Regulation of photosynthetic carbon metabolism during phosphate limitation of photosynthesis in isolated spinach chloroplasts

Intact chloroplasts isolated from spinach were illuminated in the absence of inorganic phosphate (Pi) or with optimum concentrations of Pi added to the reaction medium. In the absence of Pi photosynthesis declined after the first 1–2 min and was less than 10% of the maximum rate after 5 min. Export from the chloroplast was inhibited, with up to 60% of the ¹⁴C fixed being retained in the chloroplast, compared to less than 20% in the presence of Pi. Despite the decreased export, chloroplasts depleted of Pi had lower levels of triose phosphate while the percentage of total phosphate in 3-phosphoglycerate was increased. Chloroplast ATP declined during Pi depletion and reached dark levels after 3–4 min in the light without added Pi. At this point, stromal Pi concentration was 0.2 mM, which would be limiting to ATP synthesis. Addition of Pi resulted in a rapid burst of oxygen evolution which was not initially accompanied by net CO₂ fixation. There was a large decrease in 3-phosphoglycerate and hexose plus pentose monophosphates in the chloroplast stroma and a lesser decrease in fructose-1,6-bisphosphate. Stromal levels of triose phosphate, ribulose-1,5-bisphosphate and ATP increased after resupply of Pi. There was an increased export of ¹⁴-labelled compounds into the medium, mostly as triose phosphate. Light activation of both fructose-1,6-bisphosphatase and ribulose-1,5-bisphosphate carboxylase was decreased in the absence of Pi but increased following Pi addition.It is concluded that limitation of Pi supply to isolated chloroplasts reduced stromal Pi to the point where it limits ATP synthesis. The resulting decrease in ATP inhibits reduction of 3-phosphoglycerate to triose phosphate via mass action effects on 3-phosphoglycerate kinase. The lack of Pi in the medium also inhibits export of triose phosphate from the chloroplast via the phosphate transporter. Other sites of inhibition of photosynthesis during Pi limitation may be located in the regeneratige phase of the reductive pentose phosphate pathway.Abbreviations FBP Fructose-1,6-bisphosphate - FBPase Fructose-1,6-bisphosphatase - MP Hexose plus pentose monophosphates - PGA 3-phosphoglycerate - Pi inorganic orthophosphate - RuBP ribulose-1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase - TP Triose Phosphate     

Role of ferredoxin in the activation of sedoheptulose diphosphatase in isolated chloroplasts

Sedoheptulose 1,7-diphosphatase activity of isolated spinach chloroplasts shows a requirement for (i) reduced ferredoxin and (ii) a protein factor. Activation by ferredoxin, reduced photochemically by chloroplast fragments, was optimal at pH 7.8 and at a Mg²⁺ concentration of 5 mM. The protein factor needed for activation appears to be the same as that required by the chloroplast fructose-1,6-diphosphatase that is activated by reduced ferredoxin. The results indicate that sedoheptulose-1,7-diphosphatase, like fructose-1,6-diphosphatase, is a regulatory enzyme whose activity in chloroplasts is controlled via ferredoxin by light.     

High level expression in Escherichia coli,purification and properties of chloroplast fructose-1,6-bisphosphatase from rapeseed (Brassica napus) leaves

In chloroplasts, the light-modulated fructose-1,6-bisphosphatase catalyzes the formation of fructose 6-bisphosphate for the photosynthetic assimilation of CO₂ and the biosynthesis of starch. We report here the construction of a plasmid for the production of chloroplast fructose-1,6-bisphosphatase in a bacterial system and the subsequent purification to homogeneity of the genetically engineered enzyme. To this end, a DNA sequence that coded for chloroplast fructose-1,6-bisphosphatase of rapeseed (Brassica napus) leaves was successively amplified by PCR, ligated into the Ndel/EcoRI restriction site of the expression vector pET22b, and introduced into Escherichia coli cells. When gene expression was induced by isopropyl--d-thiogalactopyranoside, supernatants of cell lysates were extremely active in the hydrolysis of fructose 1,6-bisphosphate. Partitioning bacterial soluble proteins by ammonium sulfate followed by anion exchange chromatography yielded 10 mg of homogeneous enzyme per 1 of culture. Congruent with a preparation devoid of contaminating proteins, the Edman degradation evinced an unique N-terminal amino acid sequence [A-V-A-A-D-A-T-A-E-T-K-P-]. Gel filtration experiments and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the (recombinant) rapeseed chloroplast fructose-1,6-bisphosphatases was a tetramer [160 kDa] comprised of four identical subunits. Like other chloroplast fructose-1,6-bisphosphatases, the recombinant enzyme was inactive at 1 mM fructose 1,6-bisphosphate and 1 mM Mg²⁺ but became fully active after an incubation in the presence of either 10 mM dithiothreitol or 1 mM dithiothreitol and chloroplast thioredoxin. However, at variance with counterparts isolated from higher plant leaves, the low activity observed in absence of reductants was not greatly enhanced by high concentrations of fructose 1,6-bisphosphate (3 mM) and Mg²⁺ (10 mM). In the catalytic process, all chloroplast fructose-1,6-bisphosphatases had identical features; viz., the requirement of Mg²⁺ as cofactor and the inhibition by Ca²⁺. Thus, the procedure described here should prove useful for the structural and kinetic analysis of rapeseed chloroplast fructose-1,6-bisphosphatase in view that this enzyme was not isolated from leaves.Abbreviation DTT dithiothreitol - PCR polymerase chain reaction - EDTA (ethylenedinitrilo)tetraacetic     

Light-dependent changes of the Mg2+ concentration in the stroma in relation to the Mg2+ dependency of CO2 fixation in intact chloroplasts.

(1) Light-dependent changes of the Mg2+ content of thylakoid membranes were measured at pH 8.0 and compared with earlier measurements at pH 6.6. In a NaCl and KCl medium, the light-dependent decrease in the Mg2+ content of the thylakoid membranes at pH 8.0 is found to be 23 nmol Mg2+ per mg chlorophyll, whereas in a sorbitol medium it is 83 nmol Mg2+ per mg chlorophyll. (2) A light dependent increase in the Mg2+ content of the stroma was detected wjem chloroplasts were subjected to osmotic shock, amounting to 26 nmol/mg chlorophyll. Furthermore, a rapid and reversible light-dependent efflux of Mg2+ has been observed in intact chloroplasts when the divalent cation ionophore A 23 187 was added, indicating a light-dependent transfer of about 60 nmol of Mg2+ per mg chlorophyll from the thylakoid membranes to the stroma. (3) CO2 fixation, but not phosphoglycerate reduction, could be completely inhibited when A 23 187 was added to intact chloroplasts in the absence of external Mg2+. If Mg2+ was then added to the medium, CO2 fixation was restored. Half of the maximal restoration was achieved with about 0.2 mM Mg2+, which is calculated to reflect a Mg2+ concentration in the stroma of 1.2 mM. The further addition of Ca2+ strongly inhibits CO2 fixation. (4) The results suggest that illumination of intact chloroplasts causes an increase in the Mg2+ concentration of 1-3 mM in the stroma. Compared to the total Mg2+ content of chloroplasts, this increase is very low, but it appears to be high enough to have a possible function in the light regulation of CO2 fixation.     

Two Distinct Aldolases of Class II Type in the Cyanoplasts and in the Cytosol of the Alga Cyanophora paradoxa

Two aldolases from the alga Cyanophora paradoxa (Glaucocystophyta) can be separated by chromatography on diethylaminoethyl-Fractogel. The two aldolases are inhibited by 1 mM ethylene-diaminetetraacetate (EDTA) and, therefore, are class II aldolases. When cells of C. paradoxa were fractionated, one aldolase was associated with the cytosol fraction and the other was associated with the cyanoplast fraction. The Km(fructose-1,6-bisphosphate) was 600 [mu]M for the cytosolic aldolase and 340 [mu]M for the cyanoplast aldolase. The activity of the cytosolic aldolase was increased up to 4-fold by 100 mM K+ and slightly inhibited by Li+ and Cs+, whereas the cyanoplast aldolase was not affected by these ions. Inactivation by 1 mM EDTA could be partly restored by the addition of Co2+ or Mn2+ and to a lesser extent by Zn2+ or Mg2+. The molecular masses of the native cytosolic and cyanoplast aldolases are about 90 and 85 kD, respectively, as estimated by velocity centrifugation in sucrose gradients. Implications for the evolution of class I and II aldolases in chloroplasts of higher plants and algae will be discussed.     

Antibody inhibition of external aldolase activity in spinach chloroplast preparations

Abstract Antibodies (rabbit) have been prepared against total stroma from isolated spinach (Spinacia oleracea L. cv. Viking II) chloroplasts. These antibodies inhibited most of the aldolase activity present outside the chloroplasts in preparations of intact (80–95%) chloroplasts. They also reduced the amount of labelled fructose-1,6-bisphosphate found in the medium after ¹⁴CO₂ fixation with such preparations. Both intact and broken chloroplasts were strongly agglutinated by the antibodies. The results indicate that the external fructose-1,6-bisphosphate was formed from excreted dihydroxyacetone phosphate by the action of aldolase and triose phosphate isomerase present outside the chloroplasts. The contamination of organelle preparations with free enzymes or enzymes adsorbed on the outer surface of the organelles is probably a general phenomenon. It is suggested that antibodies can be used as a tool to detect and selectively inhibit such contaminating enzyme activities.     

The effect of high hydrostatic pressure on the modulation of regulatory enzymes from spinach chloroplasts.

High hydrostatic pressure enhanced the specific activity of regulatory enzymes of the Benson-Calvin cycle (fructose-1,6-bisphosphatase, glyceraldehyde-3-P dehydrogenase, phosphoribulokinase) which are modulated by the ferredoxin-thioredoxin system. High activity of chloroplast fructose-1,6-bisphosphatase required dithiothreitol, fructose 1,6-bisphosphate, and Ca2+. At 100 bar the A0.5 for fructose 1,6-bisphosphate (0.3 mM) was lower than that at 1 bar (1.5 mM), whereas similar variations of pressure did not alter the A0.5 for Ca2+ (55 microM). The response of chloroplast glyceraldehyde-3-P dehydrogenase exposed to 500 bar was a 4-fold increase in the NADP-linked activity; conversely, the NAD-dependent activity remained unchanged. The concerted action of high pressure and Pi (or ATP), both activators of chloroplast glyceraldehyde-3-P dehydrogenase, led to inactivation. On the other hand, the activity of phosphoribulokinase increased 10-fold when the enzyme was incubated at 1500 bar; the activation process was strictly dependent on the presence of dithiothreitol. At variance with these enzymes, bovine liver fructose-1,6-bisphosphatase, yeast glyceraldehyde-3-P dehydrogenase, and chloroplast ribulose 1,5-bisphosphate carboxylase, whose activities are not modulated by reduced thioredoxin, were inactivated by high pressure. The comparison of oligomeric enzymes revealed that the stimulation of specific activity by high pressure correlated with thioredoxin-mediated activation, and it did not depend on a particular subunit composition. Present results show that high pressure resembled thioredoxin, cosolvents, and chaotropic anions in its action on regulatory enzymes of the Benson-Calvin cycle. The comparison of physiological and non-physiological modulators suggested that thioredoxin-mediated modifications of noncovalent interactions is an important event in light-dependent regulation of chloroplast enzymes.     

Purification and Properties of an NADPH-Aldose Reductase (Aldehyde Reductase) from Euonymus japonica Leaves

Intact chloroplasts isolated from Euglena gracilis exhibit high rates of light-driven protein synthesis, whereas protein synthesis by isolated proplastids is absolutely dependent upon the addition of an exogenous energy source in the form of equimolar ATP and Mg²⁺. ATP and Mg²⁺ also stimulate translation by chloroplasts. The greatly increased rates of protein synthesis obtained by supplementing proplastids with ATP and Mg²⁺ have allowed the first clear characterization of proplastid translation products. Two-dimensional polyacrylamide gel electrophoretic analysis of proteins synthesized in organello shows that, while many translation products are common to both plastid types, most are unique to either the proplastid or the chloroplast. Pulse-chase experiments using both proplastids and chloroplasts indicate similar rates of turnover of newly synthesized proteins in both types of plastids. Thus, the differences seen between proplastid and chloroplast translation products are apparently not due to turnover. Immunoprecipitation of large subunit of ribulose-1,5-bisphosphate carboxylase (LS) from pulse-chase experiments indicates that LS is made in both proplastids and in chloroplasts and that the rate of LS turnover is similar in both types of plastids.     

Studies on the hysteretic properties of chloroplast fructose-1,6-bisphosphatase

Chloroplast fructose-1,6-bisphosphatase hysteresis in response to modifiers was uncovered by carrying out the enzyme assays in two consecutive steps. The activity of chloroplast fructose-1,6-bisphosphatase, assayed at low concentrations of both fructose-1,6-bisphosphatase and Mg2+, was enhanced by preincubating the enzyme with dithiothreitol, thioredoxin f, fructose 1,6-bisphosphate, and Ca2+. In the time-dependent activation process, fructose 1,6-bisphosphate and Ca2+ could be replaced by other sugar biphosphates and Mn2+, respectively. Once activated, chloroplast fructose-1,6-bisphosphatase hydrolyzed fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate in the presence of Mg2+, Mn2+, or Fe2+. The A0.5 for fructose 1,6-bisphosphate (activator) was lowered by reduced thioredoxin f and remained unchanged when Mg2+ was varied during the assay of activity. On the contrary, the S0.5 for fructose 1,6-bisphosphate (substrate) was unaffected by reduced thioredoxin f and depended on the concentration of Mg2+. Ca2+ played a dual role on the activity of chloroplast fructose-1,6-bisphosphatase; it was a component of the concerted activation and an inhibitor in the catalytic step. Provided dithiothreitol was present, the activating effectors were not required to maintain the enzyme in the active form. Considered together these results strongly suggest that the regulation of fructose-1,6-bisphosphatase in chloroplast occurs at two different levels, the activation of the enzyme and the catalysis.     

Regulation of the activation of chloroplast fructose-1,6-bisphosphatase: inhibition by spermidine and spermine

The activation of chloroplast fructose-1,6-bisphosphatase by fructose-1,6-bisphosphate, Ca2+, DTT and chloroplast thioredoxin-f is prevented by either spermidine or spermine; on the contrary, other amino compounds do not replace polyamines in this reversible effect. On the other hand, neither spermidine nor spermine modify the catalysis of chloroplast fructose-1,6-bisphosphatase. The effect of spermidine, but not the effect of spermine, is reversed by increasing the concentration of Ca2+ in the activation; higher concentrations of Fructose-1,6-bisphosphate or thioredoxin-f do not restore the control activity. The present results suggest that other regulatory mechanisms may control the activation of fructose-1,6-bisphosphatase in chloroplasts.     

Catalytic properties of phosphoglucomutase from pea chloroplasts.

Electrophoretically homogeneous phosphoglucomutase (PGM) with specific activity of 3.6 units/mg protein was isolated from pea (Pisum sativum L.) chloroplasts. The molecular mass of this PGM determined by gel-filtration is 125 +/- 4 kD. According to SDS-PAGE, the molecular mass of subunits is 65 +/- 3 kD. The Km for glucose-1-phosphate is 18.0 +/- 0.5 microM, and for glucose-1, 6-diphosphate it is 33 +/- 0.7 microM. At glucose-1-phosphate and glucose-1,6-diphosphate concentrations above 0.5 and 0.2 mM, respectively, substrate inhibition is observed. The enzyme has optimum activity at pH 7.9 and 35 degrees C. Mg2+ activates the PGM. Mn2+ activates the enzyme at concentrations below 0.2 mM, while higher concentrations have an inhibitory effect. The activity of the PGM is affected by 6-phosphogluconate, fructose-6-phosphate, NAD+, ATP, ADP, citrate, and isocitrate.     

Study of properties of NADP malate dehydrogenase from corn leaves]

Kinetic properties of purified chloroplast isoenzyme of the "malic" enzyme from corn leaves were studied. The enzyme had optimum activity at pH 8.0 and 36 degrees C. Under standart conditions the Michaelis constants for the "malic" enzyme with Mn2+ as cofactor are 0.091 mM for malate and 0.04 mM for NADP. In case of Mg2+ as cofactor they are 0.66 and 0.02 mM respectively. Respective Km values for the cofactors Mn2+ and Mg2+ are 0.018 and 0.091 mM. The activity of the "malic" enzyme was inhibited by reduced NADP and NAD, ATP, ADP, fructose-1,6-diphosphate, oxaloacetic, oxalic, glyoxylic, glycolic and alpha-ketoglutaric acids, as well as by phosphate anions and pyrophosphate. The inhibitory effect of all metabolites and ions is more pronounced in case of Mn, rather than Mg, used as cofactors for the reaction. A possibility of metabolic regulation of NADP-"malic" enzyme activity in the leaves of C4-plants, is discussed.     

Regulation of sedoheptulose-1,7-bisphosphatase by sedoheptulose-7-phosphate and glycerate,and of fructose-1,6-bisphosphatase by glycerate in spinach chloroplasts

Using partially purified sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea L.) chloroplasts the effects of metabolites on the dithiothreitoland Mg²⁺-activated enzyme were investigated. A screening of most of the intermediates of the Calvin cycle and the photorespiratory pathway showed that physiological concentrations of sedoheptulose-7-phosphate and glycerate specifically inhibited the enzyme by decreasing its maximal velocity. An inhibition by ribulose-1,5-bisphosphate was also found. The inhibitory effect of sedoheptulose-7-phosphate on the enzyme is discussed in terms of allowing a control of sedoheptulose-1,7-bisphosphate hydrolysis by the demand of the product of this reaction. Subsequent studies with partially purified fructose-1,6-bisphosphatase from spinach chloroplasts showed that glycerate also inhibited this enzyme. With isolated chloroplasts, glycerate was found to inhibit CO₂ fixation by blocking the stromal fructose-1,6-bisphosphatase. It is therefore possible that the inhibition of the two phosphatases by glycerate is an important regulatory factor for adjusting the activity of the Calvin cycle to the ATP supply by the light reaction.Abbreviations DTT dithiothreitol - FBPase fructose-1,6-bisphosphatase - Fru-1,6-P₂ fructose-1,6-bisphosphate - Fru-6-P fructose-6-phosphate - 3-PGA 3-phosphoglycerate - Ru-1,5-P₂ ribulose-1,5-bisphosphate - Ru-5-P ribulose-5-phosphate - SBPase sedoheptulose-1,7-bisphosphatase - Sed-1,7-P₂ sedoheptulose-1,7-bisphosphate - Sed-7-P sedoheptulose-7-phosphate This work was supported by the Deutsche Forschungsgemein-schaft.     

Regulation of ribulose-1,5-bisphosphate carboxylase activity by the activase system in lysed spinach chloroplasts

Ribulose-1,5-bisphosphate (RuBP) carboxylase in lysed spinach (Spinacia oleracea L. cv virtuosa) chloroplasts that had been partly inactivated at low CO₂ and Mg²⁺ by incubating in darkness with 4 millimolar partially purified RuBP was reactivated by light. If purified RuBP was used to inhibit dark activation of the enzyme, reactivation by light was not observed unless fructose-1,6-bisphosphate, ATP, or ADP plus inorganic phosphate were also added. Presumably, ADP plus inorganic phosphate acted as an ATP-generating system with a requirement for the generation of ΔpH across the thylakoid membrane. When the RuBP obtained from Sigma Chemical Co. was used, light did not reactivate the enzyme. There was no direct correlation between ΔpH and activation. Therefore, thylakoids are required in the ribulose-1,5-bisphosphate carboxylase activase system largely to synthesize ATP. Inactivation of RuBP carboxylase in isolated chloroplasts or in the lysed chloroplast system was not promoted simply by a transition from light to dark conditions but was caused by low CO₂ and Mg²⁺.     

Elimination of the lag period in chloroplast development in a chlorophyll mutant of peanuts

The mutation of a nuclear gene in peanut (Arachis hypogaea L.) plants results in a reduced light-dependent development of chloroplast fine structure, soluble protein, ribulose-1, 5-diP carboxylase, NADP-glyceraldehyde-3-P dehydrogenase, fructose-1, 6-diP aldolase, glycerate-3-P kinase, phosphoenolpyruvate carboxylase, malate dehydrogenase, and dark respiration during the 72-hour lag period of chlorophyll synthesis in dark-grown leaves exposed to continuous light. The mutation has pleiotropic affects. Kinetic analysis shows there is also a 72-hour lag period in the light-dependent development of NADP-glyceraldehyde-3-P dehydrogenase and fructose-1, 6-diP aldolase in the mutant leaves, whereas there is no lag in the development of NAD-malate dehydrogenase and dark respiration. There is minimal development of the chloroplast during the 72-hour mutationally induced lag period, but there is pronounced cytoplasmic and mitochondrial activity during this phase. There is a 24-hour lag period in the light-dependent enlargement of the mutant leaves. At the completion of leaf enlargement, chloroplast differentiation is initiated. The mutation does not result in any chloroplast deletions, it only affects the timing of the synthesis of these components.     

Stromal free calcium concentration and light-mediated activation of chloroplast fructose-1,6-bisphosphatase

Light-mediated activation of fructose-1,6-bisphosphatase (EC 3.1.3.11) in intact spinach chloroplasts (Spinacia oleracea L.) is enhanced in the presence of 10⁻⁵ molar external free Ca²⁺. The most pronounced effect is observed during the first minutes of illumination. Ruthenium red, an inhibitor of light-induced Ca²⁺ influx, inhibits this Ca²⁺ stimulated activation. In isolated stromal preparations, the activation of fructose-1,6-bisphosphatase is already enhanced by 2 minutes of exposure to elevated Ca²⁺ concentrations in the presence of physiological concentrations of Mg²⁺ and fructose-1,6-bisphosphate. Maximal activation of the enzyme is achieved between 0.34 and 0.51 millimolar Ca²⁺. The Ca²⁺ mediated activation decreases with increasing fructose-1,6-bisphosphate concentration and with increasing pH. The data are consistent with the proposal that the illumination of chloroplasts leads to a transient increase of free stromal Ca²⁺. In dark-kept chloroplasts the steady-state concentration of free stromal Ca²⁺ is 2.4 to 6.3 micromolar as determined by null point titration. These observations support our previous proposal that light-induced Ca²⁺ influx into chloroplasts does not only influence the cytosolic concentration of free Ca²⁺ but also regulates enzymatic processes inside the chloroplast.     

Concerted action of cosolvents, chaotropic anions and thioredoxin on chloroplast fructose-1,6-bisphosphatase. Reactivity to iodoacetamide

The incubation of chloroplast fructose-1,6-bisphosphatase with both dithiothreitol and protein denaturants made sulfhydryl groups available for reaction with [1-14C]iodoacetamide (10-12 mol iodoacetamide incorporated/mol enzyme). Digestion of S-carboxyamidomethylated enzyme with trypsin and polyacrylamide gel electrophoresis, in the presence of sodium dodecylsulfate, yielded two 14C-labeled fragments whose apparent molecular mass were 10 kDa and 16 kDa. In the absence of either dithiothreitol or protein denaturants the incorporation of iodoacetamide to the enzyme was lower than 4 mol. When chloroplast fructose-1,6-bisphosphatase was initially incubated with dithiothreitol (2.5 mM) and (a) high concentrations of both fructose 1,6-bisphosphate (4 mM) and Ca2+ (0.3 mM) or (b) low concentrations of both fructose 1,6-bisphosphate (0.8 mM) and Ca2+ (0.05 mM) in the presence of either 2-propanol (15%, by vol.), trichloroacetate (0.15 M) or chloroplast thioredoxin-f (0.5 microM) and subsequently subjected to proteolysis and electrophoresis, S-carboxyamidomethylated tryptic fragments had similar molecular masses. Thus, conditions that stimulated the specific activity of chloroplast fructose-1,6-bisphosphatase caused conformational changes which favoured both the reduction of disulfide bridges and the exposure of sulfhydryl groups. In this aspect, thioredoxin exerted structural and kinetic effects similar to compounds not involved in redox reactions (organic solvents, chaotropic anions). These results indicated that the modification of hydrophobic (intramolecular) interactions in chloroplast fructose-1,6-bisphosphatase constituted the underlying mechanism in light-activation by the ferredoxin-thioredoxin system.     

Photosynthetic Characteristics of Chloroplasts Isolated from Euglena gracilis Z Grown Photoautotrophically

Photosynthetically competent chloroplasts were isolated fromcells of Euglena gracilis Z grown photoautotrophically in 1.5%CO₂. The isolated chloroplasts were intact and substantiallyfree from cytosolic, mitochondrial and microbody materials.The effects of some compounds on the activity of photosynthetic¹⁴CO₂ fixation were examined. The optimal pH and sorbitol concentrationwere 8.0 and 0.33 M, respectively. The chloroplasts requireda high level of P, (5 to 20 mM) for the maximal rate of photosynthesis.They were insusceptible to 10 mM of free Mg²⁺. ATP, ADP andAMP at 1 to 5 mM notably stimulated photosynthesis, althoughhigh concentrations of AMP were unfavorable. In the assay mediumdeveloped for this study, the chloroplasts exhibited photosyntheticactivity of 120µmoles-mg^–1 Chl-h^–1 at 30?C. Chloroplasts could also be isolated from cells grown under ordinaryair. The rate of photosynthetic ¹⁴CO₂ fixation at 1 mM NaH^l4CO₃was higher in these chloroplasts than in those isolated fromcells grown in 1.5% CO₂, whereas at 10 mM NaH^l4CO₃, the ratesof the two types of chloroplasts were nearly the same. Theseresults suggest that the CO₂ concentration given during growthof the algal cells affects the affinity for dissolved inorganiccarbon at the chloroplast level. (Received March 30, 1987; Accepted August 17, 1987)     

Activation of CO2 fixation in isolated spinach chloroplasts

1. 1. The effect of the Mg²⁺ concentration on the CO₂ fixation activity in situ in isolated and intact spinach chloroplasts upon suspension in hypotonic medium was examined. CO₂ fixation in the dark was activated 25–100 fold by 20 mM Mg²⁺ in the presence of added ATP plus either ribulose 5-phosphate or ribose 5-phosphate. 20 mM Mg²⁺-stimulated fixation only 2–3 fold in the presence of the substrate of fixation, ribulose 1,5-diphosphate. The highest Mg²⁺-stimulated rate of fixation in the dark observed with chloroplasts was 480 μmoles CO₂ fixed per mg chlorophyll per h.

2. 2. The concentration of bicarbonate at half of the maximal velocity (apparent K_m) during the Mg²⁺-stimulated fixation of CO₂ was 0.4 mM in the presence of ATP plus ribose 5-phosphate and 0.6 mM with ribulose 1,5-diphosphate.

3. 3. Dithioerythritol or light enhanced Mg²⁺-stimulated CO₂ fixation 1–3 fold in the presence of ATP plus ribose 5-phosphate but not ribulose 1,5-diphosphate.

4. 4. These results indicate that Mg²⁺ fluxes in the stroma of the chloroplast could control the activity of the phosphoribulokinase with a lesser effect on the ribulosediphosphate carboxylase. An increase in Mg²⁺ of 6–10 mM in the stroma region of the chloroplast would be enough to activate CO₂ fixation during photosynthesis.

Higher-plant chloroplast and cytosolic fructose-1,6-bisphophosphatase isoenzymes: origins via duplication rather than prokaryote-eukaryote divergence

Full-size cDNAs encoding the precursors of chloroplast fructose-1,6-bisphosphatase (FBP), sedoheptulose-1,7-bisphosphatase (SBP), and the small subunit of Rubisco (RbcS) from spinach were cloned. These cDNAs complete the set of homologous probes for all nuclear-encoded enzymes of the Calvin cycle from spinach (Spinacia oleracea L.). FBP enzymes not only of higher plants but also of non-photosynthetic eukaryotes are found to be unexpectedly similar to eubacterial homologues, suggesting a eubacterial origin of these eukaryotic nuclear genes. Chloroplast and cytosolic FBP isoenzymes of higher plants arose through a gene duplication event which occurred early in eukaryotic evolution. Both FBP and SBP of higher plant chloroplasts have acquired substrate specificity, i.e. have undergone functional specialization since their divergence from bifunctional FBP/SBP enzymes of free-living eubacteria.Abbreviations FBP fructose-1,6-bisphosphatase - SBP sedoheptulose-1,7-bisphosphatase - FBA fructose-1,6-bisphosphate aldolase