Physiological relevance of fructose 2,6-bisphosphate in the regulation of spinach leaf pyrophosphate:fructose 6-phosphate 1-phosphotransferase.

A major problem in defining the physiological role of pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90) is the 1,000-fold discrepancy between the apparent affinity of PFP for its activator, fructose 2,6-bisphosphate (Fru-2,6-P2), determined under optimum conditions in vitro and the estimated concentration of this signal metabolite in vivo. The aim of this study was to investigate the combined influence of metabolic intermediates and inorganic phosphate (Pi) on the activation of PFP by Fru-2,6-P2. The enzyme was purified to near-homogeneity from leaves of spinach (Spinacia oleracea L.). Under optimal in vitro assay conditions, the activation constant (Ka) of spinach leaf PFP for Fru-2,6-P2 in the glycolytic direction was 15.8 nM. However, in the presence of physiological concentrations of fructose 6-phosphate, inorganic pyrophosphate (PPi), 3-phosphoglycerate (3PGA), phosphoenolpyruvate (PEP), ATP and Pi the Ka of spinach leaf PFP for Fru-2,6-P2 was up to 2000-fold greater than that measured in the optimised assay and Vmax decreased by up to 62%. Similar effects were observed with PFP purified from potato (Solanum tuberosum L.) tubers. Cytosolic metabolites and Pi also influenced the response of PFP to activation by its substrate fructose 1,6-bisphosphate (Fru-1,6-P2). When assayed under optimum conditions in the gluconeogenic direction, the Ka of spinach leaf PFP for Fru-1,6-P2 was approximately 50 microM. Physiological concentrations of PPi, 3PGA, PEP, ATP and Pi increased Ka up to 25-fold, and decreased Vmax by over 65%. From these results it was concluded that physiological concentrations of metabolites and Pi increase the Ka of PFP for Fru-2,6-P2 to values approaching the concentration of the activator in vivo. Hence, measured changes in cytosolic Fru-2,6-P2 levels could appreciably alter the activation state of PFP in vivo. Moreover, the same levels of metabolites increase the Ka of PFP for Fru-1,6-P2 to an extent that activation of PFP by this compound is unlikely to be physiologically relevant.

Purification and characterization of pyrophosphate-dependent phosphofructokinase from phosphate-starved Brassica nigra suspension cells.

Previously, we reported that inorganic phosphate (Pi) deprivation of Brassica nigra suspension cells or seedlings leads to a progressive increase in the alpha: beta-subunit ratio of the inorganic pyrophosphate (PPi)-dependent phosphofructokinase (PFP) and that this coincides with a marked enhancement in the enzyme's activity and sensitivity to its allosteric activator, fructose-2,6-bisphosphate (Fru-2,6-P2). To further investigate the effect of Pi nutrition on B. nigra PFP, the enzyme was purified and characterized from Pi-starved B. nigra suspension cell cultures. Polyacrylamide gel electrophoresis, immunoblot, and gel-filtration analyses of the final preparation indicated that this enzyme exists as a heterooctamer of approximately 500 kD and is composed of a 1:1 ratio of immunologically distinct alpha (66 kD) and beta (60 kD) subunits. The enzyme's alpha subunit was susceptible to partial proteolysis during purification, but this was prevented by the presence of chymostatin and leupeptin. In the presence and absence of 5 microM Fru-2,6-P2, the forward activity of PFP displayed pH optima of pH 6.8 and 7.6, respectively. Maximal activation of the forward and reverse reactions by Fru-2,6-P2 occurred at pH 6.8. The potent inhibition of the forward activity by Pi (concentration of inhibitor producing 50% inhibition of enzyme activity [I50] = 1.3 mM) was attributed to a marked Pi-dependent reduction in Fru-2,6-P2 binding. The reverse reaction was substrate-inhibited by Pi (I50 = 13 mM) and product-inhibited by PPi (I50 = 0.9 mM). The kinetic data are consistent with the hypothesis that PFP may function to bypass the ATP-dependent PFP in Pi-starved B. nigra. The importance of the Pi nutritional status to the regulation and predicted physiological function of PFP is emphasized.

The Fungicide Phosphonate Disrupts the Phosphate-Starvation Response in Brassica nigra Seedlings.

The development of Brassica nigra seedlings over 20 d of growth was disrupted by the fungicide phosphonate (Phi) in a manner inversely correlated with nutritional inorganic phosphate (Pi) levels. The growth of Pi-sufficient (1.25 mM Pi) seedlings was suppressed when 10, but not 5, mM Phi was added to the nutrient medium. In contrast, the fresh weights and root:shoot ratios of Pi-limited (0.15 mM) seedlings were significantly reduced at 1.5 mM Phi, and they progressively declined to about 40% of control values as medium Phi concentration was increased to 10 mM. Intracellular Pi levels generally decreased in Phi-treated seedlings, and Phi accumulated in leaves and roots to levels up to 6- and 16-fold that of Pi in Pi-sufficient and Pi-limited plants, respectively. Extractable activities of the Pi-starvation-inducible enzymes phosphoenolpyruvate phosphatase and inorganic pyrophosphate-dependent phosphofructokinase were unaltered in Pi-sufficient seedlings grown on 5 or 10 mM Phi. However, when Pi-limited seedlings were grown on 1.5 to 10 mM Phi (a) the induction of phosphoenolpyruvate phosphatase and inorganic pyrophosphate-dependent phosphofructokinase activities by Pi limitation was reduced by 40 to 90%, whereas (b) soluble protein concentrations and the activities of the ATP-dependent phosphofructokinase and pyruvate kinase were unaffacted. It is concluded that Phi specifically interrupts processes involved in regulation of the Pi-starvation response in B. nigra.

Metabolic Adaptations of Plant Respiration to Nutritional Phosphate Deprivation.

Plants respond adaptively to orthophosphate (Pi) deprivation through the induction of alternative pathways of glycolysis and mitochondrial electron transport. These respiratory bypasses allow respiration to proceed in Pi-deficient plant cells because they negate the necessity for adenylates and Pi, both pools of which are severely depressed following nutritional Pi starvation.

Phosphate starvation-inducible synthesis of the alpha-subunit of the pyrophosphate-dependent phosphofructokinase in black mustard suspension cells.

PP(i)-dependent phosphofructokinase (PFP) activity, measured in the forward direction, increased approximately 19-fold when suspension cell cultures of black mustard (Brassica nigra) were subjected to 18 days of P(i) deprivation. Fructose 2,6-bisphosphate (2 microM) elicited a 10-fold activation of PFP from P(i)-deficient cells, compared to only a 2-fold activation of the enzyme from nutrient-sufficient cells. Also, PFP from P(i)-starved cells exhibited a greater affinity for the activator (Ka = 0.09 microM) than the enzyme from nutrient-sufficient cells (Ka = 0.32 microM). Western blots of extracts from P(i)-deficient cells were probed with rabbit anti-(potato tuber PFP) immune serum and revealed equal intensity staining immunoreactive polypeptides of M(r) 66,000 (alpha-subunit) and 60,000 (beta-subunit) that co-migrated with the alpha- and beta-subunits of homogeneous potato tuber PFP. By contrast, only the M(r) 60,000 beta-subunit was observed on immunoblots of extracts prepared from nutrient-sufficient cells. Quantification of immunoblots indicated that in black mustard cells experiencing transition from P(i) sufficiency to deficiency or vice versa, the relative amount of immunoreactive alpha-subunit correlated with the degree of activation of PFP by fructose 2,6-bisphosphate. These observations provide additional evidence that (i) plant PFP is an adaptive enzyme that may function in glycolysis during P(i) deprivation, and (ii) the alpha-subunit acts as a regulatory protein in controlling the catalytic activity of the beta-subunit and its regulation by fructose 2,6-bisphosphate.

Effects of Phosphorus Limitation on Respiratory Metabolism in the Green Alga Selenastrum minutum.

The effects of phosphorus nutrition on several physiological and biochemical parameters of the green alga, Selenastrum minutum, have been examined. Algal cells were cultured in chemostats under conditions of either Pi limitation or nutrient sufficiency. Pi limitation resulted in: (a) a 5-fold lower rate of respiration, (b) a 3-fold decline in rates of photosynthetic carbon dioxide fixation and oxygen evolution, (c) a 3-fold higher rate of dark carbon dioxide fixation, (d) significant increases in activities of phosphoenolpyruvate (PEP) carboxylase and PEP phosphatase (128% and 158% of nutrient sufficient activities, respectively), (e) significant reductions in activities of nonphosphorylating NADP-glyceraldehyde-3-phosphate dehydrogenase and NAD malic enzyme, and (f) no change in levels of ATP:fructose-6-phosphate 1-phosphotransferase, phosphorylating NAD-glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and pyruvate kinase. The intracellular concentrations of Pi, ATP, AMP, soluble protein, and chlorophyll were also significantly reduced in response to Pi limitation. As well, the level of ADP was about 11-fold lower in the Pi-limited cells as compared to the nutrient sufficient controls. It was predicted that because of this low level of ADP, pyruvate kinase catalyzed conversion of PEP to pyruvate may be restricted in Pi-limited cells. During Pi limitation, PEP carboxylase and PEP phosphatase may function to "bypass" the ADP dependent pyruvate kinase, as well as to recycle Pi for its reassimilation into cellular metabolism.