| Pyruvate-dependent γ-aminobutyrate transaminase (GABA:pyruvate-T; EC 2.6.1.19) from tobacco (Nicotiana tabacum L. cv. Samsun) leaf was partially purified 1530-fold by FPLC anion-exchange chromatography of proteins from isolated mitochondria. GABA:pyruvate-T had a broad pH optimum ranging from 8–10. The partially purified enzyme had a K m of 1.5 mM for GABA and 300 μM for pyruvate. Both pyruvate- and 2-oxoglutarate-dependent GABA-T activities were present in crude extracts and lysed mitochondrial preparations, and could be separated from each other by anion exchange chromatography. Two-oxoglutarate-dependent activity was not detected with the partially purified enzyme. Pyruvate-dependent activity was further purified by a combination of affinity- and gel filtration-chromatography, native polyacrylamide gel electrophoresis (PAGE), isoelectric focussing and denaturing sodium dodecyl sulphate PAGE. The isoelectric point of the native protein was 4.8 and the subunit size was 55 kDa. The data indicate the existence of a pyruvate-specific mitochondrial GABA-T. Polymerase chain reaction experiments with heterologous probes, and screening of a tobacco-leaf cDNA library with Arabidopsis dbESTs homologous to other non-plant gaba-t genes did not result in identification of a tobacco gaba-t gene. However, a small peptide sequence from the purified tobacco GABA-T enzyme had 94% identity to an Arabidopsis data base expressed sequence tag, which in turn had more than 30% identity to many known non-plant gaba-t sequences. This sequence will facilitate the isolation of a full length plant gaba-t sequence. |
