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PURIFICATION OF L‐GLUTAMIC ACID DECARBOXYLASE FROM CATFISH BRAIN
Author(s) -
Thomas Su Y. Y.,
Wu J.Y.,
Lam Dominic M. K.
Publication year - 1979
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.1471-4159.1979.tb11719.x
Subject(s) - polyacrylamide gel electrophoresis , chromatography , chemistry , gel electrophoresis , precipitin , glutamate decarboxylase , biochemistry , size exclusion chromatography , enzyme , sephadex , isoelectric focusing , biology , antibody , immunology
—L‐Glutamic acid decarboxylase (GAD) from brain of the channel catfish ( Ictalurus punctatus ) has been purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, calcium phosphate gel and preparative polyacrylamide gel electrophoresis. The purity of the enzyme preparation was established by showing that on both 7.5% regular and 3.7–15% gradient polyacrylamide gel electrophoresis the enzyme migrated as a single protein band which contained all the enzyme activity. The molecular weight of the purified GAD was estimated by gel filtration and gradient polyacrylamide gel to be 84,000 ± 2000 and 90,000 ± 4000, respectively. SDS‐polyacrylamide gel electrophoresis revealed three major proteins with molecular weights of 22,000 ± 2000, 40,000 ± 5000 and 90, 000 ± 6000 which may represent a monomer, dimer, and tetramer. Antibodies against the purified enzyme were obtained from rabbit after four biweekly injections with a total of 80 μg of the enzyme. A double immunodiffusion test using these antibodies and a crude extract from catfish brains showed only a single, sharp precipitin band which still retained the enzyme activity, suggesting that the precipitin band was indeed a GAD‐anti‐GAD complex. In an enzyme inhibition study, a maximum inhibition of 60–70% was obtained at a ratio of GAD protein/anti‐GAD serum of about 1:1.6. Furthermore, the precipitate from the GAD‐anti‐GAD incubation mixture also contained the enzyme activity, suggesting that the antibody was specific to GAD and that the antigen used was homogeneous. Advantages and drawbacks of the purification procedures described here and those used for mouse brain preparations are also discussed.