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Phosphorylase-b

(1,4 a-D-Glucan:Orthophosphate a-D-glucosyltransferase; EC 2.4.1.1.)

Phosphorylases catalyze the breakdown of glycogen in liver and muscle. Similar enzymes, occurring in plants, catalyze the hydrolysis of starch. The hydrolysis of glycogen is represented by the following reaction:

                    PHOSPHORYLASE
(a-1,4-Glucosyl)n + Pi ------> (a-1,4 Glucosyl)n-1 + a-D-Glucose-1-P
The phosphorylase of skeletal muscle occurs in two forms, the active form (phosphorylase-a) and a much less active form (phosphorylase-b). These two forms are interconvertible. Rabbit muscle phosphorylase has been extensively studied. Phosphorylase-a has a molecular weight of 370,000 and phosphorylase-b has a molecular weight of 185,000.


1.Source: Rabbit Muscle
   Form: Freeze-dried 
   Solubility: Soluble in distilled water or dilute buffer 
   Stability: -20 C; -4 F 
   Activity (approx.): 60 U/mg protein 
   Protein (approx.): 90% (biuret) 
   Catalog No.: 104A0060 
$1.20/mg


That amount of enzyme which will liberate one micromole of glucose-1-phosphate from glycogen and orthophosphate, in the presence of 5'-AMP, per minute at pH 6.8 at 37°C.


The assay method which is described (Methods of Enzymatic Analysis, Bergmeyer, H.U. ed; Vol 1, 505, 1974, Academic Press, New York).


  1. 0.05 M Potassium phosphate buffer, pH 6.8 containing 0.1 mM EDTA.
  2. 0.1 M Magnesium chloride in distilled water.
  3. NADP (10 mg/ml) in distilled water. Prepare fresh.
  4. Glycogen (6 mg/ml) in buffer. Prepare fresh.
  5. 1 mM adenosine 5'-monophosphate (sodium salt) in buffer. Prepare fresh.
  6. Phosphoglucomutase (PGM) solution. Prepare a solution in cold 1% bovine serum albumin to yield a concentration of 20 U/ml. Must be prepared fresh.
  7. Glucose-6-P-dehydrogenase (G6P-DH) solution. Prepare a solution in cold 1% bovine serum albumin to yield a final concentration of 100 U/ml. Must be prepared fresh.
  8. Phosphorylase (enzyme) solution. Prepare a solution in cold 1% bovine serum albumin to yield a final concentration of 2-4 U/ml. Must be prepared fresh immediately prior to assay. (Note: the enzyme preparation contains 5'-AMP which is needed for its catalytic activity.)



  1. Set spectrophotometer (equipped with strip chart recorder and temperature control) at 340 nm and 37°C.
  2. Into a cuvette pipette the following reagents:
    Potassium phosphate buffer with EDTA 2.4 ml
    Magnesium chloride 0.1 ml
    NADP 0.1 ml
    Glycogen 0.1 ml
    Adenosine 5'-monophosphate 0.1 ml
    Phosphoglucomutase 0.1 ml
    Glucose-6-P-dehydrogenase 0.1 ml
    Mix and incubate at 37¡C for 5 minutes in the spectrophotometer to attain temperature equilibration.
  3. Establish blank rate, if any, at 340 nm.
  4. Initiate the reaction by adding 0.1 ml phosphorylase (enzyme) solution. Record the change in absorbance at 340 nm for 5-8 minutes.
  5. Calculate µE340/min



Activity (U/mg) = (ΔE340nm/min)(Total Vol.)(Enz. Diln.)
(6.22)(Enz. Vol.)(mg Enz./ml)
 

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