Activities synthesizing and degrading fructose 2,6-bisphosphate in spinach leaves reside on different proteins.

Activities catalyzing the synthesis and degradation of fructose 2,6-bisphosphate-6-phosphofructo-2-kinase (ATP:D-fructose-6-phosphate-2-phosphotransferase, EC 2.7.1.105) and fructose-2,6-bisphosphatase (D-fructose-2,6-bisphosphate 2-phosphohydrolase, EC 3.1.3.46)-were isolated from spinach leaves by an improved procedure and separated on the basis of both charge and molecular weight. The separated activities showed no detectable cross-contamination, indicating, in contrast to all previous data, that they are not present on a single bifunctional protein of the classical type in liver. The fructose-2,6-bisphosphatase-a newly discovered phosphatase enzyme-differed from previous mixed preparations by showing greater specificity but lower affinity for fructose 2,6-bisphosphate, greater sensitivity to inhibition by inorganic phosphate, and in being sensitive to inhibition by Mg(2+). The 6-phosphofructo-2-kinase was found to be inhibited by low levels of inorganic pyrophosphate and, in addition, to be regulated by the metabolites described previously. Similar results were obtained with preparations from lettuce leaves. The results support the view that, through individual regulation of the activities catalyzing its synthesis and breakdown, cytosolic metabolites are key factors in controlling the fructose 2,6-bisphosphate content of leaves.