Date of Award:


Document Type:


Degree Name:

Doctor of Philosophy (PhD)


Chemistry and Biochemistry

Committee Chair(s)

Elizabeth A. Boeker (Committee Chair)


Elizabeth A. Boeker


Thomas F. Emery


Thomas M. Farley


Anne J. Anderson


Daniel L. Comins


An efficient and simple purification procedure for cow brain glutaminase (L- glutamine amidohydrolase, EC 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 >106 respectively. It appeared that activation of cow brain glutaminase by phosphate was due to formation of dimers or higher molecular weight polymers.



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