Michael M. Crerar PhD (York) E-mail: mcrerar@yorku.ca Research Areas: Molecular Biology and Biochemistry, Cell Biology, Genetics |
To obtain a better understanding of protein allostery, as well as gene control in muscle, we are examining glycogen phosphorylase regulation at both enzymatic and genetic levels. The enzyme catalyzes glycogen breakdown and as such plays a major role in carbohydrate metabolism. In mammals, the enzyme exists as a family of isozymes which are catalytically similar but encoded by distinct, structurally related genes. Muscle (M), brain (B) and liver (L) isozymes exist which derive their names from the tissues where they predominate. The B isozyme is also present in foetal tissues and is replaced by the M or L isozymes during development.
Kinetic and structural analysis carried out primarily with the rabbit M isozyme indicates that phosphorylase is a complex allosteric enzyme. A variety of ligands bind to distinct sites on the enzyme and lead to either enzyme activation or inhibition. Interestingly, the M, B and L isozymes respond differently to allosteric activation by either phosphorylation or AMP. As a start towards understanding the structural determinants that are involved, we have used chimeric M/B isozymes to implicate certain regions of the protein in various aspects of differential activation. To examine this in finer detail, we are now analysing the effects of single and multiple amino acid exchanges in these regions. Using comparative protein sequence analysis, we have analyzed the evolution of allosteric control of phosphorylases from a wide variety of organisms. This analysis indicates that structural pathways in the enzyme involved in activation by phosphorylation/AMP likely differ from those involved in inhibition by glucose-6-P, an early allosteric control. To functionally assess the independence of these control pathways, we plan to examine the effect of mutating potentially important residues in the rabbit M isozyme. Phosphorylases from lower organisms vary dramatically in their responses to positive control mechanisms and we plan to examine the negative control of these enzymes as an additional step towards understanding the structural interactions within the enzyme involved in allosteric control.
We have also been involved in isolating and analysing the genes for all three mammalian isozymes from the rat. We have demonstrated that these genes are located on separate chromosomes in the mouse and have shown that mRNA accumulation governs in large part the tissue specific and developmental control of this gene family. We have isolated and sequenced the 5' flanking region of the rat M phosphorylase gene and plan to identify muscle specific regulatory elements in this region by analysing the ability of cloned gene constructs to confer regulated expression in differentiating muscle cell cultures in vitro. We are also interested in analysing the regulatory factors which control the down-regulation of the B isozyme gene during muscle development.
