Purification, Characterization, and Compartimentalization of Aspartate Aminotransferase Isoforms from Sphagnum

Abstract

Summary Four aspartate aminotransferase (EC 2.6.1.1) isoforms, designated AspAT I, II, III, and IV, were separated from extracts of the peat moss Sphagnum fallax (Klinggr.) by anion-exchange chromatography, displaying identical molecular weights and differences in charge. Aspartate aminotransferase III, the predominant isoform, has been purified to homogeneity as well from Sphagnum fallax as Sphagnum cuspidatum (Hoffm.). Native molecular weights of 94,000 and 95,000 with subunit molecular weights of 46,000 and 45,000 were determined for S. fallax and S. cuspidatum , respectively, indicating that the holoenzymes are dimers. The pH optimum was broad with 80% of the total activity between pH 6.4 and pH 8.9 in the direction of glutamate formation and between pH 7.8 and pH 9.5 in the direction of aspartate formation. Aspartate aminotransferase III from both species represented highly specific transaminases displaying similar K m values for their substrates aspartate, 2-oxoglutarate, glutamate, and oxaloacetate. At optimal pH and at saturated substrate concentrations the reaction was reversible, with a ratio of 3.4 to 1 in favour of aspartate formation. To study the compartmentalization of the AspAT isoforms we isolated chloroplasts and mitochondria on discontinuous gradients of Percoll, yielding 23% and 9% of the purified organelles, respectively. Aspartate aminotransferase III was located in the cytosolic fraction, whereas AspAT IV was found in the mitochondria exclusively. In contrast to other C3 plants, no AspAT activity could be detected in purified chloroplasts.