Measurement of 2-carboxyarabinitol 1-phosphate in plant leaves by isotope dilution

The level of 2-carboxyarabinitol 1-phosphate (CA1P) in leaves of 12 species was determined by an isotope dilution assay. ¹⁴C-labeled standard was synthesized from [2-¹⁴C]carboxyarabinitol 1,5-bisphosphate using acid phosphatase, and was added at the initial point of leaf extraction. Leaf CA1P was purified and its specific activity determined. CA1P was found in dark-treated leaves of all species examined, including spinach (Spinacea oleracea), wheat (Triticum aestivum), Arabidopsis thaliana, and maize (Zea mays). The highest amounts were found in bean (Phaseolus vulgaris) and petunia (Petunia hybrida), which had 1.5 to 1.8 moles CA1P per mole ribulose 1,5-bisphosphate carboxylase catalytic sites. Most species had intermediate amounts of CA1P (0.2 to 0.8 mole CA1P per mole catalytic sites). Such intermediate to high levels of CA1P support the hypothesis that CA1P functions in many species as a light-dependent regulator of ribulose 1,5-bisphosphate carboxylase activity and whole leaf photosynthetic CO₂ assimilation. However, CA1P levels in spinach, wheat, and A. thaliana were particularly low (less than 0.09 mole CA1P per mole catalytic sites). In such species, CA1P does not likely have a significant role in regulating ribulose 1,5-bisphosphate carboxylase activity, but could have a different physiological role.     

Metabolism of 2-carboxyarabinitol 1-phosphate and regulation of ribulose-1,5-bisphosphate carboxylase activity

Metabolism of 2-carboxy-D-arabinitol 1-phosphate (CA1P) is an important component in the light-dependent regulation of ribulose-1,5-bisphosphate carboxylase (Rubisco) activity and whole leaf photosynthetic CO₂ assimilation in many species, and functions as one mechanism for regulating Rubisco activity when photosynthesis is light-limited. Species differ in their capacity to accumulate CA1P, ranging from those which can synthesize levels of this compound approaching or in excess of the Rubisco catalytic site concentration, to those which apparently lack the capacity for CA1P synthesis. CA1P is structurally related to the six carbon transition state intermediate of the carboxylation reaction and binds tightly to the carbamylated catalytic site of Rubisco, making that site unavailable for catalysis. Under steady-state, the concentration of CA1P in the leaf is highest at low photon flux density (PFD) or in the dark. Degradation of CA1P and recovery of Rubisco activity requires light and is stimulated by increasing PFD. The initial degradation reaction is catalyzed by an enzyme located in the chloroplast stroma, CA1P phosphatase, which yields carboxyarabinitol (CA) and inorganic phosphate as its products. The pathway of CA metabolism in the plant remains to be determined. Synthesis of CA1P occurs in the dark, and in Phaseolus vulgaris this process has been shown to be stimulated by low PFD. The pathway of CA1P synthesis and its relationship to the degradative pathway remains unknown at the present time. The discovery of the existence of this previously unknown carbon pathway in photosynthesis indicates that we still have much to learn concerning the regulation of Rubisco activity and photosynthesis.Abbreviations CA 2-carboxy-D-arabinitol - CA1P 2-carboxy-D-arabinitol 1-phosphate - CABP 2-carboxy-D-arabinitol-1,5-bisphosphate (transition state analog) - PFD photon flux density - P₁ inorganic phosphate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) - RuBP ribulose-1,5-bisphosphate     

Regulation of 2-carboxyarabinitol 1-phosphatase

The regulation of 2-carboxyarabinitol 1-phosphatase (CA 1-Pase) by phosphorylated effectors was studied with enzyme purified from tobacco (Nicotiana tabacum) leaves. CA 1-Pase activity was most stimulated by fructose 1,6-bisphosphate, exhibiting an A_0.5 value of 1.9 millimolar and a 10-fold enhancement of catalysis. With ribulose-1,5-bisphosphate, the A_0.5 was 0.6 millimolar, and maximal stimulation of activity was 5.3-fold. Among the monophosphates, 3-phosphoglycerate and phosphoglycolate were more potent positive effectors than glyceraldehyde 3-phosphate, glucose 1-phosphate, glucose 6-phosphate, and dihydroxyacetone phosphate. Stimulation of CA 1-Pase by ribulose-1,5-bisphosphate and fructose 1,6-bisphosphate increased V_max but did not appreciably alter K_m (2-carboxyarabinitol 1-phosphate) values. Inorganic phosphate appeared to inhibit CA 1-Pase noncompetitively with respect to 2-carboxyarabinitol 1-phosphate, exhibiting a K_i of 0.3 millimolar. The results suggest that these positive and negative effectors bind to a regulatory site on CA 1-Pase and may have a physiologial role in the light regulation of this enzyme. Related experiments with CA 1-Pase inactivated by dialysis in the absence of dithiothreitol show that partial reactivation can be achieved in the presence of a range of reducing reagents, including dithiothreitol, cysteine, and reduced glutathione. This could imply an ancillary involvement of sulfhydryl reduction during light activation of CA 1-Pase in vivo. The enzyme was thermally stable up to 35°C, in contrast to ribulose-1,5-bisphosphate carboxylase/oxygenase activase which lost activity above 30°C. The activation energy for CA 1-Pase was calculated to be 56.14 kilojoules per mole.     

Light-dependent kinetics of 2-carboxyarabinitol 1-phosphate metabolism and ribulose-1,5-bisphosphate carboxylase activity in vivo

The light-dependent kinetics of the apparent in vivo synthesis and degradation of 2-carboxyarabinitol 1-phosphate (CA1P) were studied in three species of higher plants which differ in the extent to which this compound is involved in the light-dependent regulation of ribulose-1,5-bisphosphate carboxylase (Rubisco) activity. Detailed studies with Phaseolus vulgaris indicate that both the degradation and synthesis of this compound are light-stimulated, although light is absolutely required only for CA1P degradation. We hypothesize that the steady state level of CAIP at any particular photon flux density (PFD) represents a pseudo-steady state balance between ongoing synthesis and degradation of this compound. The rate of CA1P synthesis in P. vulgaris and the resultant reduction in the total catalytic constant of Rubisco were maximal at 200 micromoles quanta per square meter per second following a step decrease from a saturating PFD, and substantially faster than the rate of synthesis in the dark. Under these conditions an amount of CA1P equivalent to approximately 25% of the Rubisco catalytic site content was synthesized in less than 1 minute. The rate of synthesis was reduced at higher or lower PFDs. In Beta vulgaris, the rate of CA1P synthesis at 200 micromoles quanta per square meter per second was substantially slower than in P. vulgaris. In Spinacea oleracea, an apparent noncatalytic tight-binding of RuBP to deactivated sites on the enzyme was found to occur following a step decrease in PFD. When dark acclimated leaves of P. vulgaris were exposed to a step increase in PFD, the initial rate of CA1P degradation was also found to be dependent on PFD up to a maximum of approximately 300 to 400 micromoles quanta per square meter per second. The rate of degradation of this compound was similar in B. vulgaris. In S. oleracea, a step increase in PFD resulted in noncatalytic RuBP binding to Rubisco followed by an apparent release of RuBP and activation of the enzyme. The in vivo rate of change of Rubisco activity in response to an increase or decrease in PFD was similar between species despite the differences between species in the mechanisms used for the regulation of this enzyme's activity.     

Sphingosine 1-phosphate phosphatase 2 is induced during inflammatory responses

Sphingosine 1-phosphate (S1P) levels in cells and, consequently, its bioactivity as a signalling molecule are controlled by the action of enzymes responsible for its synthesis and degradation. In the present report, we examined alterations in expression patterns of enzymes involved in S1P-metabolism (sphingosine kinases including their splice variants, sphingosine 1-phosphate phosphatases, and sphingosine 1-phosphate lyase) under certain inflammatory conditions. We found that sphingosine kinase type 1 (SPHK1) mRNA could be triggered in a cell type-specific manner; individual SPHK1 splice variants were induced with similar kinetics. Remarkably, expression and activity of S1P phosphatase 2 (SPP2) was found to be highly upregulated by inflammatory stimuli in a variety of cells (e.g., neutrophils, endothelial cells). Bandshift analysis using oligonucleotides spanning predicted NFkappaB sites within the SPP2 promoter and silencing of NFkappaB/RelA via RelA-directed siRNA demonstrated that SPP2 is an NFkappaB-dependent gene. Silencing of SPP2 expression in endothelial cells, in turn, led to a marked reduction of TNF-alpha-induced IL-1beta mRNA and protein and to a partial reduction of induced IL-8, suggesting a pro-inflammatory role of SPP2. Notably, up-regulation of SPP2 was detected in samples of lesional skin of patients with psoriasis, an inflammatory skin disease. This study provides detailed insights into the regulation of SPP2 gene expression and suggests that SPP2 might be a novel player in pro-inflammatory signalling.     

3-phosphoglycerate phosphatase activity in chloroplast preparations as a result of contamination by Acid phosphatase

The presence of a nonspecific acid phosphatase which had high activity with 3-phosphoglycerate as substrate has recently been reported in Spinacia oleracea L. chloroplasts (Mulligan, Tolbert 1980 Plant Physiol 66: 1169-1173). The subcellular localization of this activity has been reinvestigated by differential centrifugation of spinach leaf homogenates. The fraction sedimenting at 1,200g comprised mostly intact chloroplasts and contained more than half the chlorophyll but only 5% of the 3-phosphoglycerate phosphatase activity present in the homogenate. The fraction of the homogenate pelleting at 5,000g contained broken chloroplasts and had considerable 3-phosphoglycerate phosphatase activity. Further purification of the 1,200g pellet fraction on a Percoll step gradient yielded greater than 95% intact chloroplasts, yet the phosphatase activity was reduced more than 15-fold on a chlorophyll basis by this purification.

When the intact chloroplast and cytoplasmic fractions of mesophyll protoplasts were separated by silicone oil filtering centrifugation, the chloroplast fraction contained more than 90% of the chlorophyll but had less than 12% of the 3-phosphoglycerate phosphatase activity. By contrast, more than 60% of the 2-phosphoglycolate phosphatase was recovered in this chloroplast fraction supporting previous evidence that this phosphatase is localized in the chloroplast stroma.

It is concluded that 3-phosphoglycerate phosphatase activity is not localized in the chloroplast but that the activity present in chloroplast preparations results from contamination by acid phosphatase, which either binds to the thylakoid membranes during preparation or is present as some other contaminant in the preparation. Inasmuch as the enzyme acts on a broad range of substrates its presence in chloroplast preparations, particularly when the percentage of intact chloroplasts is low, could produce artifacts in metabolic studies such as measurement of phosphorylation.

     

Regulation of intracellular phosphatidylinositol-4-phosphate by the Sac1 lipid phosphatase

Phosphatidylinositol 4-phosphate (PtdIns(4)P) regulates diverse cellular processes, such as actin cytoskeletal organization, Golgi trafficking and vacuolar biogenesis. Synthesis and turnover of PtdIns(4)P is mediated by a set of specific lipid kinases and phosphatases. Here we show that the polyphosphoinositide phosphatase Sac1p has a central role in compartment-specific regulation of PtdIns(4)P. We have found that sac1Delta mutants show pleiotropic, synthetically lethal interactions with mutations in genes required for vacuolar protein sorting (Vps). Disruption of the SAC1 gene also caused a defect in the late endocytic pathway. These trafficking phenotypes correlated with a dramatic accumulation of PtdIns(4)P at vacuolar membranes. In addition, sac1 mutants displayed elevated endoplasmic reticulum PtdIns(4)P. The accumulation of PtdIns(4)P at the endoplasmic reticulum and vacuole and the endocytic defect could be compensated by mutations in the PtdIns 4-kinase Stt4p. Our results indicate that elimination of Sac1p causes accumulation of a Stt4p-specific PtdIns(4)P pool at internal membranes which impairs late endocytic and vacuolar trafficking. We conclude that Sac1p functions in confining PtdIns(4)P-dependent processes to specific intracellular membranes.     

Long-term kinetics of the light-dependent regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in plants with and without 2-carboxyarabinitol 1-phosphate

The light-dependent modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity was studied in two species: Phaseolus vulgaris L., which has high levels of the inhibitor of Rubisco activity, carboxyarabinitol 1-phosphate (CA1P), in the dark, and Chenopodium album L., which has little CA1P. In both species, the ratio of initial to fully-activated Rubisco activity declined by 40–50% within 60 min of a reduction in light from high a photosynthetic photon flux density (PPFD; >700 mol · m^–2 · s^–1) to a low PPFD (65 ± 15 mol · m^–2 · s^–1) or to darkness, indicating that decarbamylation of Rubisco is substantially involved in the initial regulatory response of Rubisco to a reduction in PPFD, even in species with potentially extensive CA1P inhibition. Total Rubisco activity was unaffected by PPFD in C. album, and prolonged exposure (2–6 h) to low light or darkness was accompanied by a slow decline in the activity ratio of this species. This indicates that the carbamylation state of Rubisco from C. album gradually declines for hours after the large initial drop in the first 60 min following light reduction. In P. vulgaris, the total activity of Rubisco declined by 10–30% within 1 h after a reduction in PPFD to below 100 mol · m^–2 · s^–1, indicating CA1P-binding contributes significantly to the reduction of Rubisco capacity during this period, but to a lesser extent than decarbamylation. With continued exposure of P. vulgaris leaves to very low PPFDs (< 30 mol · m^–2 · s^–1), the total activity of Rubisco declined steadily so that after 6–6.5 h of exposure to very low light or darkness, it was only 10–20% of the high-light value. These results indicate that while decarbamylation is more prominent in the initial regulatory response of Rubisco to a reduction in PPFD in P. vulgaris, binding of CA1P increases over time and after a few hours dominates the regulation of Rubisco activity in darkness and at very low PPFDs.Abbreviations CA1P 2-carboxyarabinitol 1-phosphate - CABP 2-carboxyarabinitol 1,5-bisphosphate - k_cat substrate-saturated turnover rate of fully carbamylated enzyme - PPFD photosynthetically active photon flux density (400–700 nm) - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate     

Transient inhibition by ribose 5-phosphate of photosynthetic O2 evolution in a reconstituted chloroplast system.

Photosynthetic oxygen evolution by a reconstituted chloroplast system utilising sn-phospho-3-glycerol (3-phosphoglycerate) ceases upon the addition of ribose 5-phosphate even though the presence of this metabolite permits a rapid and immediate CO2 fixation. The period of cessation is appreciable at 0.1 mM ribose 5-phosphate. It is lengthened as the amount of added ribose 5-phosphate is increased and by the addition of dithiothreitol, a known activator of ribulose-5-phosphate kinase. Ribulose 1,5-bisphosphate is without effect. A similar interruption of O2 evolution may also be brought about by the addition of ADP or by ADP-generating systems such as glucose plus hexokinase. Spectrophotometric experiments indicate that the reoxidation of NADPH in the presence of sn-phospho-3-glycerol is similarly affected. The transient inhibition by ribose 5-phosphate is not observed in the presence of an active ATP-generating system or in the presence of sufficient DL-glyceraldehyde to inhibit ribulose-5-phosphate kinase activity. It is concluded that ribose 5-phosphate inhibits photosynthetic O2 evolution by adversely affecting the steady-state ATP/ADP ratio and consequently the reduction of sn-phospho-3-glycerol to glyceraldehyde 3-phosphate. The results are discussed in their relation to ADP regulation of photosynthetic carbon assimilation and metabolite transport.     

Purification and characterization of specific 3-deoxy-D-manno-octulosonate 8-phosphate phosphatase from Escherichia coli B

A phosphatase specific for the hydrolysis of 3-deoxy-d-manno-octulosonate (KDO)-8-phosphate was purified approximately 400-fold from crude extracts of Escherichia coli B. The hydrolysis of KDO-8-phosphate to KDO and inorganic phosphate in crude extracts of E. coli B, grown in phosphate-containing minimal medium, could be accounted for by the enzymatic activity of this specific phosphatase. No other sugar phosphate tested was an alternate substrate or inhibitor of the purified enzyme. KDO-8-phosphate phosphatase was stimulated three- to fourfold by the addition of 1.0 mM Co(+) or Mg(2+) and to a lesser extent by 1.0 mM Ba(2+), Zn(2+), and Mn(2+). The activity was inhibited by the addition of 1.0 mM ethylenediaminetetraacetic acid, Cu(2+), Ca(2+), Cd(2+), Hg(2+), and chloride ions (50% at 0.1 M). The pH optimum was determined to be 5.5 to 6.5 in both tris(hydroxymethyl)aminomethane-acetate and HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer. This specific phosphatase had an isoelectric point of 4.7 to 4.8 and a molecular weight of 80,000 +/- 6,000 as determined by molecular sieving and Ferguson analysis. The enzyme appeared to be composed of two identical subunits of 40,000 to 43,000 molecular weight. The apparent K(m) for KDO-8-phosphate was determined to be 5.8 +/- 0.9 x 10(-5) M in the presence of 1.0 mM Co(2+), 9.1 +/- 1 x 10(-5) M in the presence of 1.0 mM Mg(2+), and 1.0 +/- 0.2 x 10(-4) M in the absence of added Co(2+) or Mg(2+).     

Role of sphingosine-1-phosphate phosphatase 1 in epidermal growth factor-induced chemotaxis

Sphingosine-1-phosphate (S1P) is the ligand for a family of specific G protein-coupled receptors that regulate a wide variety of cellular functions, including cytoskeletal rearrangements and cell motility. Because of the pivotal role of S1P, its levels are low and tightly regulated in a spatial-temporal manner through its synthesis catalyzed by sphingosine kinases and degradation by an S1P lyase and specific S1P phosphatases (SPP). Surprisingly, down-regulation of SPP-1 enhanced migration toward epidermal growth factor (EGF); conversely, overexpression of SPP-1, which is localized in the endoplasmic reticulum, attenuated migration toward EGF. To determine whether the inhibitory effect on EGF-induced migration was because of decreased S1P or increased ceramide as a consequence of acylation of increased sphingosine by ceramide synthase, we used fumonisin B1, a specific inhibitor of ceramide synthase. Although fumonisin B1 blocked ceramide production and increased sphingosine, it did not reverse the negative effect of SPP-1 expression on EGF- or S1P-induced chemotaxis. EGF activated the epidermal growth factor receptor to the same extent in SPP-1-expressing cells, yet ERK1/2 activation was impaired. In agreement, PD98059, an inhibitor of the ERK-activating enzyme MEK, decreased EGF-stimulated migration. We next examined the possibility that intracellularly generated S1P might be involved in activating a G protein-coupled S1P receptor important for EGF-directed migration. Treatment with pertussis toxin to inactivate Galpha(i) suppressed EGF-induced migration. Moreover, expression of regulator of G protein signaling 3, which inhibits S1P receptor signaling and completely prevented ERK1/2 activation mediated by S1P receptors, not only reduced migration toward S1P but also markedly reduced migration toward EGF. Collectively, these results suggest that metabolism of S1P by SPP-1 is important for EGF-directed cell migration.     

2-Carboxy-D-arabinitol 1-phosphate (CA1P) phosphatase: evidence for a wider role in plant Rubisco regulation

The genes for CA1Pase (2-carboxy-D-arabinitol-1-bisphosphate phosphatase) from French bean, wheat, Arabidopsis and tobacco were identified and cloned. The deduced protein sequence included an N-terminal motif identical with the PGM (phosphoglycerate mutase) active site sequence [LIVM]-x-R-H-G-[EQ]-x-x-[WN]. The corresponding gene from wheat coded for an enzyme with the properties published for CA1Pase. The expressed protein lacked PGM activity but rapidly dephosphorylated 2,3-DPG (2,3-diphosphoglycerate) to 2-phosphoglycerate. DTT (dithiothreitol) activation and GSSG inactivation of this enzyme was pH-sensitive, the greatest difference being apparent at pH 8. The presence of the expressed protein during in vitro measurement of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) activity prevented a progressive decline in Rubisco turnover. This was due to the removal of an inhibitory bisphosphate that was present in the RuBP (ribulose-1,5-bisphosphate) preparation, and was found to be PDBP (D-glycero-2,3-pentodiulose-1,5-bisphosphate). The substrate specificity of the expressed protein indicates a role for CA1Pase in the removal of 'misfire' products of Rubisco.     

Transient inhibition by ribose 5-phosphate of photosynthetic O2 evolution in a reconstituted chloroplast system

Photosynthetic oxygen evolution by a reconstituted chloroplast system utilising sn-phospho-3-glycerol (3-phosphoglycerate) ceases upon the addition of ribose 5-phosphate even though the presence of this metabolite permits a rapid and immediate CO₂ fixation. The period of cessation is appreciable at 0.1 mM ribose 5-phosphate. It is lengthened as the amount of added ribose 5-phosphate is increased and by the addition of dithiothreitol, a known activator of ribulose-5-phosphate kinase. Ribulose 1,5-bisphosphate is without effect. A similar interruption of O₂ evolution may also be brought about by the addition of ADP or by ADP-generating systems such as glucose plus hexokinase. Spectrophotometric experiments indicate that the reoxidation of NADPH in the presence of sn-phospho-3-glycerol is similarly affected.The transient inhibition by ribose 5-phosphate is not observed in the presence of an active ATP-generating system or in the presence of sufficient dl-glyceraldehyde to inhibit ribulose-5-phosphate kinase activity.It is concluded that ribose 5-phosphate inhibits photosynthetic O₂ evolution by adversely affecting the steady-state ATP/ADP ratio and consequently the reduction of sn-phospho-3-glycerol to glyceraldehyde 3-phosphate. The results are discussed in their relation to ADP regulation of photosynthetic carbon assimilation and metabolite transport.     

Degradation of glyceraldehyde-3-phosphate dehydrogenase triggered by 4-hydroxy-2-nonenal and 4-hydroxy-2-hexenal

Lipid peroxidation products such as 4-hydroxy-2-nonenal (HNE) may be responsible for various pathophysiological events under oxidative stress, since they injure cellular components such as proteins and DNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a key enzyme of glycolysis and has been reported to be a multifunctional enzyme, is one of the enzymes inhibited by HNE. Previous studies showed that GAPDH is degraded when incubated with acetylleucine chloromethyl ketone (ALCK), resulting in the liberation of a 23-kDa fragment. In this study, we examined whether GAPDH incubated with HNE or other aldehydes of lipid peroxidation products are degraded similarly to that with ALCK. The U937 cell extract was incubated with these aldehydes at 37 degrees C and analyzed by Western blotting using anti-GAPDH antibodies. Incubation with HNE or 4-hydroxy-2-hexenal (HHE) decreased GAPDH activity and GAPDH protein level, and increased the 23-kDa fragment, in time- and dose-dependent manners, but that with other aldehydes did not. Gel filtration using the Superose 6 showed that the GAPDH-degrading activity was eluted in higher molecular fractions than proteasome activity. The enzyme activity was detected at the basic range of pH and inhibited by serine protease inhibitors, diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, but not by other protease inhibitors including a proteasome inhibitor, MG-132, and a tripeptidyl peptidase II (TPP II) inhibitor, AAF-CMK. These results suggest that GAPDH modified by HNE and HHE is degraded by a giant serine protease, releasing the 23-kDa fragment, not by proteasome or TPP II.