Carbamoylphosphate Synthetase I of Rat‐Liver Mitochondria

Abstract

An improved purification of carbamoylphosphate synthetase I from rat liver mitochondria is described. The enzyme is essentially homogeneous with enhanced specific activity and stability. The enzyme is stable at alkaline pH in concentrated solution of salts. Kinetic studies indicate two type of inactivation, reversible and irreversible, depending on pH. The monomeric unit of carbamoylphosphate synthetase I consists of one polypeptide chain. The protein migrates in dodecylsulfate/polyaacrylamide gel as a single component corresponding to a molecular weight of about 155000. The molecular weight of the polypeptide chain determined by sedimentation equilibrium in 6 M guanidine hydrochloride containing dithioerythritol is 158000 ± 5000. The weight‐average molecular weight of the native enzyme, 188000, suggests that the monomeric form is the predominant form of the enzyme, under the conditions described. Glutamine is inactive as a substrate. With ammonium, carbamoyl phosphate synthesis is optimal in the pH range 6.8–7.6. Mg 2+ and Mn 2+ are equally effective metal ions in the direction of carbamoyl phosphate synthesis, but excess Mn 2+ is inhibitory. At less than saturating concentrations of substrate, considerable activation of the enzyme is observed when metal ions are in excess of ATP; when MgATP 2− concentration is held constant at 1 mM and 10 mM, plots of v versus free Mg 2+ concentration are hyperbolic, and extrapolate to zero. These results suggests that free divalent metal ion is required in the forward reaction. The apparent K m of the substrates is similar to other preparations of the enzyme. The determination of the maximal activity of carbamoylphosphate synthetase in rat liver mitochondria is reported, providing a measure of the total enzyme concentration that is about 1–1.5 mM. The purification procedure described also provides an easy method to obtain ornithine transcarbamylase in partially purified form.

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