Carboxylate Inhibition of Apoaspartate Aminotransferase

Summary

The pyridoxamine 5′-phosphate (PMP) activation of the apoenzyme of pig heart cytosolic aspartate aminotransferase (EC 2.6.1.1, AspAT) as a function of buffer, pH, salt and thiol was investigated. At 100 mM, the cationic buffers used, i.e., bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris) and imidazole at pH 6.0 and Tris at pH 8.3, allowed maximum activation with or without 500 mM NaCl and 2.5 mM glutathione. Anionic buffers, i.e., acetate, butyrate, aspartate and glutamate at pH 5.5 and N-(2-acetamido)-2-iminodiacetate (ADA) and cacodylate at pH 6.0 permitted less activation. Malonate, succinate, gluta-rate, and adipate at pH 5.5, citrate at pH 6.0 and P_i at pH 7.0 were completely inhibitory. At lower concentrations, oxalate, malonate and adipate were the most inhibitory of the dicarboxy-lates. In the absence of NaCl and glutathione, activation was diminished in anionic buffers at pH 5.5 and 6.0. The protective effect of the additives was due to NaCl. Dicarboxylates were generally more inhibitory at low pH than at high pH. For P_i, the opposite pattern was obtained.