Journal of Comparative Physiology B

, Volume 163, Issue 6, pp 499–507 | Cite as

Purification and characterization of glycogen phosphorylase A and B from the freeze-avoiding gall moth larvae Epiblema scudderiana

  • C. P. Holden
  • K. B. Storey


The active a and inactive b forms of glycogen phosphorylase from cold-hardy larvae of the gall moth, Epiblema scudderiana, were purified using DEAE+ ion exchange and 3′-5′-AMP-agarose affinity chromatography. Maximum activities for glycogen phosphorylases a and b were 6.3±0.74 and 2.7±0.87 μmol glucose-1-P·min-1·g wet weight-1, respectively, in -4°C-acclimated larvae. Final specific activities of the purified enzymes were 396 and 82 units·mg protein-1, respectively. Both enzymes were dimers with native molecular weights of 215000±18000 for glycogen phosphorylase a and 209000±15000 for glycogen phosphorylase b; the subunit molecular weight of both forms was 87000±2000. Both enzymes showed pH optima of 7.5 at 22°C and a break in the Arrhenius relationship with a two- to four-fold increase in activation energy below 10°C. Michaelis constant values for glycogen at 22°C were 0.12±0.004 mg·ml-1 for glycogen phosphorylase a and 0.87±0.034 mg·ml-1 for glycogen phosphorylase b; the Michaelis constant for inorganic phosphate was 6.5±0.07 mmol·l-1 for glycogen phosphorylase a and 23.6 mmol·l-1 for glycogen phosphorylase b. Glycogen phosphorylase b was activated by adenosine monophosphate with a Ka of 0.176±0.004 mmol·l-1. Michaelis constant and Ka values decreased by two- to fivefold at 5°C compared with 22°C. Glycerol had a positive effect on the Michaelis constant for glycogen for glycogen phosphorylase a at intermediate concentrations (0.5 mol·l-1) but was inhibitory to both enzyme forms at high concentrations (2 mol·l-1). Glycerol production as a cryoprotectant in E. scudderiana larvae is facilitated by the low temperature-simulated glycogen phosphorylase b to glycogen phosphorylase a conversion and by positive effects of low temperature on the kinetic properties of glycogen phosphorylase a. Enzyme shut-down when polyol synthesis is complete appears to be aided by strong inhibitory effects of glycerol and KCl on glycogen phosphorylase b.

Key words

Cryoprotectant synthesis Insect cold hardiness Glycerol metabolism Regulation of glycogenolysis Gall moth, Epiblema 



activation energy


glycogen phosphorylase a


glycogen phosphorylase b


Hill coefficient


concentration of inhibitor that reduces enzymes velocity by 50%


concentration of activator that produces half-maximal activation of enzyme activity


Michaelis-Menten substrate affinity constant


molecular weight


polyethylene glycol


morganic phosphate


sodium dodecyl sulphate polyacrylamide gel electrophoresis


enzyme maximal velocity


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  1. Atkinson A, Gatenby AD, Lowe AG (1973) Phosphatase assay. Biochim Biophys Acta 320:195–204Google Scholar
  2. Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 12:248–253Google Scholar
  3. Childress CC, Sacktor B (1970) Regulation of glycogen metabolism in insect flight muscle. J Biol Chem 245:2927–2936Google Scholar
  4. Churchill TA, Storey KB (1989a) Seasonal variation in the temperature-stimulated interconversion of glycogen and glycerol pools in a freeze avoiding moth larvae. Cryo Lett 10:127–136Google Scholar
  5. Churchill TA, Storey KB (1989b) Regulation of glycerol biosynthesis in a freeze avoiding insect. J Comp Physiol B 159:461–472Google Scholar
  6. Churchill TA, Storey KB (1989c) Metabolic consequences of rapid cycles of temperature change for freeze-avoiding vs freeze-tolerant insects. J Insect Physiol 35:579–586Google Scholar
  7. Dombradi V, Matko J, Kiss Z, Kiss L, Friedrich P, Bot G (1986) Structural and functional properties of Drosophila melanogaster phosphorylase: comparison with the rabbit skeletal muscle enzyme. Comp Biochem Physiol 84B:537–543Google Scholar
  8. Fischer EH, Krebs EG (1959) Muscle phosphorylase b. Methods Enzymol 49A:369–376Google Scholar
  9. Hayakawa Y (1985) Activation mechanism of insect fat body phosphorylase by cold. Insect Biochem 15:123–128Google Scholar
  10. Hayakawa Y, Chino H (1981) Temperature-dependent interconversion between glycogen and trehalose in diapausing pupae of Philosamia cynthia ricini and pryeri. Insect Biochem 11:41–47Google Scholar
  11. Hayakawa Y, Chino H (1982) Temperature-dependent activation or inactivation of glycogen phosphorylase and synthase of fat body of the silkworm, Philosamia cynthia: the possible mechanism of the temperature-dependent interconversion between glycogen and trehalose. Insect Biochem 12:361–366Google Scholar
  12. Huang CY, Graves DJ (1970) Correlation between subunit interactions and enzymatic activity of phosphorylase a. Method for determining equilibrium constants from initial rate measurements. Biochemistry 9:660–671Google Scholar
  13. Keller PJ, Cori GT (1953) Enzymatic conversion of phosphorylase a to phosphorylase b. Biochem Biophys Acta 12:235–238Google Scholar
  14. Krebs EG, Fischer EH (1955) The phosphorylase b to a converting enzyme of rabbit skeletal muscle. Biochim Biophys Acta 20:150–157Google Scholar
  15. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685Google Scholar
  16. Marrewijk WJA van, Broek AThM van den, Beenakkers AMTh (1985) Glycogen phosphorylase in the fat body of Locusta migratoria. Insect Biochem 15:341–347Google Scholar
  17. Meinke M, Edstrom R (1991) Muscle glycogenolysis: regulation of the cyclic interconversion of phosphorylase a and phosphorylase b. J Biol Chem 266:2259–2266Google Scholar
  18. Morishima I, Sakurai S (1985) Purification and characterization of glycogen phosphorylase b from fat body of the silkworm, Bombyx mori. Comp Biochem Physiol 81B:453–458Google Scholar
  19. Morishima I, Ueno T (1990) Structural and kinetic properties of glycogen phosphorylase a from fat body of the silkworm, Bombyx mori. Comp Biochem Physiol 96B:591–595Google Scholar
  20. Newgard CB, Hwang PK, Fletterick RJ (1989) The family of glycogen phosphorylases: structure and function. Crit Rev Biochem Mol Biol 24:69–99Google Scholar
  21. Rickards J, Kelleher MJ, Storey KB (1987) Strategies of freeze avoidance in the larvae of the goldenrod gall moth, Epiblema scudderiana: winter profiles of a natural population. J Insect Physiol 33:443–450Google Scholar
  22. Shimada K (1982) Glycerol accumulation in developmentally arrested pupae of Papilio machaon obtained by brain removal. J Insect Physiol 28:975–978Google Scholar
  23. Stalmans W, Hers H-G (1975) The stimulation of liver phosphorylase b by AMP, fluoride and sulfate. Eur J Biochem 54:341–350Google Scholar
  24. Storey KB (1982) Phosphofructokinase from the overwintering gall fly larva, Eurosta solidaginis: control of cryoprotectant polyol synthesis. Insect Biochem 12:501–505Google Scholar
  25. Storey KB, Storey JM (1991) Biochemistry of cryoprotectants. In: Denlinger D, Lee RE (eds) Insects at low temperature. Chapman and Hall, New York, pp 64–93Google Scholar
  26. Titani K, Koide A, Ericsson LH, Kumar S, Wade R, Walsh KA, Neurath H, Fisher E (1977) Complete amino acid sequence of rabbit muscle glycogen phosphorylase. Proc Natl Acad Sci USA 74:4762–4766Google Scholar
  27. Vaandrager SH, van Marrewijk WJA, Beenakkers AMTh (1987) Kinetic properties of glycogen phosphorylases a, ab, and b from flight muscles of the locust, Locusta migratoria. Insect Biochem 17:695–700Google Scholar
  28. Yamashita O, Suzuki K, Hasegawa K (1975) Glycogen phosphorylase activity in relation to diapause initiation in Bombyx eggs. Insect Biochem 5:707–718Google Scholar
  29. Yi S-X, Yin C-M, Nordin JH (1987) The in vitro biosynthesis and secretion of glycerol by larval fat bodies of chilled Ostrinia nubilalis. J Insect Physiol 33:523–528Google Scholar
  30. Ziegler R, Ashida M, Fallon AM, Wimer LT, Silver Wyatt S, Wyatt GR (1979) Regulation of glycogen phosphorylase in a fat body of Cecropia silkmoth pupae. J Comp Physiol 131:321–332Google Scholar

Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • C. P. Holden
    • 1
    • 2
  • K. B. Storey
    • 1
    • 2
  1. 1.Department of BiologyCarleton UniversityOttawaCanada
  2. 2.Department of ChemistryCarleton UniversityOttawaCanada

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