A single-vial biphasic liquid extraction assay for choline acetyltransferease using (/sup 3/H)choline

Journal Article · Tue Sep 15 04:00:00 EDT 1981 · Anal. Biochem.; (United States)

DOI:https://doi.org/10.1016/0003-2697(81)90372-9· OSTI ID:6097126

Rand, J B ^[1]; Johnson, C D

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1. Univ. of Pittsburgh, PA

A single-vial liquid extraction assay for choline acetyltransferase that uses (/sup 3/H)choline as the labeled substrate has been devised. (/sup 3/H)Choline is incubated with an excess of acetyl-CoA in a small reaction vial which also serves as a scintillation vial. After a suitable reaction period, unreacted (/sup 3/H)choline is quickly and quantitatively converted to phosphoryl-(/sup 3/H)choline by the addition of an excess of choline kinase. This treatment is followed by the addition of scintillation fluid containing sodium tetraphenylboron after which the vial is capped, shaken, and counted. A two-phase system is produced in which product (/sup 3/H)choline is selectively extracted into the scintillation fluid, where is is counted. Phosphoryl-(/sup 3/H)choline remains in the aqueous phase and is not counted. This assay is rapid, simple, and quite sensitive. In comparison to assays using acetyl-CoA as the labeled substrate, it is less sensitive to interference by other enzymes and thus more suitable for measuring choline acetyltransferase in crude extracts and in the initial stages of purificaton. Similar single-vial radiometric assays are described for choline kinase and acetyl-CoA hydrolases.

• OSTI ID: 6097126
• Journal Information: Anal. Biochem.; (United States), Journal Name: Anal. Biochem.; (United States) Vol. 116:2; ISSN ANBCA
• Country of Publication: United States
• Language: English

Related Subjects

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59 BASIC BIOLOGICAL SCIENCES
62 RADIOLOGY AND NUCLEAR MEDICINE
ACETYLCHOLINE
ALCOHOLS
AMINES
AMMONIUM COMPOUNDS
AUTONOMIC NERVOUS SYSTEM AGENTS
BIOCHEMICAL REACTION KINETICS
CHEMICAL REACTIONS
CHOLINE
COENZYMES
DRUGS
ENZYMES
ESTERS
HYDROLASES
HYDROXY COMPOUNDS
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LIPOTROPIC FACTORS
NEUROREGULATORS
ORGANIC COMPOUNDS
PARASYMPATHOMIMETICS
PHOSPHORYLATION
QUATERNARY COMPOUNDS
RADIOASSAY
REACTION KINETICS
TRACER TECHNIQUES
TRANSFERASES
TRITIUM COMPOUNDS