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CHOLINE ACETYLTRANSFERASE–EVIDENCE FOR ACETYL TRANSFER BY A HISTIDINE RESIDUE
Author(s) -
MaltheSorenssen D.
Publication year - 1976
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1976.tb05149.x
Subject(s) - choline acetyltransferase , enzyme , biochemistry , chemistry , choline , cysteine , histidine , enzyme assay , residue (chemistry) , acetyltransferase , sepharose , polyacrylamide gel electrophoresis , chromatography , acetylcholine , biology , acetylation , gene , endocrinology
— Choline acetyltransferase from bovine brain has been extensively purified to a specific activity of 2.5 μmol ACh/min mg protein. Attempts to isolate an acetyl enzyme intermediate after incubation of the enzyme with [1‐ 14 C]acetyl‐CoA were unsuccessful. Such an intermediate could only be isolated using a 30‐fold less purified enzyme preparation. The protein, binding 14 C in this preparation, did not correspond to choline acetyltransferase as shown by disc‐electrophoresis. The highly purified enzyme could, however, be labelled when choline acetyltransferase was immobilized on a mercuribenzoate sepharose gel and incubated with [1‐ 14 C]acetyl‐CoA. Subsequently, the immobilized labelled enzyme or the labelled enzyme which had been released by cysteine from the gel. formed ACh after incubation with choline. The labelling and the following formation of [ 14 C]ACh was pH dependent. Masking htstidine residues of the enzyme with diethylpyrocarbonate almost abolished the labelling of the immobilized enzyme and completely abolished the formation of [ 14 C]ACh. Enzyme inhibited with 5.5′‐dithiobis(2‐nitrobenzoate) was partially reactivated when the thionitrobenzoatederivative was cleaved by KCN treatment to a thiocyanatederivalive. A reaction mechanism for ChAT is proposed based on the present data.