophobic membrane-binding domain of human erythrocyte acetylcholinesterase

Journal Article · Tue Jun 03 04:00:00 EDT 1986 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00359a004· OSTI ID:5127461

Roberts, W L; Rosenberry, T L

The hydrophobic, membrane-binding domain of purified human erythrocyte acetylcholinesterase was labeled with the photoactivated reagent 3-(trifluoromethyl)-3-(m-(/sup 125/I)iodophenyl)diazirine. The radiolabel was incorporated when the enzyme was prepared in detergent-free aggregates, in detergent micelles, or in phospholipid liposomes, but the highest percentage of labeling occurred in the detergent-free aggregates. Papain digestion of the enzyme released the hydrophobic domain, and polyacrylamide gel electrophoresis in sodium dodecyl sulfate or gel exclusion chromatography demonstrated that the label was localized exclusively in the cleaved hydrophobic domain fragment. This fragment was purified in a three-step procedure. Digestion was conducted with papain attached to Sepharose CL-4B, and the supernatant was adsorbed to acridinium affinity resin to remove the hydrophilic enzyme fragment. The nonretained fragment associated with Triton X-100 micelles was then chromatographed on Sepharose CL-6B, and finally detergent was removed by chromatography on Sephadex LH-60 in an ethanol-formic acid solvent. The fragment exhibited an apparent molecular weight of 3100 on the Sephadex LH-60 column when compared with peptide standards. However, amino acid analysis of the purified fragment revealed only 1 mol each of histidine and glycine per mole of fragment in contrast to the 25-30 mole of amino acids expected on the basis of the molecular weight estimate. This result suggests a novel non-amino acid structure for the hydrophobic domain of human erythrocyte acetylcholinesterase.

• Research Organization: Case Western Reserve Univ. School of Medicine, Cleveland, OH
• OSTI ID: 5127461
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 11; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACETYLCHOLINE
AMINES
AMINO ACID SEQUENCE
AMINO ACIDS
AMMONIUM COMPOUNDS
AUTONOMIC NERVOUS SYSTEM AGENTS
BETA DECAY RADIOISOTOPES
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY FLUIDS
CARBOXYLESTERASES
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CELL MEMBRANES
CHOLINESTERASE
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
ENZYMES
ERYTHROCYTES
ESTERASES
ESTERS
HYDROLASES
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPES
LABELLING
MATERIALS
MEMBRANES
MOLECULAR STRUCTURE
NEUROREGULATORS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
PARASYMPATHOMIMETICS
QUATERNARY COMPOUNDS
RADIOISOTOPES
STRUCTURAL CHEMICAL ANALYSIS