Doctoral Dissertations

Date of Award

3-1988

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major Professor

Wesley D. Wicks

Committee Members

Jorge Churchich, John Koontz, Carl Wust

Abstract

Synthesis of the rat liver enzyme, tyrosine aminotransferase (TAT), is regulated at both the transcriptional and post-transcriptional level by cAMP through the cAMP dependent protein kinase (A-kinase).

The first part of this work deals with an attempt to isolate A-kinase substrates which may be involved in the induction of TAT synthesis at the transcriptional level. Antibodies to a synthetic peptide whose amino acid sequence represented the phosphorylation site found in L-type pyruvate kinase were developed in rabbits. This phosphorylation site is a good consensus sequence for a class of A-kinase phosphorylation sites. Although high titer antibodies to the synthetic peptide were developed, they showed little or no interaction with intact pyruvate kinase and would not cross-react with other A-kinase substrates or with any specific soluble proteins found in cultured hepatoma cells. It was concluded that the conformation of intact proteins containing A-kinase phosphorylation sites was different from the conformation of the peptide and that the anti-peptide antibody could not induce a conformational change in intact proteins that would support strong binding.

The second part of this work deals with attempts to define the point at which cAMP affects TAT synthesis post-transcriptionally to cause a 4 to 5 fold decrease in the enzyme's synthesis. As a direct test of the possibility that cAMP exerts this effect by destabilizing TAT mRNA, the rate of decay of the mRNA was measured using a transcriptional inhibitor, Northern blot analysis and an internal standard consisting of prelabeled rRNA. It was found that the half-life of TAT mRNA (2.0 ± 0.2 hr) was not changed by treatment of cultured hepatoma cells under conditions which increase intracellular cAMP. The in vitro translatability of TAT mRNA was then assessed in mRNA dependent reticulocyte lysates. It was found that the ability of TAT mRNA to direct protein synthesis was the same regardless of whether or not the RNA was isolated from cells treated to raise intracellular levels of cAMP. It was proposed that cAMP may exert its post-transcriptional effects at the level of translation but not through a change in TAT mRNA functionality.

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