1.  Yin and Yang. Match the terms on the left with the descriptions on the right. ✓ 3

2.  Team effort. What enzymes are required for the synthesis of a glycogen particle starting from glucose 6-phosphate? ✓ 3 ✓ 4

3.  ATP is behind everything! UDP-glucose is the activated precursor for glycogen synthesis, but ultimately ATP is the power behind glycogen synthesis. Prove it by showing the reactions required to convert glucose 6-phosphate into a unit of glycogen with the concomitant regeneration of UTP. ✓ 3

4.  Force it forward. The following reaction accounts for the synthesis of UDP-glucose. This reaction is readily reversible. How is it made irreversible in vivo? ✓ 3

5.  If you insist. Why does activation of the phosphorylated b form of glycogen synthase by high concentrations of glucose 6-phosphate make good biochemical sense? ✓ 4

6.  Initiate and extend. Describe the separate roles of glycogenin and glycogen synthase in glycogen synthesis. ✓ 4

7.  An ATP saved is an ATP earned. The complete oxidation of glucose 6-phosphate derived from free glucose yields 30 molecules ATP, whereas the complete oxidation of glucose 6-phosphate derived from glycogen yields 31 molecules of ATP. Account for this difference. ✓ 5

8.  Dual roles. Phosphoglucomutase is crucial for glycogen breakdown as well as for glycogen synthesis. Explain the role of this enzyme in each of the two processes.

9.  Working at cross-purposes. Write a balanced equation showing the effect of the simultaneous activation of glycogen phosphorylase and glycogen synthase. Include the reactions catalyzed by phosphoglucomutase and UDP-glucose pyrophosphorylase. ✓ 5

10.  Achieving immortality. Glycogen synthase requires a primer. The primer was once thought to be provided when the existing glycogen granules are divided between the daughter cells produced by cell division. In other words, parts of the original glycogen molecule were simply passed from generation to generation. Would this strategy have been successful in passing glycogen stores from generation to generation? How are new glycogen molecules now known to be synthesized? ✓ 4

11.  Synthesis signal. How does insulin stimulate glycogen synthesis? ✓ 4

12.  Excessive storage. Suggest an explanation for the fact that the amount of glycogen in type I glycogen-storage disease (von Gierke disease) is increased. ✓ 4

13.  Metabolic mutants. Predict the major consequence of each of the following mutations. ✓ 5

(a) Loss of the AMP-binding site in muscle phosphorylase.

(b) Mutation of Ser 14 to Ala 14 in liver phosphorylase.

(c) Overexpression of phosphorylase kinase in the liver.

(d) Loss of the gene that encodes the inhibitor of protein phosphatase 1.

(e) Loss of the gene that encodes the glycogen-targeting subunit of protein phosphatase 1.

(f) Loss of the gene that encodes glycogenin.

14.  More metabolic mutants. Briefly predict the major consequences of each of the following mutations affecting glycogen utilization. ✓ 5

(a) Loss of GTPase activity of the G-protein α subunit.

(b) Loss of phosphodiesterase activity.

15.  Same symptoms, different cause. Von Gierke disease is frequently the result of a defect in glucose 6-phosphatase. Suggest another mutation in glucose metabolism that causes symptoms similar to those of von Gierke disease.

16.  Again, von Gierke. People suffering from von Gierke disease release a small amount of glucose into the blood after the injection of glucagon. How is this result possible?

17.  I know I’ve seen that face before. UDP-glucose is the activated form of glucose used in glycogen synthesis. However, we have already met other similar activated forms of carbohydrate in our consideration of metabolism. Where else have we seen UDP-carbohydrate?

18.  Carbohydrate conversion. Write a balanced equation for the formation of glycogen from galactose. ✓ 3

19.  Removing all traces. In human liver extracts, the catalytic activity of glycogenin was detectable only after treatment with α-amylase, an enzyme that hydrolyzes α-1,4-glucosidic bonds. Why was α-amylase necessary to reveal the glycogenin activity?

20.  Telltale products. A sample of glycogen from a patient with liver disease is incubated with orthophosphate, phosphorylase, the transferase, and the debranching enzyme (α-1,6-glucosidase). The ratio of glucose 1-phosphate to glucose formed in this mixture is 100. What is the most likely enzymatic deficiency in this patient? ✓ 3

21.  Glycogen isolation 1. The liver is a major storage site for glycogen. Purified from two samples of human liver, glycogen was either treated or not treated with α-amylase and subsequently analyzed by SDS-PAGE and western blotting with the use of antibodies to glycogenin (Chapter 5). The results are presented in the following illustration:



(a) Why are no proteins visible in the lanes without amylase treatment?

(b) What is the effect of treating the samples with α-amylase? Explain the results.

(c) List other proteins that you might expect to be associated with glycogen. Why are other proteins not visible?

22.  Glycogen isolation 2. The gene for glycogenin was transfected into a cell line that normally stores only small amounts of glycogen. The cells were then manipulated according to the following protocol, and glycogen was isolated and analyzed by SDS-PAGE and western blotting by using an antibody to glycogenin with and without α-amylase treatment (Chapter 5). The results are presented in the following illustration.



The protocol: Cells cultured in growth medium and 25 mM glucose (lane 1) were switched to medium containing no glucose for 24 hours (lane 2). Glucose-starved cells were re-fed with medium containing 25 mM glucose for 1 hour (lane 3) or 3 hours (lane 4). Samples (12 mg of protein) were either treated or not treated with α-amylase, as indicated, before being loaded on the gel.

(a) Why did the western analysis produce a “smear”—that is, the high-molecular-weight staining in lane 1(-)?

(b) What is the significance of the decrease in high-molecular-weight staining in lane 2(-)?

(c) What is the significance of the difference between lanes 2(-) and 3(-)?

(d) Suggest a plausible reason why there is essentially no difference between lanes 3(-) and 4(-)?

(e) Why are the bands at 66 kDa the same in the lanes treated with α-amylase, despite the fact that the cells were treated differently?