Factors Influencing the Inactivation of Chloroplastic Fructose-1,6-Bisphosphatase

Abstract

The activity of chloroplastic fructose-1,6-bisphosphatase was found to be stable in the crude leaf homogenates of wheat and rye for an hour or more if stored at 2°C (on ice). If the homogenate was gel filtered over Sephadex G-25 and subsequently stored on ice, there was a time-dependent loss of the light-activated component of fructose-1,6-bisphosphatase activity. A similar response was found for the enzyme purified from spinach and activated by reduced thioredoxin or by dithioerythritol alone. The lost activity could be recovered if the purified enzyme was allowed to warm to room temperatures. Attempts to stabilise the enzyme in gel-filtered crude extracts by the inclusion of substrate, several other metabolites, or alteration of the ionic strength of the extraction medium were unsuccessful. A reduction in the extraction pH for fructose-1,6-bisphosphatase below 8.3, by 1-2 pH units, resulted in a moderate stabilisation of enzyme activity after gel filtration and cold storage. Sucrose density gradient ultracentrifugation revealed a dissociation of the tetrameric form of the enzyme into its dimer component after gel filtration and cold storage. As was the case for inactivation, depolymerisation was more prevalent at the higher pH levels. We hypothesise that the cold instability of fructose-1,6-bisphosphatase which is induced by gel filtration over Sephadex G-25 is a result of depolymerisation of the tetrameric enzyme. The mechanism by which gel filtration induces the cold lability of this enzyme is unclear although it is apparently not due to removal of free low-molecular-weight substances from the medium.