Purification and properties of the glutamine- and N-acetyl-L-glutamate-dependent carbamoyl phosphate synthetase from liver of Squalus acanthias

Abstract

The L-glutamine- and N-acetyl-L-glutamate-dependent carbamoyl phosphate synthetase III present in liver of spiny dogfish (Squalus acanthias) has been purified to a high state of purity. The purified enzyme has a Mr congruent to 160,000 and is subject to self-association which is facilitated by the presence of MgATP, L-glutamine, and N-acetyl-L-glutamate. The enzyme exhibits hysteretic properties. The time course of the reaction is characterized by a lag or a burst in activity, depending upon preincubation conditions, which can last up to 30 min. The lag period can be eliminated by preincubating the enzyme at 26 degrees C (but not at 4 degrees C) in the presence of the above three ligands. Mg2+ in excess of that required to complex ATP as MgATP and N-acetyl-L-glutamate are both required for full activity. The requirement of K+ for activity can be replaced by NH4+, but not by Na+. Ammonia can act as a substrate in place of L-glutamine, but the maximal rate is much less than that which can be obtained with L-glutamine. The glutamine-dependent activity is inhibited by ammonia. Apparent Km values under optimal conditions for N-acetyl-L-glutamate, L-glutamine, MgATP, bicarbonate, and ammonia are 0.013 mM, 0.16 mM, 0.35 mM, 1.7 mM, and 2 mM, respectively. The apparent Km for N-acetyl-L-glutamate decreases when the concentration of L-glutamine increases, and vice versa. The apparent Km values for these two ligands are increased when urea is present at normal physiological concentrations (0.4 M), and the activity of the enzyme is significantly affected by changes in urea concentration. Compounds known to act as allosteric effectors on other glutamine-dependent carbamoyl phosphate synthetases had little or no effect on this enzyme. The properties of the enzyme are consistent with the view that the function of this carbamoyl phosphate synthetase III is related to the synthesis of urea which is retained in these species as a mechanism for osmoregulation.