Stromal protein phosphorylation in spinach (Spinacia oleracea) chloroplasts.

When intact spinach chloroplasts were supplied with [32P]Pi, stromal protein phosphorylation was found to occur in the dark. On illumination the thylakoid protein kinase was activated and the amount of label found in thylakoid proteins quickly exceeded that incorporated into stromal protein, such that the latter was found to account for only 10-15% of the total radioactivity bound to chloroplast proteins after 5 min illumination. The rate of phosphorylation of stromal polypeptides was unchanged by light. After SDS/polyacrylamide-gel electrophoresis, more than 15 labelled polypeptides of stromal origin were observed. A polypeptide with an Mr of approx. 70 000 had the highest specific activity of labelling. Both the large and small subunits of the ribulose-1,5-bisphosphate carboxylase were phosphorylated. The level of phosphorylation of stromal protein was increased by CO2 fixation in intact chloroplasts. This increase was not observed in the absence of NaHCO3 or in the presence of the phosphoribulokinase inhibitor DL-glyceraldehyde. These effects appeared to be largely due to changes in the phosphorylation state of the large and small subunits of ribulose-1,5-bisphosphate carboxylase. Studies with the reconstituted chloroplast system showed that the thylakoid protein kinase(s) played no part in the phosphorylation of stromal protein. The rate and level of phosphorylation of stromal protein was unaffected by the activation state of the thylakoid protein kinase and was unchanged when thylakoids were omitted from the reaction medium. The phosphorylation of stromal proteins is therefore catalysed by a discrete soluble protein kinase.     

Heterogeneous photosystem 2 activity in isolated spinach chloroplasts

A study was made of the fluorescence induction curves from gently-broken spinach chloroplasts inhibited with DCMU. It was found that there were four kinetically different phases associated with such curves of which only the fastest did not appear to follow exponential kinetics. A comparison of the effects of various concentrations of DCMU on the rate of oxygen evolution and on the fluorescence induction curve did not support the hypothesis that any of the kinetic phases was simply an artefact caused by incomplete inhibition of electron transport. It was also found that 5 min of dark incubation did not maximally oxidize the electron acceptors to photosystem 2 since some acceptors were only oxidized following far-red illumination, suggesting a heterogeneity among these acceptors with respect to their re-oxidation properties. Investigation of the effect of the Q₄₀₀ oxidation state on the fluorescence induction curve revealed that it only influenced the slowest kinetic phase and that Q₄₀₀ did not seem to be associated with the other phases.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1 - 1 dimethylurea - PS 1 photosystem 1 - PS2 photosystem 2 - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid - EDTA ethylene-diaminetetraacetic acid - F_max maximum yield of fluorescence emission - F₀ initial yield of fluorescence emission - F_v variable yield of fluorescence emission - N.E. non-exponential kinetics     

Diurnal oscillation of amylolytic activity in spinach chloroplasts

Chloroplasts isolated from spinach (Spinacia oleracea L., cv. vital^R) plants grown under controlled light/dark and temperature regimes, contained the phosphorolytic and amylolytic pathways for starch breakdown. The latter consists at least of α- and β-amylase and maltase. Only low amylolytic activity was observed in chloroplasts isolated during the light phase. In chloroplasts prepared during the dark phase, this activity was almost twice as high. These diurnal oscillations of the amylolytic activity were maintained when the plants were kept in prolonged darkness or continuous light. The amylolytic system exhibited a sharp pH optimum between 5.8 and 6.0. Phosphorylase activity, when assayed with saturating concentrations of inorganic phosphate, did not show diurnal fluctuations.     

CDP phosphotransferase activity in spinach intact chloroplasts: Possible involvement of nucleoside diphosphate kinase II

Nucleotides formation after addition of [γ³²P]-ATP has been analysed in isolated chloroplasts in the presence of exogenous CDP, UDP and GDP. The highest level of phosphotransfer was observed on CDP and UDP after 10 min incubation. Interestingly, the phosphorylation increase of chloroplastic CDP in organello correlated with the time-dependent dephosphorylation of a 18-kDa polypeptide, thereby indicating that CDP, the major endogenous phosphorylated NDP, is likely to be a potent in vivo substrate for this phosphoprotein. The 18-kDa polypeptide was immunoprecipitated with antibodies directed against human nucleoside diphosphate kinase (NDPK) A/B and spinach NDPK-II, both belonging to the ubiquitous family of NDPKs (EC 2.7.4.6). Using recombinant NDPK-II, we could not show a preference for CDP in vitro, suggesting either that CDP is the most available NDPK-II substrate in intact chloroplasts or a chloroplastic factor modulates the enzyme affinity for nucleoside diphosphate substrates in vivo.     

Stimulation by thioredoxin of shikimate kinase from spinach chloroplasts

The activity of shikimate kinase (EC 2.7.1.71) from spinach ( Spinacia oleracea L.) chloroplasts was increased up to 8.5-fold by addition of thioredoxin and dithio-threitol.     

Ribulose bisphosphate oxygenase activity from freshly ruptured spinach chloroplasts

Suspensions of freshly lysed spinach chloroplasts, in which ribulose bisphosphate carboxylase displays an in vivo K_m [CO], exhibited a ribulose bisphosphate-dependent uptake of oxygen. The kinetic properties of this oxygenase activity were examined at air levels of CO₂ (10 μm) and O₂ (240 μm). The pH optimum was 8.6–8.8 and the K_M [ribulose bisphosphate] was 45 μm. At 240 μm O₂, the oxygenase activity is inhibited one-half by 25 μm CO₂. The apparent K_m(O₂) is large, somewhere between 1 and 2 atm. The phosphoglycolate phosphatase activity of the chloroplasts was in great excess, suggesting that phosphoglycolate formed by the oxygenase would be quickly hydrolyzed to glycolate for possible metabolism by photorespiration.A comparison of the pH dependence of both the carboxylase and oxygenase activities at air levels of CO₂ and O₂ suggests that the pH of the chloroplast stroma could regulate their relative activities and that the oxygenase activity is sufficient to account for glycolate production during photosynthesis. It is predicted that at pH 7.8, about 40% of the carbon assimilated by the Calvin cycle would go through glycolate.     

Presence of adenine phosphoribosyltransferase and adenosine kinase in chloroplasts of spinach leaves

Activity of adenine phosphoribosyltransferase and adenosine kinase was detected in purified spinach chloroplasts by using differential centrifugation and discontinuous Percoll density gradients. This is the first report of purine salvage enzymes being located in chloroplasts. The role of adenine and adenosine salvage in chloroplasts is discussed.     

Adenylate kinase bound to the envelope membranes of spinach chloroplasts.

The activity of adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) in both the forward (2ADP → ATP + AMP) and backward (ATP + AMP → 2ADP) reactions was found to be associated with the envelope membranes which were isolated from spinach chloroplasts. Sonication and repeated washing in a medium of high ionic strength were unable to release the enzymes from the envelope membranes. Adenylate kinase bound to the envelope is stable in the cold and inactivated by heat and acid treatments. The enzyme requires magnesium ion as an activator. The pH-activity profile of the forward reaction catalyzed by membrane-bound adenylate kinase gave a maximal activity at pH 8.5. The apparent Michaelis constant, K_m, value for ADP in the forward reaction was estimated to be 1.3 ± 0.2 × 10^?4m. A Lineweaver-Burk plot of the forward reaction gave a straight line when the reciprocal of the reaction rate was plotted versus the reciprocal, and not the square of the reciprocal, of the concentration of substrate ADP. This favors the view that the adenylate kinase bound to the chloroplast envelope has a single or equivalent binding site of Mg-ADP^?. The probable involvement of adenylate kinase bound to the chloroplast envelope in controlling the energy pool and adenylate translocation in chloroplasts is suggested.     

Ferrochelatase of spinach chloroplasts

Spinach chloroplasts catalyse the incorporation of Fe(2+) into protoporphyrin, mesoporphyrin and deuteroporphyrin to form the corresponding haems. This ferrochelatase activity was detected by pyridine haemochrome formation with acetone-dried powders of chloroplasts, or from the formation of [(59)Fe]haems by intact chloroplasts. Decreasing the mitochondrial contamination of the chloroplasts by density-gradient centrifugation did not cause any loss of activity: spinach ferrochelatase appears to be principally a chloroplast enzyme. The characteristics of the enzyme were examined by using [(59)Fe]haem assay. The activity was pH-dependent: for both mesohaem and protohaem formation there were two pH maxima, a major peak at about pH7.8 and a smaller peak at about pH9.2. Lineweaver-Burk plots showed that the K(m) for Fe(2+) incorporation into protoporphyrin was 8mum and that for Fe(2+) incorporation into mesoporphyrin was 36mum. At non-saturating Fe(2+) concentrations the K(m) for protoporphyrin was 0.2mum and that for mesoporphyrin was 0.4mum. Ferrochelatase was not solubilized by treatment of chloroplasts with ultrasound but was solubilized by stirring in 1% (w/v) Tween 20 at pH10.4. Unlike the rat liver mitochondrial enzyme, chloroplast ferrochelatase was not stimulated by treatment with selected organic solvents. The spinach enzyme was inactive in aerobic conditions and it was shown by using an oxygen electrode that under such conditions the addition of Fe(2+) to buffer solutions caused a rapid uptake of dissolved oxygen, believed to be due to the oxidation of Fe(2+) to Fe(3+); Fe(3+) is not a substrate for ferrochelatase.     

Requirement of galactolipids for Photosystem I activity in lyophilized spinach chloroplasts

1. The effect of monogalactosyl diacylglycerol and digalactosyl diacylglycerol on reconstitution of Photosystem I activity in heptane-extracted and galactolipase-treated spinach chloroplasts was investigated.2. Both galactolipids, in a molar ratio with chlorophyll of 2.5, partially restored Photosystem I activity in heptane-extracted chloroplasts. An addition o saturating amounts of plastocyanin caused complete reactivation of Photosystem I.3. Similarly, with galactolipase-treated chloroplasts, both galactolipids partially restored Photosystem I activity and additional amounts of plastocyanin were required for complete reactivation.4. The action of galactolipids on partial reconstitution of Photosystem I supports the suggestion of their structural role in the restoration of thylakoid membranes.     

Purification and characterization of 33 kilodalton protein of spinach chloroplasts

A protein was prepared from spinach chloroplasts in a highly purified form. The isoelectric point of the protein was 5.2. The apparent molecular weight was estimated to be 33 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea, and 34 000 by gel filtration column chromatography with Sephadex G-100. The protein was provisionally named '33 kilodalton protein' according to the molecular weight. The absorption spectrum of the protein did not show any absorption band in the visible region. No histidine was found in the amino acid analysis of the protein. The 33 kilodalton protein was released from the thylakoid membrane by EDTA-treatment and also by sonic oscillation. The protein was bound to System II particles, but not to System I particles.