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

Abstract

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 Mg2 . 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

Ion-Exchange Chromatography Separates Activities Synthesizing and Degrading Fructose 2,6-Bisphosphate from C3 and C4 Leaves but Not from Rat Liver

“…We have previously shown (12), in agreement with others (10), that the two forms ofspinach leafFru 6-P,2K differ in their properties.…”

“…The absence of significant label also suggests that partially purified spinach leaf Fru 2,6-P2ase does not catalyze the hydrolysis of Fru 2,6-P2 by a mechanism involving the formation of a stable phosphoenzyme intermediate as appears to be the case for the bifunctional mammalian enzyme. The ability of Fru 6-P,2K I to catalyze phosphoenzyme formation when prepared in a different buffer system is of questionable significance as this form ofthe enzyme is considered to be minor and a degraded product of the major form (10,12).…”

“…85, 1987 that the larger columns were eluted with a total gradient of 400 ml. Subsequent purification of the active fractions by Mono Q anion-exchange chromatography was also as previously described (12). In the indicated experiments with spinach, the same procedure was also followed except that buffer A was replaced with the buffer described by (12) but also for another C3 species (pea) and two C4 species (amaranthus and sorghum) (Fig.…”

“…Rats were killed by decapitation, the livers excised immediately, and 20 g of the tissue was extracted as described for leaves. Subsequent filtration and centrifugation of the crude extracts was as described previously for spinach leaves (12). The resulting supernatant fractions were loaded onto DEAE-cellulose (DE-52) columns (1.5 x 15 cm for spinach and rat liver, 2.1 x 15 cm for amaranthus, sorghum, lettuce, and pea), which were developed as described previously (12) except Plant Physiol.…”

Fructose 2,6-Bisphosphate Hydrolyzing Enzymes in Higher Plants

“…A low (below 1 AM) Km enzyme is present in most eukaryotic cells, including those from higher plants (4,12,22,27,28), and is easily detected, even in crude preparations, by its ability to form a [32P]phosphoenzyme intermediate when incubated in the presence of 1 ,M [2-32P]Fru-2,6-P2 (12,23). A high (100-300 AtM) Km FBPase 2 is present in yeast (8) and in higher plants (2,11,13,14,18), with the reservation, however, that the specificity of this enzyme has only been carefully investigated by Frangois et al (8), and MacDonald et al (13,14).…”