Inactivation of gamma-aminobutyric acid aminotransferase by various amine buffers.
It is hypothesized that buffers capable of forming a Schiff base with the PLP of gamma-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.
Hopkins
M H
MH
Department of Chemistry, Northwestern University, Evanston, Illinois 60208-3113.
Bichler
K A
KA
Su
T
T
Chamberlain
C L
CL
Silverman
R B
RB
D000612:4-Aminobutyrate Transaminase / Q000037:antagonists & inhibitors*; D000445:Aldehyde Oxidoreductases / Q000302:isolation & purification*; D000818:Animals; D001921:Brain / Q000201:enzymology; D011732:Pyridoxal Phosphate / Q000378:metabolism; D012545:Schiff Bases; D050644:Succinate-Semialdehyde Dehydrogenase; D013552:Swine; D014325:Tromethamine / Q000031:analogs & derivatives / Q000378:metabolism*
D012545:Schiff Bases; D014325:Tromethamine; D011732:Pyridoxal Phosphate; C034249:1,3-bis(tris(hydroxymethyl)methylamino)propane; D000445:Aldehyde Oxidoreductases; D050644:Succinate-Semialdehyde Dehydrogenase; D000612:4-Aminobutyrate Transaminase