Inactivation of γ-Aminobutyric Acid Aminotransferase by Various Amine Buffers

Abstract

It is hypothesized that buffers capable of forming a Schiff base with the PLP of y-aminobutyric acid aminotransferase (GABA-AT) may lead to denaturation and inactivation of the enzyme. On the basis of this hypothesis three new methods for the selective destruction of GABA-AT in GABAse (a commercial bacterial source of a mixture of GABA-AT and succinic semialdehyde dehydrogenase [SSDH]) and from pig brain are described: (1) dialysis against a primary or secondary amine buffer; (2) gel filtration with a primary or secondary amine buffer as eluent; (3) inactivation with gabaculine followed by dialysis or gel filtration with pyrophosphate buffer. The SSDH activity in GABAse, which remains unchanged by all of these methods, may then be used in a coupled assay to measure the activity of GABA-AT from different sources. These results also suggest that the use of primary and secondary amine buffers should be avoided when inhibitors are being tested with GABA-AT.

Key Words: :

• γ-Aminobutyric acid aminotransferase
• amine buffers
• Tris
• Bis-tris propane
• Bis-tris
• pyrophosphate
• succinic semialdehyde dehydrogenase