Photoaffinity labeling of the Torpedo californica nicotinic acetylcholine receptor with an aryl azide derivative of phosphatidylserine
- Univ. of California, Santa Cruz (USA)
A photoactivatable analogue of phosphatidylserine, {sup 125}I-labeled 4-azidosalicylic acid-phosphatidylserine ({sup 125}I ASA-PS), was used to label both native acetylcholine receptor (AchR)-rich membranes from Torpedo californica and AchR membranes affinity purified from Torpedo reconstituted into asolectin vesicles. The radioiodinated arylazido group attaches directly to the phospholipid head group and thus probes for regions of the AchR structure in contact with the negatively charged head group of phosphatidylserine. All four subunits of the AchR incorporated the label, with the {alpha} subunit incorporating approximately twice as much as each of the other subunits on a per mole basis. The regions of the AchR {alpha} subunit that incorporated {sup 125}I ASA-PS were mapped by Staphylococcus aureus V8 protease digestion. The majority of label incorporated into fragments representing a more complete digestion of the {alpha} subunit was localized to 11.7- and 10.1-kDa V8 cleavage fragments, both beginning at Asn-339 and of sufficient length to contain the hydrophobic region M4. An 18.7-kDa fragment beginning at Ser-173 and of sufficient length to contain the hydrophobic regions M1, M2, and M3 was also significantly labeled. In contrast, V8 cleavage fragments representing roughly a third of the amino-terminal portion of the {alpha} subunit incorporated little or no detectable amount of probe.
- OSTI ID:
- 6698903
- Journal Information:
- Biochemistry; (USA), Vol. 29:5; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
MEMBRANE PROTEINS
AUTORADIOGRAPHY
RECEPTORS
LABELLING
SERINE
DERIVATIZATION
ACETYLCHOLINE
AZIDES
CELL MEMBRANES
ELECTROPHORESIS
IODINE 125
LIPIDS
AMINES
AMINO ACIDS
AMMONIUM COMPOUNDS
AUTONOMIC NERVOUS SYSTEM AGENTS
BETA DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
ESTERS
HYDROXY ACIDS
INTERMEDIATE MASS NUCLEI
IODINE ISOTOPES
ISOTOPES
MEMBRANES
NEUROREGULATORS
NITROGEN COMPOUNDS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
PARASYMPATHOMIMETICS
PROTEINS
QUATERNARY COMPOUNDS
RADIOISOTOPES
550201* - Biochemistry- Tracer Techniques