14CO2 fixation and glycolate metabolism in the dark in isolated maize (Zea mays L.) bundle sheath strands

Bundle sheath strands capable of assimilating up to 68 μmoles CO₂ per mg chlorophyll per hr in the dark have been isolated from fully expanded leaves of Zea mays L. This dark CO₂-fixing system is dependent on exogenous ribose-5-phosphate, ADP or ATP, and Mg²⁺ for maximum activity. The principal product of dark fixation in this system is 3-phosphoglycerate, indicating that the CO₂-fixing reaction is mediated by ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39). The rate of dark CO₂ uptake in the strands in the presence of saturating levels of ribose-5-phosphate plus ADP is inhibited by oxygen. The inhibitory effect of oxygen is rapidly and completely reversible, and is relieved by increased levels of CO₂. Glycolate is synthesized in this dark system in the presence of [U-¹⁴C]ribose-5-phosphate, ADP, oxygen, and an inhibitor of glycolate oxidase (EC 1.1.3.1). Glycolate formation is completely abolished by heating the strands, and the rate of glycolate synthesis is markedly reduced by either lowering the oxygen tension or increasing the level of CO₂.These results, obtained with intact cells in the absence of light, indicate that the direct inhibitory effect of oxygen on photosynthesis is associated with photosynthetic carbon metabolism, probably at the level of ribulose-1,5-bisphosphate carboxylase, and not with photophosphorylation or photosynthetic electron transport. Furthermore, the findings indicate that the synthesis of glycolate from exogenous substrate can readily occur in the absence of photosynthetic electron transport, an observation consistent with the ribulose-1, 5-bisphosphate “oxygenase” scheme for glycolate formation during photosynthesis.     

Light-Dark Regulation of Starch Metabolism in Chloroplasts: II. Effect of Chloroplastic Metabolite Levels on the Formation of ADP-Glucose by Chloroplast Extracts

The rate of ADP-glucose formation from [¹⁴C]glucose 6-phosphate and ATP by the soluble fraction of lysed chloroplasts is studied as a function of the levels of metabolites (3-phosphoglycerate, orthophosphate, hexose monophosphate, and ATP) as determined in whole chloroplasts of Spinacia oleracea in light and dark.     

CO(2) Assimilation and Malate Decarboxylation by Isolated Bundle Sheath Chloroplasts from Zea mays

Conditions for optimal CO₂ fixation and malate decarboxylation by isolated bundle sheath chloroplasts from Zea mays were examined. The relative rates of these processes varied according to the photosynthetic carbon reduction cycle intermediate provided. Highest rates of malate decarboxylation, measured as pyruvate formation, were seen in the presence of 3-phosphoglycerate, while carbon fixation was highest in the presence of dihydroxyacetone phosphate; only low rates were measured with added ribose-5-phosphate. Chloroplasts exhibited a distinct phosphate requirement and this was optimal at a level of 2 millimolar inorganic phosphate in the presence of 2.5 millimolar 3-phosphoglycerate, dihydroxyacetone phosphate, or ribose-5-phosphate. Malate decarboxylation and CO₂ fixation were stimulated by additions of AMP, ADP, or ATP with half-maximal stimulation occurring at external adenylate concentrations of about 0.15 millimolar. High concentrations (>1 millimolar) of AMP were inhibitory. Aspartate included in the incubation medium stimulated malate decarboxylation and CO₂ assimilation. In the presence of aspartate, the apparent Michaelis constant (malate) for malate decarboxylation to pyruvate by chloroplasts decreased from 6 to 0.67 millimolar while the calculated V_max for this process increased from 1.3 to 3.3 micromoles per milligram chlorophyll. Aspartate itself was not metabolized. It was concluded that the processes mediating the transport of phosphate, 3-phosphoglycerate, and dihydroxyacetone phosphate transport on the one hand, and also of malate might differ from those previously described for chloroplasts from C₃ plants.     

Role of mitochondria in hepatic fructose metabolism

During metabolism of fructose at concentrations exceeding 5 mM, isolated liver cells accumulate fructose 1-phosphate and lose ATP. At added bicarbonate concentrations below 10 mM in the incubation medium, the addition of atractyloside (or carboxyatractyloside) causes a significant net accumulation of 2-phosphoglycerate, resulting in an increase in the ratio 2-phosphoglycerate: 3-phosphoglycerate from below 1 to greater than 5. Digitonin fractionation revealed that virtually all this 2-phosphoglycerate is associated with the mitochondrial fraction, where it achieves a concentration estimated to be about 40 mM. The amount of 2-phosphoglycerate that accumulates is directly related to the initial concentration of fructose. With DL-glyceraldehyde in place of fructose, an even greater accumulation of 2-phosphoglycerate occurs, and this is also dependent upon both the presence of atractyloside and low bicarbonate. Formation of 2-phosphoglycerate is also observed when isolated mitochondria from rat liver are incubated together with glyceraldehyde and an energy source. The obligatory role of atractyloside for the accumulation of 2-phosphoglycerate within intact cells indicates the involvement of the mitochondrial adenylate translocator in this process, possibly as a carrier directly responsible for 2-phosphoglycerate egress from the mitochondrial matrix. If this is so, competition between 2-phosphoglycerate and ATP for egress from the matrix would be predicted to further exaggerate the fructose-induced depletion of cytosolic ATP.     

Regulation of NADP-Dependent Glyceraldehyde 3-Phosphate Dehydrogenase Activity in Spinach Chloroplasts*

Activation of NAD(P)-glyceraldehyde 3-phosphate dehydrogenase (NADP-GAPDH, EC 1.2.1.13) can be achieved in isolated chloroplasts in the light, or in the dark upon addition of dithiothreitol (DTT) and/or 3-phosphoglycerate plus ATP. Activation in darkened chloroplasts is only partial with DTT or 3-phosphoglycerate plus ATP alone, but complete when both effectors are added. In the light, full activation is only achieved upon addition of ATP. The time-course of activation appears to depend upon the actual concentration of 1,3-bisphosphoglycerate (1,3bisPGA) inside the chloroplasts. The K_a values for 1,3bisPGA are in the same range as has been determined for the purified enzyme, namely around 20 μM for the dark form (in the absence of DTT) and around 1 μM for the light form or in the presence of DTT. In contrast, the K_a value for ATP is 1 to 2 mM for both the oxidized and the reduced enzyme forms. The observed activation of NADP-GAPDH is strongly paralleled by an increase of 3PGA, and consequently of 1,3bisPGA in the illuminated chloroplast, while the ATP level remains constant or declines. Activation by 1,3bisPGA is accompanied by dissociation of the 600 kDa form to the 150 kDa form, while reduction alone does not induce a shift in molecular mass as documented by fast gel filtration on Superdex 200. Thus partial activation by DTT in the dark is due to an increased activity of the 600 kDa form, while the activation state in the light is the result of a partial conversion of the 600 kDa form into the more active 150 kDa form. The principle of this activation is a fast reduction of the enzyme by the ferredoxin/thioredoxin system, resulting in a lowered K_avalue for 1,3bisPGA, and thus adjusting the properties of the enzyme to the stromal 1,3bisPGA level. The occurrence of a 300 kDa oligomer mainly during inactivation has also been observed. From these results a model is constructed that describes the reversible interconversion of various activation and aggregation states of NADP-GAPDH as observed upon light/dark transitions in isolated spinach chloroplasts.     

Purification and characterization of pea chloroplastic phosphoriboisomerase

Pea (Pisum sativum L.) chloroplastic phosphoriboisomerase (EC 5.3.1.6) can be purified to apparent homogeneity in less than 2 days time with a 53% yield. Important steps in the purification include heat treatment and pseudoaffinity chromatography on Red H-3BN Sepharose. The purified isomerase has a subunit molecular mass of 26.4 kD. The N-terminal sequence has been determined through 34 residues. pH optima are 7.8 (ribose-5-phosphate) and 7.7 (ribulose-5-phosphate); K_m values are 0.9 millimolar (ribose-5-phosphate) and 0.6 millimolar (ribulose-5-phosphate). The enzyme is inhibited by erythrose-4-phosphate, sedoheptulosebisphosphate, glyceraldehyde-3-phosphate, and 3-phosphoglycerate at concentrations close to those found in photosynthesizing chloroplasts. Countercurrent phase partitioning experiments indicate that the pea chloroplastic phosphoriboisomerase interacts physically with phosphoribulokinase.     

Glucose requirement for postischemic recovery of perfused working heart

The quantitative importance of glycolysis in cardiomyocyte reenergization and contractile recovery was examined in postischemic, preload-controlled, isolated working guinea pig hearts. A 25-min global but low-flow ischemia with concurrent norepinephrine infusion to exhaust cellular glycogen stores was followed by a 15-min reperfusion. With 5 mM pyruvate as sole reperfusion substrate, severe contractile failure developed despite normal sarcolemmal pyruvate transport rate and high intracellular pyruvate concentrations near 2 mM. Reperfusion dysfunction was characterized by a low cytosolic phosphorylation potential [( ATP]/[( ADP][Pi]) due to accumulations of inorganic phosphate (Pi) and lactate. In contrast, with 5 mM glucose plus pyruvate as substrates, but not with glucose as sole substrate, reperfusion phosphorylation potential and function recovered to near normal. During the critical ischemia-reperfusion transition at 30 s reperfusion the cytosolic creatine kinase appeared displaced from equilibrium, regardless of the substrate supply. When under these conditions glucose and pyruvate were coinfused, glycolytic flux was near maximum, the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase reaction was enhanced, accumulation of Pi was attenuated, ATP content was slightly increased, and adenosine release was low. Thus, glucose prevented deterioration of the phosphorylation potential to levels incompatible with reperfusion recovery. Immediate energetic support due to maximum glycolytic ATP production and enhancement of the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase reaction appeared to act in concert to prevent detrimental collapse of [ATP]/[( ADP][Pi]) during creatine kinase dysfunction in the ischemia-reperfusion transition. Dichloroacetate (2 mM) plus glucose stimulated glycolysis but failed fully to reenergize the reperfused heart; conversely, 10 mM 2-deoxyglucose plus pyruvate inhibited glycolysis and produced virtually instantaneous de-energization during reperfusion. The following conclusions were reached. (1) A functional glycolysis is required to prevent energetic and contractile collapse of the low-flow ischemic or reperfused heart (2). Glucose stabilization of energetics in pyruvate-perfused hearts is due in part to intensification of glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase activity. (3) 2-Deoxyglucose depletes the glyceraldehyde-3-phosphate pool and effects intracellular phosphate fixation in the form of 2-deoxyglucose 6-phosphate, but the cytosolic phosphorylation potential is not increased and reperfusion failure occurs instantly. (4) Consistent correlations exist between cytosolic ATP phosphorylation potential and reperfusion contractile function. The findings depict glycolysis as a highly adaptive emergency mechanism which can prevent deleterious myocyte deenergization during forced ischemia-reperfusion transitions in presence of excess oxidative substrate.     

Adenine Nucleotide Levels, the Redox State of the NADP System, and Assimilatory Force in Nonaqueously Purified Mesophyll Chloroplasts from Maize Leaves under Different Light Intensities

Recently, a nonaqueous fractionation method of obtaining highly purified mesophyll chloroplasts from maize leaves was established. This method is now used to determine adenine nucleotide levels, the redox states of the NADP system, Pi levels and dihydroxyacetone phosphate/3-phosphoglycerate ratios in mesophyll chloroplasts of Zea mays L. leaves under different light intensities. The sum of the ATP, ADP, and AMP levels was estimated to be 1.4 millimolar and the ATP/ADP ratio was 1 in the dark and 2.5 to 4 in the light. The adenine nucleotides were equilibrated by adenylate kinase. The total concentration of NADP(H) in the chloroplasts was 0.3 millimolar in the dark and 0.48 millimolar in the light. The ratio of NADPH/NADP was 0.1 to 0.18 in the dark and 0.23 to 0.48 in the light. The Pi level was estimated to be 20 millimolar in the dark and 10 to 17 millimolar in the light. The 3-phosphoglycerate reducing system was under thermodynamic equilibrium in the light. The calculated assimilatory forces were 8 per molar and 40 to 170 per molar in the dark and the light, respectively. There was no relationship between the degree of activation of pyruvate, Pi dikinase, and adenylate energy charge, or ATP/ADP ratio or ADP level under various light intensities. Only a weak relationship was found between the degree of activation of NADP-malate dehydrogenase and the NADPH/NADP ratio or NADP(H) level with increasing light intensity. A possible regulatory mechanism which is responsible for the regulation of activation of pyruvate,Pi dikinase and NADP-malate dehydrogenase is discussed.     

Carbon metabolism of chloroplasts in the dark: Oxidative pentose phosphate cycle versus glycolytic pathway

The conversion of U-labelled [¹⁴C]glucose-6-phosphate into other products by a soluble fraction of lysed spinach chloroplasts has been studied. It was found that both an oxidative pentose phosphate cycle and a glycolytic reaction sequence occur in this fraction. The formation of bisphosphates and of triose phosphates was ATP-dependent and occurred mainly via a glycolytic reaction sequence including a phosphofructokinase step. The conversion, of glucose-6-phosphate via the oxidative pentose phosphate cycle stopped with the formation of pentose monophosphates. This was found not to be because of a lack in transaldolase (or transketolase) activity, but because of the high concentration ratios of hexose monophosphate/pentose monophosphate used in our experiments for simulating the conditions in whole chloroplasts in the dark. Some regulatory properties of both the oxidative pentose phosphate cycle and of the glycolytic pathway were studied.Abbreviations DHAP dihydroxyacetone phosphate - GAP 3-phosphoglyceraldehyde - PGA 3-phosphoglycerate - HMP hexose monophosphates - including F6P fructose-6-phosphate - G6P glucose-6-phosphate - GIP glucose-1-phosphate - 6-PGL phosphogluconate - PMP pentose monophosphates - including R5P ribose-5-phosphate - Ru5P ribulose-5-phosphate - X5P xylulose-5-phosphate - E4P erythrose-4-phosphate - S7P sedoheptulose-7-phosphate - FBP fructose-1,6-bisphosphate - SBP sedoheptulose-1,7-bisphosphate - RuBP ribulose-1,5-bisphosphate     

Post-mortem glycolysis in ox skeletal muscle. Effect of pre-rigor freezing and thawing on the intermediary metabolism

1. Ox sternomandibularis muscle was ;slow-frozen' by placing it in air at -22 degrees or ;fast-frozen' by immersion in liquid air or acetone-solid carbon dioxide. In all cases muscles were frozen pre-rigor. Changes in length, pH and the concentrations of P(i), creatine phosphate, hexose monophosphate (glucose 1-phosphate+glucose 6-phosphate+fructose 6-phosphate), fructose diphosphate (fructose 1,6-diphosphate+(1/2) triose phosphate), lactate, ATP, ADP, AMP and NAD(+) during freezing and during subsequent thawing were determined. In addition some measurements were made of the changes in alpha-glycerophosphate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate and pyruvate concentrations during slow freezing. 2. Appreciable shortening and marked changes in chemical composition took place during slow freezing but not during fast freezing. 3. During slow freezing the hexose monophosphate concentration fell and fructose 1,6-diphosphate and triose phosphate increased substantially. Increases also took place in 3-phosphoglycerate, 2-phosphoglycerate and phosphoenolpyruvate, but not in pyruvate. 4. On thawing, most of the chemical changes were similar to those in unfrozen muscle post mortem, but took place much more rapidly; loss of NAD(+) was particularly rapid. Fast-frozen muscle metabolized at a faster rate on thawing than did slow-frozen muscle. 5. The overall changes in length during freezing and thawing were about the same in slow-frozen as in fast-frozen muscle.     

Control of CO2 fixation. Regulation of spinach ribulose-5-phosphate kinase by stromal metabolite levels

The effect of stromal metabolites on the light-activated form of ribulose-5-phosphate kinase was studied with the enzyme rapidly extracted from illuminated spinach chlorplasts. In some instances, the effect of metabolites on the dark-inactivated enzyme extracted from darkened chloroplasts was also investigated. (1) The light-activated form of the enzyme is competitively inhibited with respect to ribulose 5-phosphate by 6-phosphogluconate, ribulose 1,5-bisphosphate, 3-phosphoglycerate and phosphate. Also, fructose 1,6-bisphosphate is inhibitory. All these compounds, except ribulose 1,5-bisphosphate, show an increasing inhibitory effect at lower pH values. Therefore, in the presence of these inhibitors, ribulose-5-phosphate kinase becomes strongly pH dependent. These compounds also exert an inhibitory effect on the dark-inactivated enzyme. (2) The assay of stromal levels of 6-phosphogluconate showed that this compound increased dramatically during a light-dark transient. (3) The dark-inactivated form of ribulose-5-phosphate kinase is strongly inhibited by ADP, the inhibition being competitive with respect to ATP. (4) A simulation of stromal metabolite levels in the enzyme activity assay indicates that in illuminated chloroplasts ribulose-5-phosphate kinase attains only about 4% of its maximal activity. When the fully light-activated enzyme is assayed under conditions occurring in the stroma in the dark, the activity is further decreased by a factor of 20. The same assay with the dark-inactivated enzyme yields an activity of virtually zero. (5) These results demonstrate that in the chloroplasts ribulose-5-phosphate kinase can not only be very efficiently switched off in the dark, but also be subjected to fine control during the illuminated state through the action of stromal metabolites.     

Relationships between protein phosphorylation and electron transport in the reconstituted chloroplast system.

Phosphorylation of the light-harvesting chlorophyll protein (LHCP) by the thylakoid protein kinase has been examined in the reconstituted chloroplast system. The level of phosphorylation by [32P]Pi was maximal at high light intensity and in the absence of 3-phosphoglycerate; dephosphorylation resulted from a subsequent decrease in light intensity or from the addition of 3-phosphoglycerate. Addition of ribose 5-phosphate, which acts as an ATP 'sink', also caused dephosphorylation. It is concluded that the degree of phosphorylation is dependent on the redox state and energy state of the system, thereby providing a mechanism for adapting light harvesting to the demands of carbon assimilation.     

Some effects of inorganic phosphate on O2 evolution by isolated chloroplasts

1. After an initial lag, isolated spinach chloroplasts evolved O₂ in illuminated reaction mixtures containing bicarbonate but no added phosphate. This evolution soon ceased but could be restarted by the addition of phosphate.

2. The phosphate requirement could be met by orthophosphate, inorganic pyrophosphate, ATP or ADP but not by AMP. Approx. 3 molecules of O₂ were evolved for each molecule of orthophosphate added and approx. 6 for each molecule of pyrophosphate.

3. With CO₂ as the sole added substrate the extent of the initial lag in O₂ evolution was not greatly affected by small quantities of added orthophosphate but as the concentration of orthophosphate was increased there was a progressive increase in the lag and a progressive decrease in the maximum rate. Pyrophosphate failed to produce these effects at a 100 times the concentration and in the presence of pyrophosphate the orthophosphate inhibition was less severe. There was little or no orthophosphate inhibition in the presence of substrate quantities of 3-phosphoglycerate or ribose 5-phosphate and CO₂.

4. There was also a requirement for phosphate by chloroplasts evolving O₂ in the presence of 3-phosphoglycerate or ribose 5-phosphate plus CO₂. In the presence of endogenous phosphate only, added ribose 5-phosphate suppressed the O₂ evolution which normally followed the addition of 3-phosphoglycerate.

5. The results provide direct support for the proposed phosphate requirement of the photosynthetic carbon cycle and are discussed in this context. They also imply that orthophosphate, ribose 5-phosphate and 3-phosphoglycerate can penetrate the intact chloroplast envelope with considerable rapidity.     

The role of the triose-phosphate shuttle and glycolytic intermediates in fatty-acid and glycerolipid biosynthesis in pea root plastids

The capacity of the triose-phosphate shuttle and various combinations of glycolytic intermediates to substitute for the ATP requirement for fatty-acid and glycerolipid biosynthesis in pea (Pisum sativum L.) root plastids was assessed. In all cases, ATP gave the greatest rates of fatty-acid and glycerolipid biosynthesis. Rates of up to 66 and 27 nmol·(mg protein)^–1·h^–1 were observed for the incorporation of acetate and glycerol-3-phosphate into lipids in the presence of ATP. In the absence of exogenously supplied ATP, the triose-phosphate shuttle gave up to 44 and 33% of the ATP-control activity in promoting fatty-acid and glycerolipid biosynthesis from acetate and glycerol-3-phosphate, respectively. The optimum shuttle components were 2 mM dihydroxyacetonephosphate (DHAP), 2 mM oxaloacetic acid and 4 mM inorganic phosphate (referred to as the DHAP shuttle). Glyceraldehyde-3-phosphate, as a shuttle triose, was approximately 82% as effective as DHAP in promoting fatty-acid synthesis while 2-phosphoglycerate, 3-phosphoglycerate, and phosphoenolpyruvate were only 27–37% as effective as DHAP. When glycolytic intermediates were used as energy sources for fatty-acid synthesis, in the absence of both exogenously supplied ATP and the triose-phosphate shuttle, phosphoenolpyruvate, 2-phosphoglycerate, fructose-6-phosphate and glucose-6-phosphate each gave 48%, 17%, 23% and 17%, respectively, of the ATP-control activity. Other triose phosphates tested were much less effective in promoting fatty-acid synthesis. When exogenously supplied ATP was supplemented with the DHAP shuttle or glycolytic intermediates, the complete shuttle increased fatty-acid biosynthesis by 37% while DHAP alone resulted in 24% stimulation. Glucose-6-phosphate, fructose-6-phosphate and glycerol-3-phosphate similarly all improved the rates of fatty-acid synthesis by 20–30%. In contrast, 3-phosphoglycerate, 2-phosphoglycerate and phosphoenolpyruvate all inhibited fatty-acid synthesis by approximately 10% each. The addition of the DHAP shuttle and glycolytic intermediates with or without exogenously supplied ATP caused an increase in the proportion of radioactive oleate and a decrease in the proportion of radioactive palmitate synthesized. The use of these alternative energy sources resulted in higher amounts of free fatty acids and triacylglycerol, and lower amounts of diacylglycerol and phosphatidic acid. The data presented here indicate that ATP is superior in promoting in-vitro fatty-acid biosynthesis in pea root plastids; however, both the triose-phosphate shuttle and glycolytic metabolism can produce some of the ATP required for fatty-acid biosynthesis in these plastids.Abbreviations DHAP dihydroxyacetonephosphate - Fru6P fructose-6-phosphate - G3P glycerol-3-phosphate - Glc6P glucose-6-phosphate - OAA oxaloacetate - PEP phosphoenolpyruvate - 2PGA 2-phosphoglycerate - 3PGA 3-phosphoglycerate - 3PGalde glyceraldehyde-3-phosphate This research was supported by grants from the Natural Sciences and Engineering Research Council of Canada.     

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     

Properties of α-glucan phosphorylase from pea chloroplasts

A partially purified preparation of α-glucan phosphorylase was obtained from chloroplasts of Pisum sativum by ion-exchange chromatography and gel filtration. The preparation, in which no other enzyme that metabolized starch or glucose 1 -phosphate could be detected, was characterized. The optimum for phosphorolysis was pH 7.2; at pH 8.0 the activity was reduced by 50%. The preparation showed normal hyperbolic kinetics with the substrates, and catalysed the formation of [¹⁴C]glucose 1-phosphate from ¹⁴C-labelled starch grains from pea chloroplasts. None of the following, generally at 5 and 10 mM, significantly altered the rate of phosphorolysis: glucose, fructose, sucrose, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, pyruvate, ATP, ADP, AMP, 6-phosphogluconate, 2-phosphoglycollate, Mg²⁺, dithiothreitol. However, phosphorolysis was inhibited by ADPglucose. Measurements of ADPglucose in leaves and in isolated chloroplasts showed that none could be detected in the dark and suggested that the concentration in the light was high enough to cause a modest inhibition of the phosphorylase. The control of the breakdown of chloroplast starch is discussed.     

Early effects of illumination on the activity of some photosynthetic enzymes

Leaves of dark-grown corn (Zea mays) were illuminated for periods ranging from 3 minutes to 12 hours. The changes in the activities of ribose-5-phosphate isomerase, ribulose-5-phosphate kinase, and ribulose-1,5-diphosphate carboxylase were followed.

The activity of ribose-5-phosphate isomerase did not change significantly until between 12 and 24 hours of illumination. An increase in ribulose-5-phosphate kinase activity occurred after a lag of about 6 hours. The increase in carboxylase activity began after 3 minutes of illumination and increased until after 3 to 6 hours in the light, after which it began to decline. The increases in these enzymes appear to be the result of protein synthesis.

     

Metabolic regulation of pyruvate kinase isolated from autotrophically and heterotrophically grown Paracoccus denitrificans

Pyruvate kinase (ATP: pyruvate phosphotransferase (EC 2.7.1.40) was partially purified from both autotrophically and heterotrophycally grown Paracoccus denitrificans. The organism grown under heterotrophic conditions contains four times more pyruvate kinase than under autotrophic conditions. The enzyme isolated from both sources exhibited sigmoidal kinetics for both phosphoenolpyruvate (PEP) and ADP. The apparent M _m for ADP and PEP in the autotrophic enzyme were 0.63 mM ADP and 0.25 mM PEP. The effect of several low molecular weight metabolites on the pyruvate kinase activity was investigated. Ribose-5-phosphate, glucose-6-phosphate and AMP stimulated the reaction at low ADP levels; this stimulation was brought about by an alteration in the apparent K _m for ADP. The pyruvate kinases differ in their response to adenine nucleotides, but both preparations seem to be under adenylate control. The results are discussed in relation to the role of pyruvate kinase as a regulatory enzyme in P. denitrificans grown under both autotrophic and heterotrophic conditions.Non-Common Abbreviations PEP phosphoenolpyruvate - R-5-P ribose-5-phosphate - G-6-P glucose-6-phosphate - F-6-P fructose-6-phosphate - 3-PGA 3-phosphoglycerate     

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.

Maltose is the major form of carbon exported from the chloroplast at night

Transitory starch is formed in chloroplasts during the day and broken down at night. We investigated carbon export from chloroplasts resulting from transitory-starch breakdown. Starch-filled chloroplasts from spinach (Spinacia oleracea L. cv. Nordic IV) were isolated 1 h after the beginning of the dark period and incubated for 2.5 h, followed by centrifugation through silicone oil. Exported products were measured in the incubation medium to avoid measuring compounds retained inside the chloroplasts. Maltose and glucose made up 85% of the total exported products and were exported at rates of 626 and 309 nmol C mg^–1 chlorophyll h^–1, respectively. Net export of phosphorylated products was less than 5% and higher maltodextrins were not detected. Maltose levels in leaves of bean (Phaseolus vulgaris L. cv. Linden), spinach, and Arabidopsis thaliana (L.) Heynh. were low in the light and high in the dark. Maltose levels remained low and unchanged during the light/dark cycle in two starch-deficient Arabidopsis mutants, stf1, deficient in plastid phosphoglucomutase, and pgi, deficient in plastid phosphoglucoisomerase. Through the use of nonaqueous fractionation, we determined that maltose was distributed equally between the chloroplast and cytosolic fractions during darkness. In the light there was approximately 24% more maltose in the cytosol than the chloroplast. Taken together these data indicate that maltose is the major form of carbon exported from the chloroplast at night as a result of starch breakdown. We hypothesize that the hydrolytic pathway for transitory-starch degradation is the primary pathway used when starch is being converted to sucrose and that the phosphorolytic pathway provides carbon for other purposes.Abbreviations CAM crassulacean acid metabolism - Chl chlorophyll - DHAP dihydroxyacetone phosphate - FBPase fructose bisphosphatase - GAP glyceraldehyde-3-phosphate - G6P glucose 6-phosphate - PGA 3-phosphoglycerate - TPT triose phosphate translocator - WT wild type