Date of Award:

5-1979

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Chemistry and Biochemistry

Committee Chair(s)

Elizabeth A. Boeker (Committee Chair)

Committee

Elizabeth A. Boeker

Abstract

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 μmole/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 14C-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.

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