Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Donald J. Graves
In the first section of this dissertation, the role of the amino-terminal region of glycogen phosphorylase was studied by removal of this region and determination of the properties of the truncated phosphorylase analog. Limited proteolysis of phosphorylase by subtilisin Carlsberg in the presence of the phosphorylase inhibitor caffeine produced a phosphorylase analog, named phosphorylase b[subscript]sp S', missing only the amino-terminal 16 to 18 residues. Phosphorylase b[subscript]sp S' was purified by anion-exchange high performance liquid chromatography (HPLC). The maximal velocity of phosphorylase b[subscript]sp S' was 47% higher than that of phosphorylase b, while the Michaelis constants were similar. The subunit interactions in phosphorylase b[subscript]sp S' were weaker than those in phosphorylase b. That removal of the amino-terminal region of phosphorylase changes the properties of the enzyme implies that this region interacts with the rest of the protein, which conflicts with the conclusion from X-ray diffraction studies that the amino-terminal region of phosphorylase is mobile;In the second section of this dissertation, the effect of phosphorylation of one subunit of phosphorylase on the conformation of the enzyme was studied by comparing phosphorylase b (nonphosphorylated) and phosphorylase ab (one subunit phosphorylated) as substrates for phosphorylase kinase. Phosphorylase ab was purified by anion-exchange HPLC and shown to be stable under the conditions of the phosphorylase kinase assay. It was found that phosphorylase kinase had a lower Michaelis constant for phosphorylase ab than for phosphorylase b, which implies that phosphorylation of one subunit of phosphorylase b causes conformational changes that make the other subunit a better substrate for the kinase. Using an HPLC assay for phosphorylases b, ab and a during the full course of phosphorylation, it was found that when the monomeric [gamma][delta] complex of phosphorylase kinase subunits was used as the enzyme in place of the multimeric holophosphorylase kinase, the difference in the rates of phosphorylation of the two subunits decreased. This implies that holophosphorylase kinase might be capable of simultaneous phosphorylation of the two subunits of phosphorylase.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
William Richard Harris
Harris, William Richard, "Regulation of glycogen phosphorylase by its amino-terminal region " (1987). Retrospective Theses and Dissertations. 8649.