Purification and characterization of glycogen phosphorylase A and B from the freeze-avoiding gall moth larvae Epiblema scudderiana |
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Authors: | C P Holden K B Storey |
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Institution: | (1) Department of Biology, Carleton University, K1S 5B6 Ottawa, Ontario, Canada;(2) Department of Chemistry, Carleton University, K1S 5B6 Ottawa, Ontario, Canada |
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Abstract: | 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 mol 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 K
a of 0.176±0.004 mmol·l-1. Michaelis constant and K
a 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.Abbreviations
E
a
activation energy
- GPa
glycogen phosphorylase a
- GPb
glycogen phosphorylase b
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h
Hill coefficient
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I
50
concentration of inhibitor that reduces enzymes velocity by 50%
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K
a
concentration of activator that produces half-maximal activation of enzyme activity
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K
m
Michaelis-Menten substrate affinity constant
- MW
molecular weight
- PEG
polyethylene glycol
- Pi
morganic phosphate
- SDS PAGE
sodium dodecyl sulphate polyacrylamide gel electrophoresis
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V
max
enzyme maximal velocity |
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Keywords: | Cryoprotectant synthesis Insect cold hardiness Glycerol metabolism Regulation of glycogenolysis Gall moth Epiblema |
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