Carbamoyl-phosphate synthase (ammonia) of rat and axolotl liver: determination of immunological cross-reactivity without purification of the axolotl enzyme

A method has been developed to establish the degree of cross-reactivity of an antiserum raised against purified carbamoyl-phosphate synthase (ammonia) from adult rat liver, toward a homologous enzyme from another species without purification of the latter enzyme. For that purpose the ratio between enzyme activity and enzyme protein, i.e., the molecular specific activity in crude liver extracts, was determined by two independent methods. When the molecular specific activity was determined by means of radioimmunoassay using a specific antiserum raised against rat liver carbamoyl-phosphate synthase this ratio was a factor four higher for the axolotl than for the rat. Both axolotl and rat liver carbamoyl-phosphate synthase appear as a very prominent band with an apparent molecular weight of 165,000 after sodium dodecyl sulphate-polyacrylamide gelelectrophoresis. Therefore, the amount of enzyme protein could be determined by means of densitometry of this band using purified rat liver carbamoyl-phosphate synthase as a standard. The ratio between enzyme activity and enzyme protein calculated from this method appeared to be the same for axolotl and rat. From these results it can be deduced that the degree of cross-reactivity between rat and axolotl liver carbamoyl-phosphate synthase is approximately 25% when using the antiserum raised against the rat liver antigen