Partial purification of two forms of spinach leaf sucrose-phosphate synthase which differ in their kinetic properties

Abstract

Spinach leaves were supplied with mannose in the light, or were illuminated for 9h to allow sucrose to accumulate and then darkened. Extracts were then prepared. After pretreatment with mannose, sucrose-phosphate synthase was less sensitive to inhibition by phosphate. The sucrose pretreatment led to a high phosphate sensitivity. Sucrose-phosphate synthase was purified 200-fold and 60-fold from leaves which had been pretreated with mannose and sucrose, respectively, using amino-hexyl agarose chromarography, anion-exchange chromatography on Mono-Q, and gel filtration on Superose-12. The initial differences in phosphate sensitivity were retained through the purification. Kinetic analysis of the partially purified preparation revealed there are small changes in the affinity for the substrates, fructose 6-phosphate and UDP-glucose, and large changes in the sensitivity to the allosteric activator Glc6P, and inhibitor, Pi.