An efficient and simple purification procedure for cow brain glutaminase (L- glutamine amidohydrolase, EC 3.5.1.2) is described. The main steps consisted of acetone extraction and French press treatment in the presence of phosphate ions, precipitation of nucleic acids and lipids by 0.08 % protamine·S04 and high speed centrifugation (300,000xg), ammonium sulfate fractionation, and gel filtration on Sepharose 4B first as the low molecular weight and then as the aggregated form. The yield was 22% and the final preparation had a specific activity of 142 μmoles/min/mg. The purification was more than 8000-fold over crude brain homogenate. The enzyme showed one strong and one diffuse band on SDS gel electrophoresis, suggesting that the enzyme was highly purified. Phosphate activated cow brain glutaminase with a sigmoidal concentration dependence. The activation was time dependent, a function of the pretreatment, and was enhanced by high concentrations of protein. The molecular weight of cow brain glutaminase also depended on the nature of the buffer in which it is dissolved. Gel filtration on Sepharose 4B was used to determine the molecular weight. Three forms of glutaminase were observed, one each in tris/acetate, phosphate, and borate/ phosphate buffer. These were interconvertible and have molecular weights of 170,000, 300,000, and >10^6 respectively. It appeared that activation of cow brain glutaminase by phosphate was due to formation of dimers or higher molecular weight polymers.

A simple, quick, and sensitive radiometric assay for glutaminase has been established. This assay uses L-(U-14C)-glutamine as the substrate and measures the 14co2 produced when the reaction is coupled to glutamate decarboxylase. An efficient purification procedure for cow brain glutaminase has also been developed; the yield is 12%. Steps include acetone extraction, ammonium sulfate fractionation, calcium phosphate gel treatment, and differential gel filtration on Sepharose 4B. The final preparation has a specific activity of 385 Mmole/min/mg and shows a single protein band on polyacrylamide gel electrophoresis in sodium dodecyl sulfate; this band corresponds to a subunit molecular weight of 82,000. Polyacrylamide gel electrophoresis studies did not reveal any impurities; the enzyme activity coincided with the protein staining. This enzyme is stable between pH 7.5 and 9.2 and has maximal activity around 8.8. The activity of glutaminase appears to be independent of the nature of the buffer with which it was equilibrated before it was assayed. The enzyme absolutely requires phosphate for activity; the dependence is sigmoidal and has a Hill coefficient of 2.2. The phosphate concentration that gives half maximal velocity is 50 mM. At 0.2 M potassium phosphate (pH 8.8), the dependence of activity on glutamine is hyperbolic; the observed Kgln is 17 mM. Neither amnonium ion (0.1 M) nor citrate, succinate, or glutarate (57 mM) inhibit the enzyme activity. Glutamate inhibits competitively with respect to glutamine. Phosphate affects both Kgln and Kglu the same way. A radioactivity exchange study was not able to detect incorporation of 14C-glutamate into 14 c-glutamine. The results are consistent with a model in which ammonia is released irreversibly from the enzyme-substrate complex and is the first product to be released.