One of your colleagues has obtained a sample of muscle phosphorylase b that is known to be relatively inactive. She has approached you for advice on how to set up an appropriate assay. She has the following items available, not all of which are appropriate for this study. Help her out by selecting the items that she should use and what their purpose is in the assay, and then explain why each of the other items would not be useful.1. 100 uM AMP2. 100 uM GTP3. 100 uM glucose4. 100 uM glucose 6-phosphate5. Branched glycogen6. Amylose (i.e. unbranched glycogen)7. 50 mM HEPES buffer, pH 7.58. 50 mM potassium phosphate buffer, pH 7.5Write out a short explanation for each of the above items, and upload your answers by the due date.

Glycogen is storage-type homopolysaccharides that contain two types of glucose polymers: amylose: D-glucose residues are linked via α(1→4)glycosidic linkage to form long straight chains amylopectin: successive D-glucose residues are linked via α(1→4) glycosidic linkage to form long unbranched chains with branching occurring every 8 to 12 residues. The branches are linked via α(1→6) glycosidic linkage Glycogen phosphorylase releases glucose 1-phosphate from glycogen. In the active form, it is called phosphorylase a and in the inactive form, it is phosphorylase b. An increase in the cellular concentration of cAMP activates protein kinase A which then activates phosphorylase b kinase. Phosphorylase b kinase phosphorylates phosphorylase b and converts it to phosphorylase a. Phosphorylase a then breaks down glycogen to release glucose 1-phosphate. In muscle cells, the cellular concentration of AMP increases as a consequence of ATP breakdown. AMP binds to the regulatory site on phosphorylase b and activates it. Phosphorylase then catalyses the breakdown of glycogen to glucose 1-phosphate. Phosphorylase can cleave only 1→4 glycosidic bonds so it can release glucose 1-phosphate only from the amylose chain of glycogen. For conducting the assay, the reagents required are : 100 uM of AMP: to activate phosphorylase b Amylose: acts as the substrate 50 mM of potassium phosphate buffer: to maintain optimal pH and adds inorganic phosphates to the reaction mix. The assay doesn't require the following reagents: 100 uM GTP and 100 uM glucose 6-phosphate: phosphorylase catalyse the addition of inorganic phosphate to organic substrates. GTP and G6P are not required as phosphate donors. 100 uM glucose and Branched glycogen: cannot act as a substrate to assess the catalytic activity of the assay. 50 mM HEPES buffer, pH 7.5: buffering range is less than potassium phosphate buffer and cannot flourish the reaction mix with inorganic phosphates.