Purification and properties of rabbit brain and liver 4-aminobutyrate aminotransferases isolated by monoclonal-antibody immunoadsorbent chromatography.
The use of a monoclonal-antibody immunoaffinity column for the rapid isolation of 4-aminobutyrate aminotransferases (EC 2.6.1.19) from rabbit brain and liver is described. Homogeneous enzyme protein is eluted from the immunoadsorbent with 100mM-citrate buffer, pH5, and remains stable at 4 degrees C for several days. One such column (bed volume 8 ml) has been used 40 times in a 9-month period to isolate 10-15 units of enzyme activity (specific activity approx. 3.5-7.5 units/mg) per extraction. Kinetic and spectral analysis of the enzymes from the two tissues revealed a close similarity. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis showed the isolated enzyme to have a monomeric Mr of 52 000, and this was confirmed by h.p.l.c. gel exclusion at pH 5.0. The results of Sephadex G-100 chromatography at different pH values are taken to indicate that the enzyme behaves as a dimer at pH 7.0 and above, but as a monomer at pH 5.0. 4-Aminobutyrate aminotransferase isolated from the brain by the procedure of Fowler & John [(1981) Biochem. J. 197, 149-152] is more stable than the immunoaffinity-purified material, and has been shown to contain a contaminant protein of Mr 84 000 that exhibits succinic semialdehyde dehydrogenase activity.
D000612:4-Aminobutyrate Transaminase / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001921:Brain / Q000201:enzymology*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D007163:Immunosorbent Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D011817:Rabbits; D013053:Spectrophotometry; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D000612:4-Aminobutyrate Transaminase