Studies were made of requirements of a quantitative assay of glutamine-dependent carbamyl phosphate synthetase from mouse spleen. Of miscellaneous factors that affected its activity, the followings were found to need special attention during the assay: (1) extreme lability of the enzyme at a low temperature (0 to 10°C), and a marked variation of the temperature-dependent stability in the presence of substrates and of organic solvents; (2) variation of enzyme activity depending upon the concentration of the organic solvents, such as dimethyl sulfoxide and glycerol, which are necessary for enzyme stabilization in an assay mixture; (3) an absolute requirement of the enzyme activity for free magnesium ion (Mg2+). In analysis using calculated concentrations of free Mg2+ and magnesium-ATP complex (MgATP2−), 0.8 and 0.4 mM free Mg2+ gave the half maximal activation for the reactions with MgATP2− fixed at 3.0 and 10 mM, respectively. Effects of pH and substrate concentrations on the reaction velocity were studied and apparent Km values for MgATP2−, L-glutamine, and bicarbonate are described.
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Tatibana et al. (1972) studied this question.