Brain L-glutamate decarboxylase: purification and subunit structure.

Area of Science:

• Biochemistry
• Neuroscience
• Enzymology

Background:

• Glutamate decarboxylase (GDCase) is crucial for synthesizing gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the mammalian brain.
• Understanding GDCase's biochemical properties is essential for insights into neurological function and disorders.

Purpose of the Study:

• To purify and characterize glutamate decarboxylase (GDCase) from whole rat brain.
• To determine the enzyme's molecular mass, subunit composition, isoelectric point, kinetic parameters, and substrate specificity.

Main Methods:

• Multi-step protein purification involving DEAE-cellulose chromatography, hydroxylapatite chromatography, gel filtration, and preparative polyacrylamide gel electrophoresis.
• Enzyme activity assays, nondenaturing and gradient polyacrylamide gel electrophoresis, isoelectric focusing, SDS-PAGE, and immunoblotting.

Main Results:

• GDCase was purified approximately 1300-fold with a specific activity of 2.4 units/mg protein.
• The native enzyme has a molecular mass of approximately 120 kDa and an isoelectric point of 5.4.
• GDCase exists as a dimer of 40 kDa subunits, with a Km for glutamate of 1.59 x 10(-3) M and a pH optimum around 7.3.

Conclusions:

• The study successfully purified and characterized rat brain GDCase, elucidating its quaternary structure and key biochemical properties.
• The findings provide a foundation for further research into GDCase's role in GABAergic neurotransmission and its potential involvement in neurological conditions.