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A Rapid Purification of L‐Glutamic Acid Decarboxylase from the Brain of the Locust Schistocerca gregaria
Author(s) -
Stapleton Andrew,
Tyrer N. Mark,
Goosey Michael W.,
Cooper Michael E.
Publication year - 1989
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.1471-4159.1989.tb07405.x
Subject(s) - chromatography , chemistry , size exclusion chromatography , enzyme , schistocerca , gel electrophoresis , biochemistry , polyacrylamide gel electrophoresis , locust , fast protein liquid chromatography , glutamate decarboxylase , biology , botany
L‐Glutamic acid decarboxylase (GAD; EC 4.1.1.15) was purified to apparent homogeneity from the brain of the locust Schistocerca gregaria using a combination of chro‐matofocusing (Mono P) and gel filtration (Superose 12) media. The homogeneity of the enzyme preparation was established by native polyacrylamide gel electrophoresis (PAGE) with silver staining. The molecular weight of the purified enzyme was estimated from native gradient gel electrophoresis and gel filtration chromatography to be 97,000 ± 4,000 and 93,000 ± 5,000, respectively. When analysed by sodium do‐decyl sulphate‐PAGE, the enzyme was found to be composed of two distinct subunits of M r 51,000 ± 1,000 and 44,000 ± 1,500. Tryptic peptide maps of iodinated preparations of these two subunits showed considerable homology, suggesting that the native enzyme is a dimer of closely related subunits. The purified enzyme had a pH optimum of 7.0‐7.4 in 100 m M potassium phosphate buffer and an apparent K m for glutamate of 5.0 m M. The enzyme was strongly inhibited by the carbonyl‐trapping reagent aminooxyacetic acid with an I 50 value of 0.2 μ M.