Allosteric effectors and trehalose protect larval Manduca sexta fat body glycogen phosphorylase B against thermal denaturation.
In this paper we assessed the ability of modulators of the activity of glycogen phosphorylase b from the fat body of larval Manduca sexta to stabilize the enzyme against thermal denaturation. This approach has allowed us to distinguish between modulators that stabilize the enzyme, presumably through some conformational effect, from those that do not affect thermal stability. For example, 5'-AMP and 5'-IMP are both positive modulators of the enzyme and the K(m)s for AMP and IMP were similar, 0.71 and 1.09 mM, respectively. However, the V(max) for AMP (123 nmol/mg/min) was 10 times higher than the value found for IMP (12.5 nmol/mg/min) and AMP increased the thermal stability of glycogen phosphorylase b, however IMP did not increase the enzyme's thermal stability. Indeed, IMP decreased both the allosteric activation of the enzyme by AMP and the thermal protection conferred by AMP. The allosteric inhibitors ADP and ATP, which in vertebrate phosphorylase bind to the same site as AMP, both increased the thermal stability of the enzyme, however with less efficiency than AMP. Inorganic phosphate increased thermal stability, but glycogen and amylose did not. Glycerol, at 600 mM, protected the enzyme against thermal inactivation, whereas sorbitol at the same concentration did not show any effect. Among the polyols tested, trehalose was the most effective in conferring thermal stability. In fact, in the presence of 20 mM AMP and 600 mM trehalose, 90% of the enzyme activity remained after 20 min at 60 degrees C.
Meyer-Fernandes
J R
JR
Department of Biochemistry and Center for Insect Science, Biological Sciences West, University of Arizona, Tucson, USA.
Arrese
E L
EL
Wells
M A
MA
D000494:Allosteric Regulation; D000818:Animals; D005216:Fat Body / Q000201:enzymology; D006361:Heating; D018402:Manduca / Q000201:enzymology*; D010763:Phosphorylase b / Q000378:metabolism*; D011489:Protein Denaturation; D014199:Trehalose / Q000378:metabolism*
D014199:Trehalose; D010763:Phosphorylase b