Purification and characterization of fructose-2,6-bisphosphatase, a substrate-specific cytosolic enzyme from leaves.

Fructose-2,6-bisphosphatase (EC 3.1.3.46), which hydrolyzes fructose 2,6-bisphosphate to fructose 6-phosphate and Pi, has been purified to apparent homogeneity from spinach leaves and found to be devoid of fructose-6-phosphate,2-kinase activity. The isolated enzyme is a dimer (76 kDa determined by gel filtration) composed of two 33-kDa subunits. The enzyme is highly specific and displays hyperbolic kinetics with its fructose 2,6-bisphosphate substrate (Km = 32 microM). The products of the reaction, fructose 6-phosphate and Pi, along with AMP and Mg2+ are inhibitors of the enzyme. Nonaqueous cell fractionation revealed that, like the fructose 2,6-bisphosphate substrate, fructose-2,6-bisphosphatase as well as fructose-6-phosphate,2-kinase occur in the cytosol of spinach leaves.

Studies on the entry of fructose-2,6-bisphosphate into chloroplasts.

The regulatory metabolite fructose-2,6-bisphosphate (Fru-2,6-P(2)) has an important function in controlling the intermediary carbon metabolism of leaves. Fru-2,6-P(2) controls two cytosolic enzymes involved in the interconversion of fructose-6-phosphate and fructose-1,6-bisphosphate (fructose-1,6-bisphosphatase and pyrophosphate, fructose-6-phosphate 1-phosphotransferase) and thereby controls the partitioning of photosynthate between sucrose and starch. It has been demonstrated that Fru-2,6-P(2) is present mainly in the cytosol. Here we present evidence that Fru-2,6-P(2) can be taken up by isolated intact chloroplasts but at a very slow rate (about 0.01 micromoles per milligram of chlorophyll per hour). This uptake is time and concentration dependent and is inhibited by PPi. When provided a physiological concentration of Fru-2,6-P(2) (10 micromolar), chloroplasts accumulated up to 0.6 micromolar Fru-2,6-P(2) in the stroma. Elevated plastid Fru-2,6-P(2) levels had no effect on overall photosynthetic rates of isolated chloroplasts. The results indicate that, while Fru-2,6-P(2) enters isolated chloroplasts at a sluggish rate, caution should be exercised in ascribing physiological importance to effects of Fru-2,6-P(2) on chloroplast enzymes.

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.

Ion-exchange chromatography separates activities synthesizing and degrading fructose 2,6-bisphosphate from C3 and C4 leaves but not from rat liver.

Fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase were separated on the basis of charge from leaves of C3 (spinach, lettuce, and pea) and C4 (sorghum and amaranthus) plants but not from rat liver--a tissue known to contain a bifunctional enzyme with both activities. [2-32P]Fructose 2,6-bisphosphate binding experiments also suggest that the major forms of these activities reside on different proteins in leaves.

The subcellular location and characteristics of pyrophosphate-fructose-6-phosphate 1-phosphotransferase from suspension-cultured cells of soybean.

The cytoplasm was identified as the probable location of pyrophosphate-fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90) in suspension-cultured cells of soybean (Glycine max L.). The characteristics of the partially purified enzyme were investigated. The activity was strongly dependent on the presence of fructose 2,6-bisphosphate and this activator exerted its effects through a dramatic increase in the affinity of the enzyme for its substrates, fructose 6-phosphate and inorganic pyrophosphate. Saturation curves for all substrates were hyperbolic. The apparent molecular weight of the partially purified enzyme was 183000 by gel filtration chromatography and 128000 by sucrose-density-gradient centrifugation. The activation by fructose 2,6-bisphosphate was not accompanied by any measurable change in molecular weight. The possible role of this enzyme in the metabolism of non-photosynthetic sink tissues is discussed.

Partial purification and characterization of granule-bound starch synthases from normal and waxy maize.

The granule-bound starch-synthases from normal and waxy maize kernels have been solubilized, partially purified, and characterized. Two broad categories of starch synthases were revealed with representatives in the soluble phase and also on granules from both normal and waxy maize though the activity associated with granules from waxy was small. Data for native molecular weights, kinetic parameters, and immunological relatedness are used to demonstrate that the granule-bound isozymes from normal maize are different from the soluble enzymes. These distinct granule-bound enzymes are missing in waxy maize granules and a further novel form of starch synthase is revealed. These findings are discussed in relation to the type of starch produced in tissue affected by the waxy mutation.

Solubilization of the starch-granule-bound starch synthase of normal maize kernels.

The starch-granule-bound starch synthase from Zea mays has been solubilized with a recovery of between 50 and 84%. Chromatography of the solubilized enzyme on DEAE-Sepharose resolves two fractions of activity which may be distinguished by their response to citrate. Neither solubilized isoenzyme displays any significant activity with UDPglucose.