Phosphorylation of tyrosine aminotransferase by cyclic AMP dependent protein kinase

Author

Charles Spielholz

Date of Award

3-1984

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Wesley D. Wicks

Committee Members

John Koontz, Jayant G. Joshi

Abstract

Tyrosine aminotransferase (E.C. 2.6.1.5, TAT) has been shown to be a phosphoprotein in rat liver and cultured H-35 hepatoma cells (Lee and Nickol, 1974; Wicks and Su, 1978). Stellwagen and Kohli (1981) have shown that phospho-TAT isolated from cultured hepatoma cells may contain at least two tryptic phospho-peptides. They have also shown that TAT can be phosphorylated in vitro by cyclic AMP dependent protein kinase (PK-C).

Undegraded TAT was purified to near homogeneity from rat liver and was confirmed to be a substrate for beef heart PK-C in vitro. A polyclonal antibody, which was found to be specific for TAT, was used to quantitate the amount of phosphate incorporated into the enzyme. Alteration of reaction conditions indicated that phosphate incorporation was maximum with a PK-C to TAT ratio of 7:1 using 200 µM ATP for 60 minutes at 37°C. The amount of phosphate incorporated into TAT in this work was significantly higher than that obtained by Stellwagen and Kohli (1981): 0.7 moles phosphate/mole TAT subunit versus 0.2 moles phosphate/mole TAT subunit.

Phospho-peptide maps of TAT phosphorylated in vitro by PK-C and TAT obtained from cells incubated with ³²p and treated with dexamethasone with and without α-bromo-cyclic AMP were produced by the method of Cleveland et al. (1977) using a-chymotrypsin, papain and subtilisin. significant difference in phospho-peptide maps of TAT isolated from cells that were treated with 8-bromo-cyclic AMP and cells that were not. Also, the major sites phosphorylated in TAT in vitro by PK-C were not the same as the major sites phosphorylated in vivo. There was, however, some homology between minor sites of phosphorylation in vivo and in vitro. These results indicate that PK-C is not responsible for most TAT phosphorylation in vivo and that a cyclic AMP independent protein kinase is involved.

Recommended Citation

Spielholz, Charles, "Phosphorylation of tyrosine aminotransferase by cyclic AMP dependent protein kinase. " Master's Thesis, University of Tennessee, 1984.
https://trace.tennessee.edu/utk_gradthes/14721