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 glyc ogen phosphorylase b from the fat body of larval Manduca sexta to stabilize the enzyme against thermal denaturation. This approach has allowed us to d istinguish between modulators that stabilize the enzyme, presumably through some conformational effect, from those that do not affect thermal stabilit y. For example, 5'-AMP and 5'-IMP are both positive modulators of the enzym e and the K(m)s for AMP and LMP 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 stabi lity of glycogen phosphorylase b, however LMP 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 alloste ric inhibitors ADP and ATP, which in vertebrate phosphorylase bind to the s ame site as AMP, both increased the thermal stability of the enzyme, howeve r with less efficiency than AMP. Inorganic phosphate increased thermal stab ility, but glycogen and amylose did not. Glycerol, at 600 mM, protected the enzyme against thermal inactivation, whereas sorbitol at the same concentr ation 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. (C) 2000 Elsevier Science Ltd. All rights reserved.