The role of photophosphorylation in SO2 and SO 3 2- inhibition of photosynthesis in isolated chloroplasts

Sulphur dioxide inhibits noncyclic photophosphorylation in isolated envelope-free chloroplasts. This inhibition was shown to be reversible and competitive with phosphate, with an inhibitor constant of K_i=0.8mM. The same inhibition characteristics were observed when phosphoglycerate (PGA)- or ribulose-1,5-bisphosphate (RuBP)-dependent oxygen evolution was examined in a reconstituted chloroplast system in the presence of SO ₃ ^2- . Using an ATP-regenerating system (phosphocreatine-creatine kinase), it was demonstrated that the inhibition of PGA-dependent oxygen evolution is solely the result of inhibited photophosphorylation. It is concluded that at low SO₂ and SO ₃ ^2- concentrations the inhibition of photophosphorylation is responsible for the inhibition of photosynthetic oxygen evolution.Abbreviations Chl chlorophyll - PGA D-3-phosphoglyceric acid trisodium salt - Pi inorganic phosphate - RuBP D-ribulose-1,5-bisphosphoric acid tetrasodium salt     

Purification,some properties and quaternary structure of thed-ribulose 1,5-diphosphate carboxylase ofAlcaligenes eutrophus

d-Ribulose 1,5-diphosphate carboxylase has been purified from autotrophically grown cells of the facultative chemolithotrophic hydrogen bacteriumAlcaligenes eutrophus. The enzyme was homogeneous by the criteria of polyacrylamide gel electrophoresis. The molecular weight of the enzyme was 505000 determined by gel filtration and sucrose density gradient centrifugation, and a sedimentation coefficient of 18.2 S was obtained. It was demonstrated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis that the enzyme consists of two types of subunits of molecular weight 52000 and 13000.Electron microscopy on the intact and the partially dissociated enzyme lead to the construction of a model for the quaternary structure of the enzyme which is composed of 8 large and 8 small subunits. The most probable symmetry of the enzyme molecule is 4:2:2.Michaelis constant (K _m ) values for ribulose 1,5-diphosphate, Mg^2-, and CO₂ were 0.59 mM, 0.33 mM, and 0.066 mM measured under air. Oxygen was a competitive inhibitor with respect to CO₂ suggesting that the enzyme also exhibits an oxygenase activity. The oxygenolytic cleavage of ribulose 1,5-diphosphate was shown and a 1:1 stoichiometry between oxygen consumption and 3-phosphoglycerate formation observed.Abbreviations DTE dithioerythritol - EDTA ethylenediamine tetraacetate - RuDP d-ribulose 1,5-diphosphate     

Specific inhibition of oxygenase activity of ribulose-1,5-diphosphate carboxylase by hydroxylamine

Ribulose-1,5-diphosphate oxygenase activity of ribulose-1,5-diphosphate carboxylase was completely inhibited by preincubation of the enzyme with 5mM hydroxylamine in presence of the substrate ribulose-1,5-diphosphate. Inhibition by hydroxylamine was uncompetitive with respect to ribulose-1,5-diphosphate and noncompetitive with respect to magnesium. Carboxylase activity was not affected by hydroxylamine. These results suggest that the two activities of the enzyme can be regulated differentially and that inhibiting the oxygenase activity does not stimulate the carboxylase activity of the enzyme. The data further suggest that the inhibition by hydroxylamine may be through its interaction with carbonyl groups of the enzyme exposed on the binding of ribulose-1,5-diphosphate to the protein.     

Ribulose bisphosphate carboxylase from Thiobacillus A2. Its purification and properties

Ribulose bisphosphate carboxylase (EC 4.1.1.39) from Thiobacillus A2 has been purified to homogeneity on the basis of polyacrylamide gel electrophoresis and U.V. analysis during sedimentation velocity studies. The enzyme had an optimum pH of about 8.2 with Tris-HCl buffers. The molecular weight was about 521000 with an S _rel. of 16.9. K _m for RuBP was 122 M, for total CO₂ it was 4.17 mM, and for Mg²⁺ 20.0 M. The absolute requirement for a divalent cation was satisfied by Mg²⁺ which was replaceable to a certain extent by Mn²⁺. Activity was not significantly affected by SO ₄ ^2- , SO ₃ ^2- , or S₂O ₃ ^2- at 1.0 mM. At this concentration S^2- caused a 27% stimulation. All mercurials tested were inhibitory. pHMB was the most potent causing about 60% inhibition at 0.01 mM. This inhibition was reversible by low concentrations of cysteine. Cyanide was also inhibitory. Its mode of inhibition with respect to RuBP was un-competitive and with a K _i of 20 M. Lost activity could be restored partially by GSH or Cu²⁺. Although azide at the concentration tested had no significant effect on enzyme activity, 2,4-dinitrophenol at 1.0 mM caused 91% inhibition. Finally, activity was also affected by energy charge.Abbreviations ATP adenosine-5-triphosphate - GAPDH glyceraldehyde phosphate dehydrogenase - GSH (reduced) glutathione - G6P glucose-6-phosphate - NAD⁺ nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - pHMB parahydroxymercuribenzoate - 6PG 6-phosphogluconate - 3-PGA 3-phosphoglycerate - PGK phosphoglyceratekinase - RuBP ribulose-1,5-bisphosphate     

d-Ribulose 1,5-diphosphate carboxylase from the blue-green alga Aphanocapsa 6308

d-Ribulose 1,5-diphosphate carboxylase from extracts of the unicellular blue-green alga Aphanocapsa 6308 has been purified by ammonium sulphate precipitation and linear sucrose density gradient centrifugation. The molecular weight was estimated to be 525 000 and the enzyme consisted of two types of sub-unit of molecular weights 51 000 and 15 000. The small sub-units were not detected after purification involving acid precipitation but were observed if the acid precipitation step was omitted. The Michaelis constants for Mg²⁺ and CO₂, when tested under air, were 0.35 mM and 0.071 mM respectively. Oxygen acted as a competitive inhibitor with respect to CO₂, suggesting that the enzyme also acts as an oxygenase. This was confirmed by measuring ribulose diphosphate-dependent O₂ uptake. A 1:1 stoichiometry between ribulose diphosphate utilization and O₂ consumption was observed. 6-Phosphogluconate inhibited carboxylase activity both at high (20 mM) and low (1 mM) bicarbonate concentrations. The data are compared with the properties of ribulose diphosphate carboxylase from other autotrophic prokaryotes and from chloroplasts.Abbreviations RuDP d-Ribulose 1,5-diphosphate - EDTA ethylene diamine tetraacetic acid - GSH reduced glutathione - SDS sodium dodecyl sulphate - 6PGluc 6-phosphogluconate - STB supplemented Tris buffer     

Small-angle X-ray study on the structure of ribulose-1,5-bisphosphate carboxylase-oxygenase from Rhodospirillum rubrum

The quaternary structure of ribulose-1,5-bisphosphate carboxylase-oxygenase (rubisco) from Rhodospirillum rubrum, an enzyme consisting of two large subunits, L₂, was investigated by small-angle X-ray scattering. In the presence of HCO ₃ ^- and Mg²⁺, rubisco is in the active state and displays a radius of gyration of 2.96 nm, a maximum diameter of 9.5 nm and a volume of 170 nm³. A model is presented where the subunits are arranged back-to-back, rotated relative to each other by 90°, and shifted by 1.3 nm. Upon inactivation by removal of HCO ₃ ^- and Mg²⁺, the model swells slightly without any distinct changes in configuration. This contrasts with our previous observations with rubisco from Alcaligenes eutrophus, an enzyme composed of small (S) and large (L) subunits, L₈S₈, where inactivation gives rise to substantial changes in configuration.Abbreviations RuBP Ribulose-1,5-bisphosphate - 3-PGA 3-phosphoglyceric acid     

Nitrogen supply as a factor influencing photoinhibition and photosynthetic acclimation after transfer of shade-grown Solanum dulcamara to bright light

We have compared the ability of shadegrown clones of Solamum dulcamara L. from shade and sun habitats to acclimate to bright light, as a function of nitrogen nutrition before and after transfer to bright light. Leaves of S. dulcamara grown in the shade with 0.6 mM NO ₃ ^- have similar photosynthetic properties as leaves of plants grown with 12.0 mM NO ₃ ^- . When transferred to bright light for 1–2 d the leaves of these plants show substantial photoinhibition which is characterized by about 50% decrease in apparent quantum yield and a reduction in the rate of photosynthesis in air at light saturation. Photoinhibition of leaf photosynthesis is associated with reduction in the variable component of low-temperature fluorescence emission, and with loss of in-vitro electron transport, especially of photosystem II-dependent processes.We find no evidence for ecotypic differentiation in the potential for photosynthetic acclimation among shade and sun clones of S. dulcamara, or of differentiation with respect to nitrogen requirements for acclimation. Recovery from photoinhibition and subsequent acclimation of photosynthesis to bright light only occurs in leaves of plants provided with 12.0 mM NO ₃ ^- . In these, apparent quantum yield is fully restored after 14 d, and photosynthetic acclimation is shown by an increase in light-saturated photosynthesis in air, of light-and CO₂-saturated photosynthesis, and of the initial slope of the CO₂-response curve. The latter changes are highly correlated with changes in ribulose-bisphosphate-carboxylase activity in vitro. Plants supplied with 0.6 mM NO ₃ ^- show incomplete recovery of apparent quantum yield after 14 d, but CO₂-dependent leaf photosynthetic parameters return to control levels.Symbols and abbreviations F_o initial level of fluorescence at 77 K - F_m maximum level of fluorescence at 77 K - F_v variable components of fluorescence at 77 K (F_v=F_m-F_o) - PSI, PSII photosystem I and II, respectively - RuBP ribulose-1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase-oxygenase (EC 4.1.1.39)     

Further proof for the catalytic role of the larger subunit in the spinach leaf ribulose-1,5-diphosphate carboxylase

The catalytically active oligomeric form of the larger subunit, A_m, obtained from spinach leaf ribulose-1,5-diphosphate carboxylase by pretreatment with p-mercuribenzoate at pH 7.5 followed by incubation at pH 9.0, was free of the smaller subunit based on C-terminal amino acid analyses. Valine was the predominant C-terminus of the A_m preparations, the release of tyrosine being negligibly small [cf. Sugiyama and Akazawa, $\text{Biochemistry}$ 9 (1970) 4499]. The pH optimum of the ribulose-1,5-diphosphate carboxylase reaction by A_m was about 8.5, in comparison to the native enzyme which showed an alkaline pH optimum only in the absence of Mg²⁺. The substrate saturation curve of the catalytic subunit with respect to bicarbonate followed the Michaelis-Menten equation, as contrasted to the anomalous reaction kinetics of the native ribulose-1,5-diphosphate carboxylase molecule reported previously. These overall results indicate that the allosteric properties of spinach ribulose-1,5-diphosphate carboxylase are possibly conveyed by a unique structural conformation that requires the presence of the smaller subunit in association with the larger catalytic subunit component of the enzyme molecule.     

Properties and regulation of ribulose diphosphate carboxylase from Thiobacillus novellus

Ribulose-diphosphate carboxylase from Thiobacillus novellus has been purified to homogeneity as observed by polyacrylamide gel electrophoresis and U. V. light observation during sedimentation velocity analysis. The optimum pH for the enzyme with Tris-HCl buffers was about 8.2. Concentrations of this buffer in excess of 80 mM were inhibitory. The apparent K _m RuDP was about 14.8 M with a Hill value of 1.5, for HCO ₃ ^- the apparent K _m was about 11.7 mM with an n value of 1.18 and for Mg²⁺ about 0.61 mM. The enzyme was specific for this cation. Relatively high concentrations of either Hg²⁺ or pCMB were required before significant inhibition was observed. Activity declined slowly during a 4-hr incubation period in either 3.0 M or 8.0 M urea. Incubation for 12 hrs resulted in complete loss of activity which was not prevented by 10 mM Mg²⁺ and was not reversed by dialysis and subsequent addition of 10 mM cysteine. Polyacrylamide gel electrophoresis revealed a loss of the major band and the appearance of 2 new bands. SDS polyacrylamide gel electrophoresis gave an average M.W. of 73 500±2500 for the slower moving band and 12250 ±2500 for the faster moving. However, incubation in urea for up to 40 hrs revealed a decrease in the M.W. of the slower moving band to about 60000. The E _a for the enzyme was calculated to be about 18.85 kcal mole^-1, with the possibility of a break between 40 and 50°C. The Q ₁₀ was 3.07 between 20 to 30°C whereas between 30 to 40°C it was 3.31. Only phosphorylated compounds caused significant inhibition of enzyme activity. They included ADP, FDP, F6P, G6P, PEP, 6PG, 2-PGA, R1P, R5P and Ru5P.Abbreviations ATP adenosine-5-triphosphate - FDP fructose-1,6-diphosphate - F6P fructose-6-phosphate - G6P glucose-6-phosphate - GPDH glyceraldehyde-3-phosphate dehydrogenase - NADH nicotinamide adenine dinucleotide (reduced) - OAA oxalacetate - pCMB parachlormercuribenzoate - PEP phosphoenolpyruvate - 6PG 6-phosphogluconate - 2-PGA 2-phosphoglycerate - 3-PGA 3-phosphoglycerate - PGK 3-phosphoglyceric phosphokinase - R1P ribose-1-phosphate - R5P ribose-5-phosphate - RuDP ribulose-1,5-diphosphate - Ru5P ribulose-5-phosphate - SDS sodium dodecyl sulfate     

Chlorophyll Accumulation, CO2 Fixation Enzymes, and Hill Activity, in Greening Roots of Lens culinaris

The carboxylation of ribulose-1,5-diphosphate was demonstrated in vitro with extracts of ctiolated seedling roots. The presence of ribulose-1,5-diphosphate carboxylase was characterized in the subcellular fraction enriched in amyloplasts. Synthesis of chlorophyll, development of CO² fixation capacities and of Hill activity upon illumination have been studied with roots of Lens culinaris seedlings. The marked increases in CO² fixation with ribulose-1,5-diphosphate as the substrate and in Hill activity that occur after a lag phase seem to be related to cytological changes during the greening of roots.     

Carbon dioxide fixation in Pyrobotrys stellata

The green alga Pyrobotrys stellata Korshik., an obligate phototroph, is unable to utilise carbon dioxide for growth, although assimilation of acetate is dependent on the photosynthetic process. The incorporation of ¹⁴CO₂ from ¹⁴C-bicarbonate into the cells of P. stellata is only 3% of that in Chlorella pyrenoidosa Chick. The activity of the key enzyme of the Calvin cycle, ribulose-1-5-diphosphate carboxylase, is very low in P. stellata, being only 7% of that in C. pyrenoidosa. The determination of the products of ¹⁴CO₂ fixation in intact cells confirms that ribulose-1-5-diphosphate activity is very low in P. stellata, since little carbon-14 is found in 1–3 diphosphoglyceric acid, the product of carboxylation of ribulose-1-5-diphosphate. It is concluded that the inability of P. stellata to utilize carbon dioxide for growth in the light is probably the result of the low ribulose-1-5-diphosphate carboxylase activity in the organism.     

Metabolic regulation in Pseudomonas oxalaticus OX1

Metabolic control associated with diauxic growth of Pseudomonas oxalaticus in batch cultures on mixtures of formate and oxalate was investigated by measuring intracellular enzyme and coenzyme concentrations and Q _{O 2}values during transition experiments from oxalate to formate and vice versa. In transition from oxalate to formate oxalyl-CoA reductase concentration declined after the exhaustion of oxalate and ribulose-1,5-diphosphate carboxylase and ¹⁴CO₂ fixation appeared upon addition of formate. In the reciprocal transition, ribulose-1,5-diphosphate carboxylase and ¹⁴CO₂ fixation rate declined sharply after formate exhaustion, and oxalyl-CoA reductase appeared only after addition of oxalate. The intracellular NAD and NADP concentrations measured in the same experiments are reported. At substrate exhaustion the proportion of NAD in the reduced form fell from 15–20% to 2%. On addition of formate to an oxalate-starved culture there was an immediate increase in the proportion of NADH to 50%; such an increase was not observed in the reverse experiment.Abbreviations RuDP ribulose-1,5-diphosphate - HEPES 2-(N-2 hydroxyethylpiperazin-N-yl) ethane sulphonic acid     

Enzyme activities in an artificial stroma medium

When spinach leaf tissue was subjected to evaporative dehydration, photosynthetic capacity at very high (5%) CO₂ concentration and saturating irradiance (300 W·m^-2), decreased in parallel to the relative water content (RWC). A 50% inhibition was observed at 60–40% RWC. In order to examine whether the inhibition was caused by increased solute concentrations in chloroplasts or cytoplasm, an artificial stroma medium (ASM) was set up containing all major osmotically relevant solutes measured in isolated intact spinach chloroplasts. Subsequently, the response of enzyme activities to normal and to increased concentrations of ASM was examined. Inhibition of enzymes by a concerted increase of all solutes was well correlated to the in-vivo response of photosynthesis to dehydration (60% inhibition at double-strength ASM). Inhibitory solutes were mainly divalent inorganic anions, such as sulfate and phosphate. Inhibition of ribulose-1,5-bisphosphate carboxylase by these ions as studied in more detail. Inhibition of the enzyme by sulfate and phosphate was competitive with respect to ribulose-1,5-bisphosphate, but not with respect to CO₂. The K_I for sulfate was 2.1 mmol·l^-1 and for phosphate 0.57 mmol·l^-1. Sugars and amino acids at the concentrations found in spinach chloroplasts did not prevent inhibition of enzymes by anions. The results indicate that increased anion concentrations in cells and organelles are responsible for primary, quickly reversible effects of moderate dehydration on plant tissues.Abbreviations ASM artificial stroma medium - RuBP ribulose 1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate-carboxylase/oxygenase - RWC relative water content     

Role of intracellular carbonic anhydrase in inorganic-carbon assimilation by Porphyridium purpureum

Air-grown cells of Porphyridium purpurem contain appreciable carbonic-anhydrase activity, comparable to that in air-grown Chlamydomonas reinhardtii, but activity is repressed in CO₂-grown cells. Assay of carbonic-anhydrase activity in intact cells and cell extracts shows all activity to be intracellular in Porphyridium. Measurement of inorganic-carbon-dependent photosynthetic O₂ evolution shows that sodium ions increase the affinity of Porphyridium cells for HCO ₃ ^- . Acetazolamide and ethoxyzolamide were potent inhibitors of carbonic anhydrase in cell extracts but at pH 5.0 both acetazolamide and ethoxyzolamide had little effect upon the concentration of inorganic carbon required for the half-maximal rate of photosynthetic O₂ evolution (K_0.5[CO₂]). At pH 8.0, where HCO ₃ ^- is the predominant species of inorganic carbon, the K_0.5 (CO₂) was increased from 50 M to 950 M in the presence of ethoxyzolamide. It is concluded that in air-grown cells of Porphyridium. HCO ₃ ^- is transported across the plasmalemma and intracellular carbonic anhydrase increases the steady-state flux of CO₂ from inside the plasmalemma to ribulose-1,5-bisphosphate carboxylase-oxygenase by catalysing the interconversion of HCO ₃ ^- and CO₂ within the cell.Abbreviations AZ acetazolamide - EZ ethoxyzolamide - K_0.5[CO₂] half-maximal rate of photosynthetic O₂ evolution     

Purification and properties of ribulose 1,5-bisphosphate carboxylase from sunflower leaves

Extracts from sunflower leaves possess a high ribulose-1,5-bisphosphate (RuBP) carboxylase capacity but this enzyme activity is not stable. A purification procedure, developed with preservation of carboxylase activity by MgSO₄, yielded purified RuBP carboxylase with high specific activity (40 nkat mg^-1 protein). Measurement of kinetic parameters showed high Km values (RuBP, HCO ₃ ^- ) and high Vmax of the reaction catalyzed by this sunflower enzyme; the results are compared with those obtained for soybean carboxylase. Enzyme characteristics are discussed in relation to stabilization and activation procedures and to the high photosynthesis rates of this C₃ species.     

Metabolism of glucose by unicellular blue-green algae

Summary A facultative photo- and chemoheterotroph, the unicellular bluegreen alga Aphanocapsa 6714, dissimilates glucose with formation of CO₂ as the only major product. A substantial fraction of the glucose consumed is assimilated and stored as polyglucose (probably glycogen). The oxidation of glucose proceeds through the pentose phosphate pathway. The first enzyme of this pathway, glucose-6-phosphate dehydrogenase, is partly inducible. In addition, the rate of glucose oxidation is controlled, at the level of glucose-6-phosphate dehydrogenase function, by the intracellular level of an intermediate of the Calvin cycle, ribulose-1,5-diphosphate, which is a specific allosteric inhibitor of this enzyme. As a consequence, the rate of glucose oxidation is greatly reduced by illumination, an effect reversed by the presence of DCMU, an inhibitor of photosystem II.Two obligate photoautotrophs, Synechococcus 6301 and Aphanocapsa 6308, produce CO₂ from glucose at extremely low rates, although their levels of pentose pathway enzymes and of hexokinase are similar to those in Aphanocapsa 6714. Failure to grow with glucose appears to reflect the absence of an effective glucose permease. A general hypothesis concerning the primary pathways of carbon metabolism in blue-green algae is presented.Abbreviations A (U)DPG ADP-glucose or UDP-glucose - G-1-P glucose-1-phosphate - G-6-P glucose-6-phosphate - G_(int.) intracellular glucose - F-6-P fructose-6-phosphate - 6-PG 6-phosphogluconate - Ru-5-P ribulose-5-phosphate - RUDP ribulose-1,5-diphosphate - PGA 3-phosphoglycerate - GAP glyceraldehyde-3-phosphate     

Some properties of coupled hepatoma mitochondria exhibiting uncoupler-insensitive ATPase activity

Ribulose-1,5-diphosphate car?ylase from the photosynthetic bacterium Chromatium catalyses the oxidative formation of phosphoglycolate and 3-phosphoglycerate from ribulose-1,5-diphosphate at an alkaline pH (9.3) in an atmosphere of oxygen. The catalytically active oligomeric form of the large subunit of the car?ylase molecule, A_m, was proved to be functionally active in the ribulose-1,5-diphosphate oxygenase reaction without the presence of the smaller subunit.     

Comparison of Properties of the Proteolytic Degradation of Unassembled Nuclear-encoded Subunits of Ribulose-1,5-bisphosphate Carboxylase and of the Coupling Factor of Photophosphorylation CF1*

The proteolytic degradation of unassembled small subunit polypeptides of ribulose-1,5-bisphosphate carboxylase and of the δ-subunit of the coupling factor of photophosphorylation CF₁ were analyzed and compared in vitro in the presence of stroma or membrane preparations from ribosome-deficient plastids isolated from 32°C-grown rye leaves (Secale cereale L.). Extracts obtained from 70S ribosome-deficient rye leaves after radioactive labeling were used as substrate source for the unassembled polypeptides. Soluble stroma as well as membrane preparations from isolated plastids contained proteolytic activities catalyzing the degradation of both the small subunits of ribulose-1,5-bisphosphate carboxylase and CF₁-δ in vitro. Maximal in vitro degradation was observed at pH 2–3 for the unassembled small subunits, but at pH 6–7 for the purified holoprotein of ribulose-1,5-bisphosphate carboxylase, and at pH 6.0 for unassembled CF₁-δ. Degradation of unassembled small subunits of ribulose-1,5-bisphosphate carboxylase at pH 3.0 was stimulated by Cu²⁺ but not by Ca²⁺, Mg²⁺ or ATP. At pH 3.0 the degradation of unassembled small subunits of ribulose-1,5-bisphosphate carboxylase was not inhibited by various protease inhibitors but was even stimulated. At pH 7.0 its degradation was inhibited by HgCl₂ and diazoacetyl nor-leucine methyl ester + Cu-acetate. The degradation of CF₁-δ was markedly inhibited by phenylmethylsulphonyl fluoride (PMSF) and to a lesser extent by 1,10-phenanthroline. According to present results different proteolytic systems appear to be involved in the degradation of unassembled small subunits of ribulose-1,5-bisphosphate carboxylase and of unassembled CF₁-δ.     

Bicarbonate uptake in the marine macroalga Ulva sp. is inhibited by classical probes of anion exchange by red blood cells

We demonstrate in this work that HCO _inf3 ^sup– uptake in the marine macroalga Ulva sp. features functional resemblances to anion transport mediated by anion exchangers of mammalian cell membranes. The evidence is based on (i) competitive inhibition of photosynthesis by the classical red-blood-cell anion-exchange blockers 4,4-dinitrostilbene-2,2-disulfonate and 4-nitro-4-isothiocyanostilbene-2,2-disulfonate under conditions where HCO _inf3 ^sup– , but not CO₂, was the inorganic carbon form taken up; (ii) inhibition of HCO _inf3 ^– uptake by pyridoxal phospate, indicating the involvement of lysine residues in the binding/translocation of HCO _inf3 ^sup– ; and (iii) inhibition of HCO _inf3 ^sup– (but not of CO₂) uptake by exofacial trypsin treatments, indicating the functional involvement of a plasmalemma protein. It is suggested that HCO _inf3 ^sup– uptake mediated by such a putative anion transporter can be a fundamental step in providing inorganic carbon for the CO₂-concentrating system of marine marcoalgae in an environment where the HCO _inf3 ^sup– concentration is high, but the CO₂ concentration and rates of uncatalyzed HCO _inf3 ^sup– dehydration are low.Abbreviations CI ionorganic carbon - DIDS 4,4-diisothiocyanostilbene-2,2-disulfonate - DNDS 4,4-dinitrostilbene-2,2-disulfonate - NIDS 4-nitro-4-isothiocyanostilbene-2,2-disulfonate - PLP pyridoxal phosphate - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase     

Salt-induced NO<Subscript>3</Subscript><Superscript>-</Superscript> uptake inhibition in cowpea roots is dependent on the ionic composition of the salt and its osmotic effect

Salinity remarkably inhibits NO₃ ^- uptake but the mechanisms are not well understood. This study was addressed to elucidate the role of ionic and osmotic components of salinity on NO₃ ^- influx and efflux employing classic kinetics involving a low affinity transport system (LATS) and a high affinity transport system (HATS). In the presence of KCl, NaCl, and Na₂SO₄ at 100 mM concentrations, in both LATS and HATS, Michaelis constant (K_m) was similar for the three salts and maximum rate (V_max) decreased as follows: KCl > NaCl > Na₂SO₄, compared to control indicating a non-competitive interaction with NO₃ ^-. Unexpectedly, iso-osmotic solutions (osmotic potential Ψ_π = -0.450) of polyethylene glycol (PEG, 17.84 %, v/v) and mannitol (100 mM) remarkably increased K_m in both the LATS and the HATS, but V_max did not change indicating a competitive inhibition. Under the PEG and mannitol treatments, K_m and V_max were higher than under the salt treatments. The salts increased slightly NO₃ ^- efflux in the following order KCl > NaCl > Na₂SO₄. In contrast, mannitol strongly stimulated and the PEG inhibited NO₃ ^- efflux. The obtained data reveal that salinity effects were not dependent on the anion type (Cl^- versus SO₄ ^2-) indicating a non-competitive inhibition mechanism between Cl^- and NO₃ ^-. In contrast, the cation types (K⁺ versus Na⁺) had a pronounced effect. The osmotic component is important to net NO₃ ^- uptake affecting remarkably the influx in both LATS and HATS components of cowpea roots.