The role of pH in the regulation of carbon fixation in the chloroplast stroma. Studies on CO2 fixation in the light and dark

The light-dependent accumulation of radioactively labeled inorganic carbon in isolated spinach (Spinacia orwcea L.) chloroplasts was determined by silicone oil filtering centrifuption. Intact chloroplasts, darkincubated 60 seconds at pH 7.6 and 23°C with 0.5 millimolar sodium bicarbonate, contained 0.5 to 1.0 millimolar internal inorganic carbon.The stromal pool of inorganic carbon increased 5-to 7-fold after 2 to 3 minutes of light. The saturated internal bicarbonate concentration of illuminated spinach chloroplasts was 10-to 20-fold greater than that of the external medium. This ratio decreased at lower temperatures and with increasing external bicarbonate. Over one-half the inorganic carbon found in intact spinach chloroplasts after 2 minutes of light was retained during a subsequent 3-minute dark incubation at 5'C. Calculations of light-induced stromal alkalization based on the uptake of radioactively labeled bicarbonate were OA to 0.5 pH units less than measurements performed with '4qCdimethyloxazolidine-dione. About one-third of the binding sites on the enzyme ribulose 1,5-bisphosphate carboxyase were radiolabeled when the enzyme was activated in situt and 4C02 bound to the activator site was trapped in the presence of carboxypentitol bisphosphates. Deleting orthophosphate from the incubation medium eliminated inorganic carbon accumulation in the stroma. Thus, bicarbonate ion distribution across the chloroplast envelope was not strictly pH dependent as predicted by the Henderson-Hasselbach formula. This finding is potentially explained by the presence of bound CO2 in the chloroplast.that illumination should produce a 10-fold increase of bicarbonate ion within this space. The following report details efforts in this laboratory to verify this observation.In addition to being the substrate for photosynthetic carbon assimilation, CO, is an essential factor in the light-activation of the enzyme RuBP' carboxylase (14,18 MATERIALS AND METHODSThe low ambient CO2 concentration in the atmosphere may be a critical factor limiting the photosynthetic rate of agronomic crop species (6). In spite of the importance of CO2 to the carbon assimilation process, there have been relatively few studies on the acquisition of inorganic carbon by isolated chloroplasts or by chloroplast envelope preparations (21,22,27). The transfer of inorganic carbon across the chloroplast membrane has been reviewed recently (8) and this process has the following important features: (a) permeability of the chloroplast envelope is high for CO2 relative to bicarbonate (27), (b) diffusion of CO2 across the chloroplast envelope is rapid and bicarbonate accumulation saturates in less than 30 s at 9C (21, 27), and (c) distribution of bicarbonate ion across the chloroplast envelope is dependent upon the pH gradient between the internal and external medium as predicted by the Henderson-Hasselbach equation (27). This latter observation is significant inasmuch as the internal and external bicarbonate ion concentrations can be used to calculate the ...

Section: Methodscontrasting

confidence: 60%

Characteristics of Light-Dependent Inorganic Carbon Uptake by Isolated Spinach Chloroplasts

Sicher¹

1984

“…The smooth response of spontaneous rubisco activation suggests that the primary effect of pH is chemical. In the activase system, however, an optimum was observed at about pH 8.2, near the measured stromal pH in the light (21).…”

Section: Resultsmentioning

confidence: 72%

“…Rubisco activation showed an optimum at about pH 8.2, near the optimal stromal pH of 8.1 for chloroplast CO2 fixation (21).…”

Section: Discussionmentioning

confidence: 95%

Activation of Ribulosebisphosphate Carboxylase/Oxygenase at Physiological CO₂ and Ribulosebisphosphate Concentrations by Rubisco Activase

Portis

Salvucci²,

Ogren³

1986

180

120

The enzyme-catalyzed activation of ribulosebisphosphate carboxylase/ oxygenase (rubisco) was investigated in an illuminated reconstituted system containing thylakoid membranes, rubisco, ribulosebisphosphate (RuBP), MgCl2, carbonic anhydrase, catalase, the artificial electron acceptor pyocyanine, and partially purified rubisco activase. Optimal conditions for light-induced rubisco activation were found to include 100 micrograms per milliliter rubisco, 300 micrograms per milliliter rubisco activase, 3 millimolar RuBP, and 6 millimolar free Mg2' at pH 8.2. The half-time for rubisco activation was 2 minutes, and was 4 minutes for rubisco deactivation. The rate of rubisco deactivation was identical in the presence and absence of activase. The K,,t(CO2) of rubisco activation in the reconstituted system was 4 micromolar C02, compared to a K,A,(C02) of 25 to 30 micromolar CO2 for the previously reported spontaneous CO2/Mg2' activation mechanism. The activation process characterized here explains the high degree of rubisco activation at the physiological concentrations of 10 micromolar CO2 and 2 to 4 millimolar RuBP found in intact leaves, conditions which lead to almost complete deactivation of rubisco in vitro.The photosynthetic assimilation ofCO2 is initiated by rubisco.2 This enzyme exhibits complex regulatory properties, including a requirement that it be converted to an activated state in order to acquire catalytic competency (8,9,14). The activation of rubisco in vitro occurs by the spontaneous addition of activating CO2 (ACO2) to the e-amino group of a lysine residue near the active site, followed by the addition of Mg2' (Eq. 1, where E = rubisco) (9, 10). (about 10 AM). Also, direct measurements have shown that rubisco activation in illuminated leaves is not very responsive to the intercellular CO2 concentrations (12, 17), although it does respond quantitatively over a wide range of light intensities (13,17). Thus the spontaneous mechanism of rubisco activation (9, 10) as shown in Eq. 1 is insufficient to explain the activation of the enzyme in vivo.We recently demonstrated (16) that the absence of rubisco activation in a mutant of the C3 plant Arabidopsis thaliana (20) was correlated with the absence of two chloroplast polypeptides, and that a soluble chloroplast extract stimulated rubisco activation in an illuminated reconstituted system containing chloroplast thylakoid membranes and rubisco. These observations led us to conclude that rubisco activation in vivo did not occur spontaneously but required the presence of an enzyme, rubisco activase. We have now partially purified the enzyme which catalyzes rubisco activation and demonstrate that it reduces the CO2 requirement for activation to a concentration consistent with that found in leaves.MATERIALS AND METHODS Chemicals and Equipment. RuBP was synthesized from ribose 5-P (Sigma3) as described previously (7) and stored in liquid N2. Some commercial preparations have been examined and found to be unsatisfactory for reconstitution studies. Pyocyanine...

“…In the chloroplast, pyruvate kinase would be inactive in the dark, but fully active in the light, when stromal pH is increased to about pH 8 (16). Since it has also been demonstrated that fatty acid biosynthesis occurs in the light but not in the dark (17), it appears that chloroplast pyruvate kinase is a light-controlled regulatory enzyme at an early stage of chloroplast fatty acid biosynthesis.…”

Section: Resultsmentioning

confidence: 99%

Isoenzymes of Pyruvate Kinase in Etioplasts and Chloroplasts

Ireland¹,

Deluca²,

Dennis³

1979

Isoenzymes of pyruvate kinase from green leaves of castor bean and etiolated leaves of pea plants have been separated by ion filtration chromatography. One of the isoenzymes is localzed in the plastid, whereas the other is in the cytosol. The cytosolic enzyme has a pH optimum from pH 7 to pH 9, and is able to utilize nucleotides other than ADP as the phosphoryl acceptor. The plastid enzyme has a much sharper optimum at pH 8, and is less efficient at using alternative nucleotides. The plastic pyruvate kinase, unlike the cytosolic enzyme, requires the presence of dithiothreitol or 2-mercaptoethanol during isolation and storage to stabilize the activity.