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MOLECULAR CHARACTERIZATION OF CHOLINE ACETYLTRANSFERASE FROM BOVINE BRAIN CAUDATE NUCLEUS AND SOME IMMUNOLOGICAL PROPERTIES OF THE HIGHLY PURIFIED ENZYME
Author(s) -
MaltheSosrenssen D.,
Lea T.,
Fonnum F.,
Eskeland T.
Publication year - 1978
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.1471-4159.1978.tb07032.x
Subject(s) - choline acetyltransferase , enzyme , sephadex , biochemistry , chemistry , affinity chromatography , caudate nucleus , immunodiffusion , polyacrylamide gel electrophoresis , ammonium sulfate precipitation , microbiology and biotechnology , molecular mass , chromatography , biology , antibody , size exclusion chromatography , acetylcholine , immunology , endocrinology
Abstract— Choline acetyltransferase from bovine brain caudate nucleus has been purified to a specific activity of 25–30 μ mol ACh formed per min and mg protein. Disc electrophoresis at pH 9.5 of the purified enzyme showed two protein bands localized close to each other. We were not able to show if ChAT was linked to one or both bands. In SDS disc electrophoresis the enzyme preparation showed one major and one minor protein band with molecular weights of 69,000 and 34,000, respectively. Heterogeneity of the enzyme preparation was also demonstrated by immunodiffusion and immunoelectrophoresis. After ammonium sulphate precipitation no aggregation of the enzyme could be detected by gelfiltration on Ultrogel AC‐34 whilst a high molecular weight fraction was occasionally observed by gelfiltration on Sephadex G‐200. The enzyme was, however, separated into two molecular forms (A and B) on CM‐Sephadex chromatography. Both molecular forms had the same S 2 20w but differed in heat stability and affinity for acetyl‐CoA. Both forms were inactivated by an antibody preparation raised against either a purified preparation of ChAT, or A and B separately. The highly purified enzyme preparation was inactivated more than 98% by immunoprecipitation. The antibody crossreacted with ChATs from several mammalian species, but only slightly with ChAT from pigeon. The results of binding studies with affinity columns, suggest that the enzyme contains a hydrophobic lobe and a dinucleotide fold, and that a free purine rather than a free ribosyl ring of acetyl‐CoA is important for the binding of the substrate to the active site. The hydrophobic lobe may be the same as the dinucleotide fold.