Glycogen phosphorylase kinase

Calcium ions released from the SR are required to initiate contraction and activate glycogen phosphorylase kinase... 

Answer Kinase (factor(s)) PKA (cAMP) PKG (cGMP) PKC (Ca2 +, DAG) Ca2+/CaM kinase (Ca2+, CaM) cyclin-dependent kinase (cyclin) protein Tyr kinase (ligand for the receptor, such as insulin) MAPK (Raf) Raf (Ras) glycogen phosphorylase kinase (PKA). 

In this laboratory exercise, you will study the effects of phosphorylation and allosteric regulation on the activity of glycogen phosphorylase. In the first experiment, you will phosphorylate glycogen phosphorylase b in vitro using y-[32P]ATP and glycogen phosphorylase kinase. In the second experiment, you will study the effect of phosphorylation on glycogen phosphorylase activity, as well as the effect of AMP on glycogen phosphorylase b activity with the use of a coupled enzymatic (kinetic) assay (Fig. 15-3). 

Glycogen phosphorylase kinase stock solution (Sigma catalog P-2014) ... 

Set up the reactions shown in the table on the next page in 1.5-ml microcentrifuge tubes. The stock glycogen phosphorylase and the stock glycogen phosphorylase kinase solutions should be diluted to the proper concentrations with 30 mM cysteine buffer, pH 7.0. 

Determine the concentration of glycogen phosphorylase kinase present in reaction tube 3 (from step 2) using the following equation ... 

After you have completed these calculations, you will have obtained two values for the concentration of glycogen phosphorylase kinase present in this assay, one based on the 5-min time point and one based on the 10-min time point. From these data, determine the average value of glycogen phosphorylase kinase present in this assay (units/ml of enzyme). 

Glycogen phosphorylase kinase stock solution—Dissolve a 2500-unit package size of glycogen phosphorylase kinase (Sigma catalog P-2014) in 1 ml of concentrated enzyme storage buffer (gently, do not use a vortex mixer). Divide into 20-pl aliquots and store at —20°C. Dilute this 2500-unit/ml stock solution to the appropriate concentration (see protocol) just before use. Store the dilute enzyme on ice until it is ready to be used. 

Glycogen phosphorylase kinase solution—Dilute 40 /xl of the 2500-unit/ml glycogen phosphorylase kinase stock solution in 4.96 ml of 30 mM cysteine, pH 7.0. 

GPK Glycogen phosphorylase kinase, a serine/threonine-specihc protein kinase which activates glycogen... 

Protein kinases like glycogen-phosphorylase kinase act on specific amino-acid sequences, called consensus sequences. Protein-kinase-a acts on the serine/threonine residue in the sequence X-arg-arg/lys-X-ser/thr-asx. Cyclin-dependent-kinase-2 acts on the same residues in the sequence X-ser/thr-pro-X-lys/arg. Only the alcoholic amino acids serine, threonine and tyrosine can be phosphorylated. 

The answer is b. (Murray, pp 199-207. Scriver, pp 1521-1552. Sack, pp 121-138. Wilson, pp 287-31 77) In the presence of low blood glucose, epinephrine or norepinephrine interacts with specific receptors to stimulate adenylate cyclase production of cyclic AMP Cyclic AMP activates protein kinase, which catalyzes phosphorylation and activation of phosphorylase kinase. Activated phosphorylase kinase activates glycogen phosphorylase, which catalyzes the breakdown of glycogen. Phosphorylase kinase can be activated in two ways. Phosphorylation leads to complete activation of phosphorylase kinase. Alternatively, in muscle, the transient increases in levels of Ca" associated with contraction lead to a partial activation of phosphorylase kinase. Ca" " binds to calmodulin, which is a subunit of phosphorylase kinase. Calmodulin regulates many enzymes in mammalian cells through Ca" binding. 

Glycogen phosphorylase kinase Glycogen synthase Guanylate cyclase Myosin light-chain kinase NADPH oxidase Phosphofructokinase... 

In the sections below, we describe the general characteristics of these regulatory mechanisms and illustrate the first three with glycogen phosphorylase, glycogen phosphorylase kinase, and protein kinase A. 

Glycogen phosphorylase also can be activated through phosphorylation by glycogen phosphorylase kinase. Either phosphorylation or AMP binding can change the enzyme to the same fully active conformation. The phosphate is removed by protein... 

Fig. 9.9. Activation of muscle glycogen phosphorylase by AMP and by phosphorylation. Muscle glycogen phosphorylase is composed of two identical subunits. The substrate binding sites in the active catalytic site are denoted by S. AMP binds to the allosteric site, a site separate from the active catalytic site. Glycogen phosphorylase kinase can transfer a phosphate from ATP to one serine residue in each subunit. Either phosphorylation or binding of AMP causes a change in the active site that increases the activity of the enzyme. The first event at one subunit facilitates the subsequent events that convert the enzyme to the fully active form.

One of the enzymes activated by Ca -calmodulin is muscle glycogen phosphorylase kinase, which is also activated by protein kinase A (see Fig. 9.9). When a... 

Sakai, K. Matsumura, S. Okimura, Y. Yamamura, H. Nishizuka, Y. Liver glycogen phosphorylase kinase. Partial purification and characterization. J. Biol. Chem., 254, 6631-6637 (1979)... 

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