Thermal Inactivation of Avian Liver and Muscle Fructose 1,6-Diphosphatases in the Presence of Fructose 1,6-Diphosphate, AMP, and Divalent Cations

The effects at various pH levels of fructose 1,6-diphosphate (FDP), AMP, CaCl2, MnCl2, and/or MgCl2 on the thermal stability of pure forms of avian liver and muscle fructose 1,6-diphosphatases were investigated. Both enzymes were more stable when stored at 20 °C than at 2 °C. Maximum stabilities of the two enzymes when incubated at various temperatures in a Tris buffer occurred at approximately pH 6. All of the above compounds when added to a pH 7.5 Tris buffer markedly increased the thermal stabilities of both enzymes. Individually, FDP provided the highest degree of protection for the muscle enzyme whereas MnCl2 was most effective with the liver enzyme; in all cases the degree of protection was concentration dependent. At pH 7.5 combinations of MgCl2, FDP, and AMP were most effective. In the presence of these compounds initial inactivation of the muscle and liver enzymes did not occur until respective temperatures of 74 and 78 °C (15 min) were reached; these compare with initial inactivation temperatures in the absence of these compounds of 55 and 59 °C. The interaction of MgCl2 and FDP with the two enzymes at varying pH levels showed markedly different patterns. At pH7.5 and 8.8 increasing levels of MgCl2 provided increasing levels of protection whereas at pH 6.2 increasing MgCl2 concentrations resulted in an enhanced degree of inactivation. In the presence of FDP an opposite pattern was observed at the various pH levels. The presence of phosphate in the buffer tended to yield results similar to those of FDP.