Investigation of the in vitro phosphorylation of gamma aminobutyrate aminotransferase by cAMP dependent protein kinase

Author

R. Keith Carr

Date of Award

3-1984

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Wesley D. Wicks

Committee Members

R. Feinberg, R. Holton, J. Koontz

Abstract

Highly purified gamma aminobutyrate aminotransferase from porcine brain was phosphorylated in vitro using cAMP dependent protein kinase catalytic subunit. The native enzyme incorporated 0.7 moles of phosphate/mole of dimer. When the phosphorylation reaction mixture was subjected to DEAE cellulose chromatography, the phosphorylated enzyme was retained longer than the unphosphorylated enzyme. The late eluting fractions contained approximately one mole of phosphate/mole of dimer.

A variety of kinetic studies of the native enzyme and the enzyme from the phosphorylation reaction mixture demonstrate that phosphorylation does not alter the enzyme's activity. Analyses of the unphosphorylated and phosphorylated enzyme resolved by DEAE chromatography demonstrate that the inactive enzyme is preferentially phosphorylated.

The presence or absence of cofactor, pyridoxal-5-phosphate, did not alter the extent of phosphorylation. However, thermal denaturation and reduction with sodium borohydride did alter the amount of phosphate incorporated into the enzyme.

A comparison of Cleveland gel fragmentation patterns of the enzyme phosphorylated under various conditions and the stoichiometry of phosphorylation indicate that only one site per dimer is phosphorylated.

Recommended Citation

Carr, R. Keith, "Investigation of the in vitro phosphorylation of gamma aminobutyrate aminotransferase by cAMP dependent protein kinase. " PhD diss., University of Tennessee, 1984.
https://trace.tennessee.edu/utk_graddiss/12840