Intracellular localization of CA1P and CA1P phosphatase activity in leaves of Phaseolus vulgaris L.

CA1P and CA1P phosphatase occur in the chloroplasts of leaf mesophyll cells of many species. However, whether either may occur exclusively in the chloroplast has not yet been established. To examine their intracellular distribution, mature, dark-or light-treated leaves of Phaseolus vulgaris were frozen, lyophilized and then centrifuged in density gradients of heptane and tetrachloroethylene. After gradient fractionation, both CA1P and CA1P phosphatase activity co-segregated with chloroplast material. Distribution analyses using sub-cellular compartment markers indicated that both CA1P and CA1P phosphatase do occur exclusively in leaf chloroplasts.Abbreviations Bicine N,N-bis[2-hydroxyethyl]glycine - CA1P 2-carboxyarabinitol 1-phosphate - CABP 2-carboxyarabinitol 1,5-bisphosphate - Chl chlorophyll - DTT dithiothreitol - EDTA (ethylenediamine)tetraacetic acid - PEP phosphoenolpyruvate - Tris tris(hydroxymethyl)aminomethane     

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     

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.     

The orientation of substrate and reaction intermediates in the active site of ribulose-1,5-bisphosphate carboxylase

There are four possible orientations of the substrate ribulose 1,5-bisphosphate in the active site of ribulose-1,5-bisphosphate carboxylase. Distinction between these four possible orientations has been made on the basis of 31P NMR and borohydride-trapping experiments. The orientation of the reaction-intermediate analog, 2'-carboxy-D-arabinitol 1,5-bisphosphate with respect to the divalent metal ion was determined by 31P NMR studies of the quaternary complex, enzyme.CO2.Ni2+.2'-carboxyarabinitol 1,5-bisphosphate. Assignment of the phosphorus resonances of this complex was made by labeling the phosphoryl group at either C-1 or C-5 with 17O. The phosphorus atom closer to the paramagnetic metal ion, Ni2+, to which the broader of the phosphorus resonances is attributed, has been identified as that attached to C-1. When bound to the active site of carbaminated enzyme, D-ribulose 1,5-bisphosphate was reduced by sodium borohydride with absolute stereospecificity to D-arabinitol 1,5-bisphosphate. The reduction of the enzyme-bound substrate thus occurred on the Si face of the C-2 carbonyl group. These two results together establish that ribulose 1,5-bisphosphate is oriented within the active site so that 1) the phosphoryl group at C-1 is closer to the divalent metal ion than that at C-5 and 2) the Si face of the carbonyl group points to the "outside world."     

Inhibition of ribulose 1,5-bisphosphate carboxylase/oxygenase by 2-carboxyarabinitol-1-phosphate

In some plants, 2-carboxy-d-arabinitol 1-phosphate (CA 1P) is tightly bound to catalytic sites of ribulose, 1,5-bisphosphate carboxylase/oxygenase (rubisco). This inhibitor's tight binding property results from its close resemblance to the transition state intermediate of the carboxylase reaction. Amounts of CA 1P present in leaves varies with light level, giving CA 1P characteristics of a diurnal modulator of rubisco activity. Recently, a specific phosphatase was found that degrades CA 1P, providing a mechanism to account for its disappearance in the light. The route of synthesis of CA 1P is not known, but could involve the branched chain sugar, hamamelose. There appear to be two means for diurnal regulation of the number of catalytic sites on rubisco: carbamylation mediated by the enzyme, rubisco activase, and binding of CA 1P. While strong evidence exists for the involvement of rubisco activase in rubisco regulation, the significance of CA 1P in rubisco regulation is enigmatic, given the lack of general occurrence of CA 1P in plant species. Alternatively, CA 1P may have a role in preventing the binding of metabolites to rubisco during the night and the noncatalytic binding of ribulose bisphosphate in the light.     

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.     

Intermediates in the ribulose-1,5-bisphosphate carboxylase reaction

At least two intermediates of the D-ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) reaction were liberated in detectable amounts when the functioning enzyme from Rhodospirillum rubrum was quenched in acid. Using substrate labeled with 32P in C-1, [32P]orthophosphate (Pi) was found when the quenched solution was rapidly processed for extraction of Pi as the acid molybdate complex. Reaction with sodium borohydride under mildly alkaline conditions immediately after acid quenching of the carboxylase reaction decreased the amount of 32Pi that was observed by 68%. The compound whose degradation to Pi was prevented by reaction with sodium borohydride decomposed under both acid and neutral conditions with a half-time of about 5 min at 25 degrees C and was assigned to the beta-keto acid recently demonstrated for the spinach enzyme ( Schloss , J.V., and Lorimer , G.H. (1982) J. Biol. Chem. 257, 4691-4694). It was sufficiently stable upon neutralization to react productively with fresh enzyme. As substrate CO2 concentration was decreased below the steady state Km value, the proportion of the 32P that did not react with sodium borohydride increased, indicative of a second unstable intermediate that precedes the carboxylation step. The decomposition of the latter intermediate to Pi, which occurs with a t1/2 less than or equal to 6 ms, was prevented if I2 was present in the acid quench medium. These are properties expected of the 2,3- enediol form of ribulose bisphosphate. Both intermediates reach their maximum levels when product formation is most rapid and disappear when product formation is complete as expected of reaction intermediates.     

Modulation of the tight binding of carboxyarabinitol 1,5-bisphosphate to the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase

The large subunit (L) of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) from Synechococcus PCC 6301 was expressed in Escherichia coli, purified as the octamer L8, and analyzed for its ability to tightly bind the transition state analog, 2-carboxyarabinitol 1,5-bisphosphate (CABP). [14C]CABP remained tightly bound to L8 after challenging with [12C]CABP and gel filtration, indicating that L8 alone without the small subunit (S) could tightly bind CABP. Binding of CABP to L8 induced a shift in the gel filtration profile due to apparent aggregation of L8. Aggregation did not occur with the L8S8-CABP complex nor with L8-CABP in the presence of 150 mM MgCl2. If ionic strength was increased with either KCl or MgCl2 during or after the binding of [14C]CABP to L8, [14C]CABP in the complex exchanged with [12C]CABP and was lost from the protein. Ionic strength strongly affected the rate constant (k4) for [14C]CABP dissociation from the L8-[14C]CABP complex, but had little effect on k4 for the L8S8-CABP complex. The differences in CABP binding characteristics between the L8-CABP and L8S8-CABP complexes demonstrate that S is intimately involved in maintaining the stability of the tight binding of CABP to the active site. These are the same interactions stabilizing the intermediate, 3-keto-2-carboxyarabinitol 1,5-bisphosphate, to native rubisco during CO2 fixation.     

Photometric method for routine determination of kcat and carbamylation of rubisco

Ribulose bisphosphate carboxylase (rubisco) is the first enzyme in photosynthetic CO₂ assimilation. It is also the single largest sink for nitrogen in plants. Several parameters of rubisco activity are often measured including initial activity upon extraction, degree of carbamylation, catalytic constant of the enzyme (k_cat), and the total amount of enzyme present in a leaf. We report here improvements of the photometric assay of rubisco in which rubisco activity is coupled to NADH oxidation which is continuously monitored in a photometer. The initial lag usually found in this assay was eliminated by assaying rubisco activity at pH 8.0 instead of 8.2, using a large amount of phosphoglycerate kinase, and adding monovalent cations to the assay buffer. We found that when using the photometric assay, the ratio of activity found initially upon extraction divided by the activity after incubating with CO₂ and Mg²⁺ reflects the degree of carbamylation as determined by ¹⁴carboxyarabinitol bisphosphate/¹²carboxyarabinitol bisphosphate competition. We developed methods for measuring the catalytic constant of rubisco as well as the total amount of enzyme present using the photometric assay and carboxyarabinitol 1,5-bisphosphate. We believe that the photometric assay for activity will prove more useful than the ¹⁴CO₂ assay in many studies.Abbreviations CA1P 2-carboxyarabinitol 1-phosphate - GAP glyceraldehyde 3-phosphate - OD optical density - PGA 3-phosphoglycerate - rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate     

Comparative affinities of the epimeric reaction-intermediate analogs 2- and 4-carboxy-D-arabinitol 1,5-bisphosphate for spinach ribulose 1,5-bisphosphate carboxylase

2-Carboxy-3-keto-D-arabinitol 1,5-bisphosphate is a tightly bound intermediate of the carboxylase reaction of ribulosebisphosphate carboxylase/oxygenase. Two stereoisomers of an analog of this intermediate, 2-carboxy-D-arabinitol 1,5-bisphosphate (2CABP) and 4-carboxy-D-arabinitol 1,5-bisphosphate (4CABP), are exceptionally potent, virtually irreversible inhibitors of the spinach carboxylase, presumably due to their structural similarity to the gem-diol (hydrated carbonyl at C-3) form of the intermediate. Incubation of the enzyme with either leads to time-dependent loss of activity. Inhibition of the enzyme is biphasic, with initial dissociation constants of 0.47 and 0.19 microM and maximal rates for tight complex formation of 2.2 and 1.8 min-1 for 2CABP and 4CABP, respectively. These values give second-order rate constants for tight complex formation of 7.8 x 10(4) and 1.6 x 10(5) M-1 s-1. To determine the overall affinity of the spinach enzyme for 2CABP and 4CABP, the release rates were determined by dual isotope exchange (3H-inhibitor complex with free 14C-inhibitor). Exchange half-times of 1.82 and 530 days were observed for 4CABP and 2CABP, respectively. Overall dissociation constants of 28 pM (2.8 x 10(-11) M) and 190 fM (1.9 x 10(-13) M) were calculated from these dissociation rates together with the rates of association determined by inactivation kinetics. The difference in affinity of 2CABP and 4CABP corresponds to 2.9 kcal/mol, presumably reflecting the difference in interaction of the enzyme with the two hydroxyls of the intermediate's gem-diol. The kinetic behavior of these two inhibitors, in particular the rather slow maximal rates of association, are consistent with the expected behavior of analogs of a labile intermediate of an enzymic reaction that is far more stable than a transition state.     

Light-dependent modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in the genus Phaseolus

Modulation of the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in low light and darkness was measured in A) 25 genotypes from the four cultivated species of Phaseolus (P. vulgaris, P. acutifolius, P. lunatus and P. coccineus), B) 8 non-cultivated Phaseolus species, and C) the related species Macroptileum atropurpureum. The activity ratio of Rubisco (the ratio of initial and total Rubisco activities, which reflects Rubisco carbamylation), and the molar activity of fully-activated Rubisco (which primarily reflects the inhibition of Rubisco activity by carboxyarabinitol 1-phosphate, CA1P) were assayed in leaves from the cultivated species sampled at midday in full sunlight, in low light at dusk (60 to 100 mol photons m^-2s^-1), and after at least 4 h in darkness. Dark inhibition of Rubisco molar activity was compared in both cultivated and non-cultivated species. In all cultivated genotypes, a significant reduction of the activity ratio of Rubisco was measured in leaves sampled at low light; however, the molar activity of fully activated Rubisco was not greatly reduced in these low light samples. In darkened leaves, molar activities substantially declined in most Phaseolus species with 11 of 13 exhibiting greater than 60% reduction. In P. vulgaris, the reduction of molar activity was extensive (greater than 69%) in all genotypes studied, which included wild progenitors as well as ancient and advanced cultivars. These results indicate that at low light late in the day, modulation of Rubisco activity is primarily through changes in carbamylation state, with CA1P playing a more limited role. By contrast in the dark, binding of CA1P dominates the modulation of Rubisco activity in Phaseolus in a pattern that appears to be conserved within a species, but can vary significantly between species within a genus. The degree of CA1P inhibition in Phaseolus was associated with phylogenetic affinities within the genus, as the species with extensive dark-inhibition of Rubisco activity tended to be more closely related to each other than to species with reduced inhibition of Rubisco activity.Abbreviations CA1P carboxyarabinitol 1-phosphate - CABP carboxyarabinitol bisphosphate - PFD photon flux density between 400 and 700 nm - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase     

Measurement of ribulose 1,5-bisphosphate from spinach chloroplasts

A technique has been developed for the rapid and simple measurement of ribulose 1,5-bisphosphate from isolated spinach chloroplasts. The endogenous ribulose bisphosphate was detected enzymically using (14)CO(2) and ribulose bisphosphate carboxylase/oxygenase released from the chloroplasts. Ribulose 5-phosphate kinase was inhibited with 0.4 to 0.6 millimolar 2,6-dichlorophenol-indophenol and 4 micromolar carbonyl cyanide m-chlorophenylhydrazone. Phosphoenolpyruvate carboxylase activity was low with washed chloroplasts and its labeled product, [(14)C]oxalacetate, was destroyed by heating with 1.0 n HCl at 90 C. The assay method was linear from 0.05 to 0.87 nanomoles ribulose bisphosphate per milliliter. The latter value was determined with chloroplast material having 44 micrograms of chlorophyll per milliliter. This technique was simple and direct, used less chloroplast material, yet provided results comparable to a previously described enzymic technique in which ribulose bisphosphate was determined after the precipitation of chloroplast proteins by perchloric acid.     

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     

Characterization of phosphofructokinase 2 and of enzymes involved in the degradation of fructose 2,6-bisphosphate in yeast

Phosphofructokinase 2 from Saccharomyces cerevisiae was purified 8500-fold by chromatography on blue Trisacryl, gel filtration on Superose 6B and chromatography on ATP-agarose. Its apparent molecular mass was close to 600 kDa. The purified enzyme could be activated fivefold upon incubation in the presence of [gamma-32P]ATP-Mg and the catalytic subunit of cyclic-AMP-dependent protein kinase from beef heart; there was a parallel incorporation of 32P into a 105-kDa peptide and also, but only faintly, into a 162-kDa subunit. A low-Km (0.1 microM) fructose-2,6-bisphosphatase could be identified both by its ability to hydrolyze fructose 2,6-[2-32P]bisphosphate and to form in its presence an intermediary radioactive phosphoprotein. This enzyme was purified 300-fold, had an apparent molecular mass of 110 kDa and was made of two 56-kDa subunits. It was inhibited by fructose 6-phosphate (Ki = 5 microM) and stimulated 2-3-fold by 50 mM benzoate or 20 mM salicylate. Remarkably, and in deep contrast to what is known of mammalian and plant enzymes, phosphofructokinase 2 and the low-Km fructose-2,6-bisphosphatase clearly separated from each other in all purification procedures used. A high-Km (approximately equal to 100 microM), apparently specific, fructose 2,6-bisphosphatase was separated by anion-exchange chromatography. This enzyme could play a major role in the physiological degradation of fructose 2,6-bisphosphate, which it converts to fructose 6-phosphate and Pi, because it is not inhibited by fructose 6-phosphate, glucose 6-phosphate or Pi. Several other phosphatases able to hydrolyze fructose 2,6-bisphosphate into a mixture of fructose 2-phosphate, fructose 6-phosphate and eventually fructose were identified. They have a low affinity for fructose 2,6-bisphosphate (Km greater than 50 microM), are most active at pH 6 and are deeply inhibited by inorganic phosphate and various phosphate esters.     

Fructose 2,6-bisphosphate and its phosphorothioate analogue. Comparison of their hydrolysis and action on glycolytic and gluconeogenic enzymes.

Purified chicken liver 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was phosphorylated either from fructose 2,6-bis[2-32P]phosphate or fructose 2-phosphoro[35S]thioate 6-phosphate. The turnover of the thiophosphorylated enzyme intermediate as well as the overall phosphatase reaction was four times faster than with authentic fructose 2,6-bisphosphate. Fructose 2-phosphorothioate 6-phosphate was 10-100-fold less potent than authentic fructose 2,6-bisphosphate in stimulating 6-phosphofructo-1-kinase and pyrophosphate:fructose 6-phosphate phosphotransferase, but about 10 times more potent in inhibiting fructose 1,6-bisphosphatase. The analogue was twice as effective as authentic fructose 2,6-bisphosphate in stimulating pyruvate kinase from trypanosomes.     

Degradation of 2-carboxyarabinitol 1-phosphate by a specific chloroplast phosphatase

The catalytic degradation of 2-carboxyarabinitol 1-phosphate (CA 1-P), a naturally occurring inhibitor of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), was investigated by chromatographic and spectroscopic analyses of the reaction products. Carboxy-labeled [¹⁴C]CA 1-P was incubated with a partially purified tobacco (Nicotiana rustica) chloroplast protein that has been shown previously to catalyze metabolism of CA 1-P to a form incapable of inhibiting Rubisco (ME Salvucci, GP Holbrook, JC Anderson, and G Bowes [1988] FEBS Lett 231: 197-201). In the presence and absence of NADPH, ion-exchange chromatography showed a progressive conversion of [2′-¹⁴C]CA 1-P to a labeled compound which coeluted with authentic carboxyarabinitol. Parallel assays with unlabeled CA 1-P showed a concomitant decrease in the ability of reaction samples to inhibit Rubisco activity. In separate experiments, a 1:1 stoichiometry was found between the release of inorganic phosphate from [2′-¹⁴C]CA 1-P and accumulation of the ¹⁴C-labeled product. Liberation of inorganic phosphate was not observed when the tobacco enzyme was incubated with ribulose-1,5-bisphosphate, fructose-1,6-bisphosphate, glucose-1-phosphate, glucose-6-phosphate, or 6-phosphogluconate. Proton nuclear magnetic resonance spectroscopy of the labeled CA 1-P reaction product established its identity as carboxyarabinitol. We therefore propose that light-stimulated degradation of CA 1-P is catalyzed in vivo by a specific phosphatase, 2-carboxyarabinitol 1-phosphatase. Carboxyarabinitol 1-phosphatase activity was detected in the absence of NADPH, but increased threefold when 2 millimolar NADPH was present. Thus, while not required for the reaction, NADPH may play an important role in the regulation of CA 1-P degradation.     

31P NMR Identification of Metabolites and pH Determination in the Cyanobacterium Synechocystis sp. PCC 6308

The identity of a number of phosphorus-containing metabolites present in Synechocystis sp. PCC 6308 has been confirmed by ³¹P NMR spectroscopy. The presence of D-ribulose 1,5-bisphosphate (RuBP); DL-glyceraldehyde 3-phosphate (GlyP); D(−) 3-phosphoglyceric acid (3PGA); D-ribulose 5-phosphate (Ru5P); 6-phosphogluconic acid (6PGA); phosphoenolpyruvate (PEP); inorganic phosphate (Pi); uridine diphosphoglucose (UDPG); ADP and ATP were demonstrated by the pH dependence of their ³¹P NMR chemical shifts in spectra of perchloric acid cell extracts. Intracellular pH of cells was determined to be 7.5–7.7. Received: 20 September 1996 / Accepted: 26 October 1996     

Fructose-2,6-bisphosphatase and 6-phosphofructo-2-kinase are separable in yeast.

Fructose-2,6-bisphosphatase was purified from yeast and separated from 6-phosphofructo-2-kinase and alkaline phosphatase. The enzyme released Pi from the 2-position of fructose 2,6-bisphosphate and formed fructose 6-phosphate in stoichiometric amounts. The enzyme displays hyperbolic kinetics towards fructose 2,6-bisphosphate, with a Km value of 0.3 microM. It is strongly inhibited by fructose 6-phosphate. The inhibition is counteracted by L-glycerol 3-phosphate. Phosphorylation of the enzyme by cyclic-AMP-dependent protein kinase causes inactivation, which is reversible by the action of protein phosphatase 2A.     

Proteolysis and transition-state-analogue binding of mutant forms of ribulose-1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii

Trypsin digestion reduces the sizes of both the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) from the green alga Chlamydomonas reinhardtii. Incubation of either CO₂/Mg²⁺ -activated or nonactivated enzyme with the transition-state analogue carboxyarabinitol bisphosphate protects a trypsin-sensitive site of the large subunit, but not of the small subunit. Incubation of the nonactivated enzyme with ribulosebisphosphate (RuBP) provided the same degree of protection. Thus, the very tight binding that is a characteristic of the transitionstate analogue is apparently not required for the protection of the trypsin-sensitive site of the large subunit. Mutant enzymes that have reduced CO₂/O₂ specificities failed to bind carboxyarabinitol bisphosphate tightly. However, their large-subunit trypsin-sensitive sites could still be protected. The K _m values for RuBP were not significantly changed for the mutant enzymes, but the V _max values for carboxylation were reduced substantially. These results indicate that the failure of the mutant enzymes to bind the transition-state analogue tightly is primarily the consequence of an impairment in the second irreversible binding step. Thus, in all of the mutant enzymes, defects appear to exist in stabilizing the transition state of the carboxylation step, which is precisely the step proposed to influence the CO₂/O₂ specificity of Rubisco.Abbreviations and Symbols CABP 2-carboxyarabinitol 1,5-bisphosphate - enol-RuBP 2,3-enediolate of ribulose 1,5-bisphosphate - K _c K _m for CO₂ - K _o K _m for O₂ - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - V _c V _max for carboxylation - V _o V _max for oxygenation Paper No. 9313, Journal Series, Nebraska Agricultural Research DivisionThis work was supported by National Science Foundation grant DMB-8703820. We thank Drs. Archie Portis and Raymond Chollet for their helpful comments, and also thank Dr. Chollet for graciously providing CABP and [¹⁴C]CABP.     

Effects of fructose on the energy metabolism and acid-base status of the perfused starved-rat liver. A 31phosphorus nuclear magnetic resonance study.

Fructose metabolism has been studied with 31P n.m.r. in perfused livers from rats starved for 48h. The time course of changes in liver ATP, Pi and sugar phosphate (fructose l-phosphate) concentrations, and intracellular pH were followed in each perfusion after infusion of fructose to give an initial concentration of either 5mM or 10mM. Rapid falls in the concentrations of ATP and Pi and intracellular pH occurred after infusion of fructose, reaching a minimum after 4-5 min, which was lower in the 10mM group than in the 5mM group. These changes were accompanied by a rapid rise in fructose 1-phosphate, reaching a plateau also after 4-5 min. At both concentrations of fructose, after the early falls, some recovery of ATP, Pi and intracellular pH occurred; this was complete for Pi and intracellular pH in the 5mM-fructose experiments (within 12-30 min). Complete restoration of ATP to the pre-fructose value was not achieved in either the 5mM of 10mM groups. Measurements of the uptake of lactate by the liver indicated that the fall in intracellular pH was caused primarily by production of protons accompanying the formation of lactate from fructose with possibly a transient contribution generated during the rise in fructose 1-phosphate.