Formation and metabolism of glycolate in the cyanobacterium Coccochloris peniocystis

The formation and metabolism of glycolate in the cyanobacterium Coccochloris peniocystis was investigated and the activities of enzymes of glycolate metabolism assayed. Photosynthetic ¹⁴CO₂ incorporation was O₂ insensitive and no labelled glycolate could be detected in cells incubated at 2 and 21% O₂. Under conditions of 100% O₂ glycolate comprised less than 1% of the acid-stable products indicating ribulose 1,5 bisphosphate (RuBP) oxidation only occurs under conditions of extreme O₂ stress. Metabolism of [1-¹⁴C] glycolate indicated that as much as 62% of ¹⁴C metabolized was released as ¹⁴CO₂ in the dark. Metabolism of labelled glycolate, particularly incorporation of ¹⁴C into glycine, was inhibited by the amino-transferase inhibitor amino-oxyacetate. Metabolism of [2-¹⁴C] glycine was not inhibited by the serine hydroxymethyltransferase inhibitor isonicotinic acid hydrazide and little or no labelled serine was detected as a result of ¹⁴C-glycolate metabolism. These findings indicate that a significant amount of metabolized glycolate is totally oxidized to CO₂ via formate. The remainder is converted to glycine or metabolized via a glyoxylate cycle. The conversion of glycine to serine contributes little to glycolate metabolism and the absence of hydroxypyruvate reductase confirms that the glycolate pathway is incomplete in this cyanobacterium.Abbreviations AAN aminoacetonitrile - AOA aminooxyacetate - DIC dissolved inorganic carbon - INH isonicotinic acid hydrazide - PEP phosphoenolpyruvate - PEPcase phosphoenolpyruvate carboxylase - PG phosphoglycolate - PGA phosphoglyceric acid - PGPase phosphoglycolate phosphatase - PR photorespiration - Rubisco ribulose-1,5-bisphosphate carboxylase oxygenase - TCA trichloroacetic acid - RuBP ribulose-1,5-bisphosphate     

Mass-spectrometric evidence for the double-carboxylation pathway of malate synthesis by Crassulacean acid metabolism plants in light

Phyllodia of the Crassulacean acid metabolism (CAM) plant Kalanchoë tubiflora were allowed to fix ¹³CO₂ in light and darkness during phase IV of the diurnal CAM cycle, and during prolongation of the regular light period. After ¹³CO₂ fixation in darkness, only singly labelled [¹³C]malate molecules were found. Fixation of ¹³CO₂ under illumination, however, produced singly labelled malate as well as malate molecules which carried label in two, three or four carbon atoms. When the irradiance during ¹³CO₂ fixation was increased, the proportion of singly labelled malate decreased in favour of plurally labelled malate. The irradiance, however, did not change either the ratio of labelled to unlabelled malate molecules found in the tissue after the ¹³CO₂ application, or the magnitude of malate accumulation during the treatment with label. The ability of the tissue to store malate and the labelling pattern changed throughout the duration of the prolonged light period. The results indicate that malate synthesis by CAM plants in light can proceed via a pathway containing two carboxylation steps, namely ribulose-1,5-bisphosphate-carboxylase/oxygenase (EC 4.1.1.39) and phosphoenolpyruvate carboxylase (EC 4.1.1.31) which operate in series and share common intermediates. It can be concluded that, in light, phosphoenolpyruvate carboxylase can also synthesize malate independently of the proceeding carboxylation step by ribulose-1,5-bisphosphate carboxylase/oxygenase.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase (EC 4.1.1.31) - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) - TMS trimethylsilyl     

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.     

Immuno-gold localization of cytochrome f,light-harvesting complex,ATP synthase and ribulose 1,5-bisphosphate carboxylase/oxygenase

The proteins ribulose 1,5-bisphosphate carboxylase/oxygenase, ATP synthase, light-harvesting chlorophyll a/b protein, and cytochrome f, have been localized in mesophyll chloroplasts of barley (Hordeum vulgare L.) by electron microscopy of immunogold-labelled sections. The light-harvesting chlorophyll a/b protein and cytochrome f are shown to be present in the grana, both within the stacks and at the margins, and in the stromal membranes. Although the absolute amount of labelling for these proteins is greater in the grana than in the stromal membranes, when expressed as label/membrane length the partitioning appears approximately equal between appressed and non-appressed membranes for both the light-harvesting chlorophyll a/b protein and cytochrome f. ATP synthase is restricted to the non-appressed thylakoid membranes, and ribulose 1,5-bisphosphate carboxylase/oxygenase is uniformly distributed through the stromal contents.Abbreviations CF₁ ATP synthase - LHCPII light-harvesting chlorophyll a/b protein - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase     

Partial Purification and Characterization of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Large Subunit epsilonN-Methyltransferase

The large subunit (LS) of tobacco (Nicotiana rustica) ribulose-1,5-bisphosphate carboxylase/oxygenase (ribulose-P₂ carboxylase) contains a trimethyllysyl residue at position 14, whereas this position is unmodified in spinach ribulose-P₂ carboxylase. A protein fraction was isolated from tobacco chloroplasts by rate-zonal centrifugation and anion-exchange fast protein liquid chromatography that catalyzed transfer of methyl groups from S-adenosyl-[methyl-³H]-l-methionine to spinach ribulose-P₂ carboxylase. ³H-Methyl groups incorporated into spinach ribulose-P₂ carboxylase were alkaline stable but could be removed by limited tryptic proteolysis. Reverse-phase high-performance liquid chromatography of the tryptic peptides released after proteolysis showed that the penultimate N-terminal peptide from the LS of spinach ribulose-P₂ carboxylase contained the site of methylation, which was identified as lysine-14. Thus, the methyltransferase activity can be attributed to S-adenosylmethionine:ribulose-P₂ carboxylase LS (lysine) `N-methyltransferase, a previously undescribed chloroplast enzyme. The partially purified enzyme was specific for ribulose-P₂ carboxylase and exhibited apparent K_m values of 10 micromolar for S-adenosyl-l-methionine and 18 micromolar for ribulose-P₂ carboxylase, a V_max of 700 picomoles CH₃ groups transferred per minute per milligram protein, and a broad pH optimum from 8.5 to 10.0. S-Adenosylmethionine:ribulose-P₂ carboxylase LS (lysine)^εN-methyltransferase was capable of incorporating 24 ³H-methyl groups per spinach ribulose-P₂ carboxylase holoenzyme, forming 1 mole of trimethyllysine per mole of ribulose-P₂ carboxylase LS, but was inactive on ribulose-P₂ carboxylases that contain a trimethyllysyl residue at position 14 in the LS. The enzyme did not distinguish between activated (Mg²⁺ and CO₂) and unactivated forms of ribulose-P₂ carboxylase as substrates. However, complexes of activated ribulose-P₂ carboxylase with the reaction-intermediate analogue 2′-carboxy-d-arabinitol-1,5-bisphosphate, or unactivated spinach ribulose-P₂ carboxylase with ribulose-1,5-bisphosphate, were poor substrates for tobacco LS ^εN-methyltransferase.     

Interactions between ribulose-1,5-bisphosphate carboxylase and stromal metabolites. II. Corroboration of the role of this enzyme as a metabolite buffer

The capacity of ribulose-1,5-bisphosphate carboxylase to bind reversibly chloroplast metabolites which are the substrates for both thylakoid and stromal enzymes was assessed using spinach chloroplasts and chloroplast extracts and with pure wheat ribulose-1,5-bisphosphate carboxylase. Measurements of the rate of coupled electron flow to methyl viologen in ‘leaky’ chloroplasts (which retained the chloroplast envelope and stromal enzymes but which were permeable to metabolites) and also with broken chloroplasts and washed thylakoids were used to study the effects of binding ADP and inorganic phopshate to ribulose-1,5-bisphosphate carboxylase. The presence of ribulose-1,5-bisphosphate carboxylase significantly altered the values obtained for apparent K_m for inorganic phosphate and ADP of coupled electron transport. The K_m (P_i) in washed thylakoids was 60–80 μM, in ‘leaky’ chloroplasts it was increased to 180–200 μM, while in ‘leaky’ chloroplasts preincubated with KCN and ribulose 1,5-bisphosphate the value was decreased to 40–50 μM. Similarly, the K_m (ADP) of coupled electron transport in washed thylakoids was 60–70 μM, in ‘leaky’ chloroplasts it was 130–150 μM and with ‘leaky’ chloroplasts incubated in the presence of KCN and ribulose 1,5-bisphosphate a value of 45–50 μM was obtained. The ability of ribulose 1,5-bisphosphate carboxylase to reduce the levels of free glycerate 3-phosphate in the absence of ribulose 1,5-bisphosphate was examined using a chloroplast extract system by varying the concentrations of stromal protein or purified ribulose 1,5-bisphosphate carboxylase. The effect of binding glycerate 3-phosphate to ribulose-1,5-bisphosphate carboxylase on glycerate 3-phosphate reduction was to reduce both the rate an the amount of NADPH oxidation for a given amount of glycerate 3-phosphate added. The addition of ribulose 1,5-bisphosphate reinitiated NADPH oxidation but ATP or NADPH did not. Incubation of purified ribulose-1,5-bisphosphate carboxylase with carboxyarabinitolbisphosphate completely inhibited the catalytic activity of the enzyme and decreased inhibition of glycerate-3-phosphate reduction. Two binding sites with different affinities for glycerate 3-phosphate were observed with pure ribulose-1,5-bisphosphate carboxylase.     

The Association of d-Ribulose- 1,5-Bisphosphate Carboxylase/Oxygenase with Phosphoribulokinase

When Ribulose- 1,5-bisphosphate carboxylase/oxygenase was purified from spinach leaves (Spinacia oleracea) using precipitation with polyethylene glycol and MgCl₂ followed by DEAE cellulose chromatography, 75% of phosphoribulokinase and 7% of phosphoriboisomerase activities copurified with ribulose- 1,5-bisphosphate carboxylase/oxygenase. This enzyme preparation showed ribose-5-phosphate and ribulose-5-phosphate dependent carboxylase and oxygenase activities which were nearly equivalent to its corresponding ribulose- 1,5-bisphosphate dependent activity. The ribose-5-phosphate and ribulose-5-phosphate dependent reaction rates were stable and linear for much longer time periods than the ribulose- 1,5-bisphosphate dependent rates. When sucrose gradients were used to purify ribulose- 1,5-bisphosphate carboxylase/oxygenase from crude stromal extracts, phosphoribulokinase was found to cosediment with ribulose- 1,5-bisphosphate carboxylase. Under these conditions most of the phosphoriboisomerase activity remained with the slower sedimenting proteins. Ammonium sulfate precipitation resulted in separation of the ribulose- 1,5-bisphosphate carboxylase peak from phosphoribulokinase peak. Crude extracts of peas Pisum sativum and spinach contained 0.725 to 0.730 milligram of phosphoribulokinase per milligram of chlorophyll, respectively, based on an enzyme-linked immunosorbent assay.     

Regulation of photosynthesis in nitrogen deficient wheat seedlings

Nitrogen effects on the regulation of photosynthesis in wheat (Triticum aestivum L., cv Remia) seedlings were examined. Ribulose 1,5-bisphosphate carboxylase/oxygenase was rapidly extracted and tested for initial activity and for activity after incubation in presence of CO₂ and Mg²⁺. Freeze clamped leaf segments were extracted for determinations of foliar steady state levels of ribulose 1,5-bisphosphate, triose phosphate, 3-phosphoglycerate, ATP, and ADP. Nitrogen deficient leaves showed increased ATP/ADP and triose phosphate/3-phosphoglycerate ratios suggesting increased assimilatory power. Ribulose 1,5-bisphosphate levels were decreased due to reduced pentose phosphate reductive cycle activity. Nevertheless, photosynthesis appeared to be limited by ribulose 1,5-bisphosphate carboxylase/oxygenase, independent of nitrogen nutrition. Its degree of activation was increased in nitrogen deficient plants and provided for maximum photosynthesis at decreased enzyme protein levels. It is suggested that ribulose 1,5-bisphosphate carboxylase/oxygenase activity is regulated according to the amount of assimilatory power.     

Isolation and partial characterization of pyrenoids from the brown alga Pilayella littoralis (L.) Kjellm.

In order to isolate high yields of pyrenoids from the brown alga Pilayella littoralis it is necessary to pretreat them with 0.1% HgCl₂ in sea water for 3 h. Without this pretreatment there is a substantial loss of pyrenoid ground substance and yields are low. Pyrenoid fractions of high purity have been obtained using silica sol gradients. A partial characterization has shown the pyrenoid to be proteinaceous and lacking chlorophyll. SDS polyacrylamide gel electrophoresis has shown that the majority of protein present is accounted for by two polypeptides which resemble the large and small subunits of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39).Abbreviations DTT dithiothreitol - HEPES N-2-hydroxyethylniperazine N¹-2-ethanesulfonic acid - PEG polyethylene glycol - PVPP polyvinylpolypyrrolidone - RuBP ribulose-1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase - SDS sodium dodecyl sulphate     

Development and Characterization of Ribulose-1,5-bisphosphate Carboxylase : Evidence Indicating a Lack of Carboxylase Function in CO(2) Fixation in Endosperm of Germinating Castor Bean Seedlings

Ribulose-1,5-bisphosphate carboxylase activity was found in endosperm of germinating castor bean seed Ricinus communis and was localized in proplastids. The endosperm carboxylase has been extensively purified and is composed of two different subunits. The molecular weights of the native carboxylase and its subunits were 560,000, 55,000, and 15,000 daltons, respectively. The Michaelis-Menten constants, K_m, for the endosperm carboxylase with respect to ribulose 1,5-bisphosphate, bicarbonate, CO₂, and magnesium in millimolar are 0.54, 13.60, 0.92, and 0.57, respectively. The endosperm carboxylase was activated by Mg²⁺ and HCO₃⁻. The preincubation of the carboxylase with 1 millimolar HCO₃⁻ and 5 millimolar MgCl₂ resulted in activation by low and inhibition by high concentrations of 6-phosphogluconate.

In studies of dark ¹⁴CO₂ fixation by endosperm slices, [¹⁴C]malate and [¹⁴C]citrate were the predominantly labeled products after 30 seconds of exposure of the tissue to H¹⁴CO₃⁻. In pulse-chase experiments, 87% of the label is malate, and citrate was transferred to sugars after a 60-minute chase with a small amount of the label appearing in the incubation medium as ¹⁴CO₂. The minimal incorporation of the label from ¹⁴CO₂ into phosphoglyceric acid indicated a lack of the endosperm ribulose-1,5-bisphosphate carboxylase participation in the endosperm's CO₂ fixation system. The activities of key Calvin cycle enzymes were examined in the endosperms and cotyledons of dark-grown castor bean seedlings. Many of these autotrophic enzymes develop in the dark in these tissues. The synthesis of ribulose-1,5-bisphosphate carboxylase in the nonphotosynthetic endosperms is not repressed in the dark, and high levels of enzymic activity appear with germination. All of the Calvin cycle enzymes are present in the castor bean endosperm except NADP-linked glyceraldehyde 3-P dehydrogenase, and the absence of this dehydrogenase probably prevents the functioning of these series of reactions in dark CO₂ fixation.

     

Ribulose 1,5-bisphosphate carboxylase and oxygenase from Thiocapsa roseopersicina: activation and catalysis.

Ribulose 1,5-bisphosphate carboxylase/oxygenase purified from malate-grown Thiocapsa roseopersicina required Mg²⁺ for the activation of both carboxylase and oxygenase activities. Mg²⁺ was either not required or required at very low concentrations for catalysis by both enzyme activities. EDTA and dithiothreitol had no effect on ribulose 1,5-biphosphate oxygenase. The K_0.5 values with respect to Mg²⁺ for activation of the carboxylase and oxygenase activities were 8.4 and 2 mm, respectively. Ribulose 1,5-biphosphate carboxylase and oxygenase activities revealed differential sensitivities to 6-phosphogluconate. This ligand at 1 mm inhibited the carboxylase activity 30%, whereas the oxygenase activity was inhibited by 69%.     

High specific activity ribulose 1,5-bisphosphate carboxylase-oxygenase from Nicotiana tabacum

Ribulose 1,5-bisphosphate carboxylase (EC.4.1.1.39) has been obtained from Nicotiana tabacum leaf homogenates with specific activites from 0.5 to 0.8 µmol CO₂ fixed (mg protein min)^-1. These activities are reconciled with much lower, previously reported activities. The results suggest that if the tobacco enzyme is assayed under optimum conditions there is little difference in the intrinsic specific activities of tobacco and spinach ribulose 1,5-bisphosphate carboxylase. Several factors affecting activity measurements were examined.     

Exchange Properties of the Activator CO(2) of Spinach Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

The exchange properties of the activator CO₂ of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase were characterized both in vitro with the purified enzyme, and in situ within isolated chloroplasts. Carboxyarabinitol-1,5-bisphosphate, a proposed reaction intermediate analog for the carboxylase activity of the enzyme, was used to trap the activator CO₂ on the enzyme both in vitro and in situ. Modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in intact chloroplasts during a light/dark cycle was associated with a similar modulation in carboxyarabinitol-1,5-bisphosphate-trapped CO₂. The exchange kinetics of the activator CO₂ were monitored by activation of the enzyme to steady state in the presence of ¹²CO₂, followed by addition of ¹⁴CO₂ and determination of the amount of labeled CO₂ trapped on the enzyme by carboxyarabinitol-1,5-bisphosphate. Rate constants (K_obs) for exchange with both the purified enzyme (0.45 min⁻¹) and in illuminated chloroplasts (0.18 min⁻¹) were comparable to the observed rate constants for enzyme activation under the two conditions. A similar exchange of the activator CO₂ was not observed in chloroplasts in the dark. Kinetic analysis of the exchange properties of the purified enzyme were consistent with an equilibrium between active and inactive forms of the enzyme during steady state activation.     

Photosynthesis of Ulva sp: III. O(2) Effects, Carboxylase Activities, and the CO(2) Incorporation Pattern

Ulva, a common green seaweed, performs at the biochemical level as a typical C₃ plant. Over 90% of label was found in glycerate 3-phosphate following a 3 second ¹⁴C pulse in the light, and the label was subsequently transferred to sugars. Also, the level of ribulose-1,5-bisphosphate carboxylase activity in crude extracts was about 10 times higher than that of phosphoenolpyruvate carboxylase. Concerning gas exchange, photosynthetic rates of Ulva showed no O₂ sensitivity, indicating that photorespiratory CO₂ losses are repressed as in C₄ plants. This apparent anomaly could be explained by the efficient HCO₃⁻ uptake system of Ulva which might concentrate CO₂ to the chloroplasts, thus suppressing the oxygenase activity of ribulose-1,5-bisphosphate carboxylase.     

Protein composition of the carboxysomes of Thiobacillus neapolitanus

For purifying carboxysomes of Thiobacillus neapolitanus an isolation procedure was developed which resulted in carboxysomes free from whole cells, protoplasts and cell fragments. These purified carboxysomes are composed of 8 proteins and at the most of 13 polypeptides. The two most abundant proteins which make up more than 60% of the carboxysomes, are ribulose-1,5-bisphosphate carboxylase and a glycoprotein with a molecular weight of 54,000. The shell of the carboxysomes consists of four glycoproteins, one also with a molecular weight of 54,000. The other proteins are present in minor quantities. Ribulose-1,5-bisphosphate carboxylase is the only enzyme which could be detected in the carboxysomes and 3-phosphoglycerate was the only product formed during incubation with ribulose-1,5-diphosphate and bicarbonate. The supernatant of a broken and centrifuged carboxysome suspension contained the large subunit of ribulose-1,5-bisphosphate carboxylase. The small subunit of ribulose-1,5-bisphosphate carboxylase was found in the pellet together with the shell proteins which indicates that the small subunit of ribulose-1,5-bisphosphate carboxylase is connected to the shell.Abbreviations RuBisCO ribulose-1,5-bisphosphate carboxylase - PMSF phenylmethylsulfonyl fluoride - PAA gelectrophoresis, polyacrylamide gelelectrophoresis - SDS sodium dodecyl sulphate - CIE crossed immunoelectrophoresis - IEF isoelectric focusing     

Products of photosynthetic 14CO2 fixation and related enzyme activities in fruiting structures of chickpea

Fruiting structures of a number of legumes including chickpea are known to carry out photosynthetic CO₂ assimilation, but the pathway of CO₂ fixation and particularly the role of phosphoenolpyruvate carboxylase (EC 4.1.1.31) in these tissues is not clear. Activities of some key enzymes of the Calvin cycle and C₄ metabolism, rates of ¹⁴CO₂ fixation in light and dark, and initial products of photosynthetic ¹⁴CO₂ fixation were determined in podwall and seedcoat (fruiting structures) and their subtending leaf in chickpea (Cicer arietinum L.). Compared to activities of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) and other Calvin cycle enzyme, viz. NADP⁺-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13), NAD⁺-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) and ribulose-5-phosphate kinase (EC 2.7.1.19), the levels of phosphoenolpyruvate carboxylase and other enzymes of C₄ metabolism viz. NADP⁺-malate dehydrogenase (EC 1.1.1.82), NAD⁺-malate dehydrogenase (EC 1.1.1.37), NADP⁺ malic enzyme (EC 1.1.1.40), NAD⁺-malic enzyme (EC 1.1.1.39), glutamate oxaloacetate transaminase (EC 2.6.1.1) and glutamate pyruvate transaminase (EC 2.6.1.2), were generally much higher in podwall and seedcoat than in the leaf. Podwall and seedcoat fixed ¹⁴CO₂ in light and dark at much higher rates than the leaf. Short-term assimilation of ¹⁴CO₂ by illuminated fruiting structures produced malate as the major labelled product with less labelling in 3-phosphoglycerate, whereas the leaf showed a major incorporation into 3-phosphoglycerate. It seems likely that the fruiting structures of chickpea utilize phosphoenolpyruvate carboxylase for recapturing the respired carbon dioxide.     

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.     

Activity and quantity of ribulose bisphosphate carboxylase-and phosphoenolpyruvate carboxylase-protein in two Crassulacean acid metabolism plants in relation to leaf age,nitrogen nutrition,and point in time during a day/night cycle

Activity of ribulose 1,5-bisphosphate (RuBP) carboxylase in leaf extracts of the constitutive Crassulacean acid metabolism (CAM) plant Kalanchoe pinnata (Lam.) Pers. decreased with increasing leaf age, whereas the activity of phosphoenolpyruvate (PEP) carboxylase increased. Changes in enzyme activities were associated with changes in the amount of enzyme proteins as determined by immunochemical analysis, sucrose density gradient centrifugation, and SDS gel electrophoresis of leaf extracts. Young developing leaves of plants which received high amounts of NO ₃ ^- during growth contained about 30% of the total soluble protein in the form of RuBP carboxylase; this value declined to about 17% in mature leaves. The level of PEP carboxylase in young leaves of plants at high NO ₃ ^- was an estimated 1% of the total soluble protein and increased to approximately 10% in mature leaves, which showed maximum capacity for dark CO₂ fixation. The growth of plants at low levels of NO ₃ ^- decreased the content of soluble protein per unit leaf area as well as the extractable activity and the percentage contribution of both RUBP carboxylase and PEP carboxylase to total soluble leaf protein. There was no definite change in the ratio of RuBP carboxylase to PEP carboxylase activity with a varying supply of NO ₃ ^- during growth. It has been suggested (e.g., Planta 144, 143–151, 1978) that a rhythmic pattern of synthesis and degradation of PEP carboxylase protein is involved in the regulation of -carboxylation during a day/night cycle in CAM. No such changes in the quantity of PEP carboxylase protein were observed in the leaves of Kalanchoe pinnata (Lam.) Pers. or in the leaves of the inducible CAM plant Mesembryanthemum crystallinum L.Abbreviations CAM Crassulacean acid metabolism - RuBP ribulose 1,5-bisphosphate - PEP phosphoenolpyruvate - G-6-P glucose-6-phosphate     

Immunological determination of phosphoenolpyruvate carboxylase and the large and small subunits of ribulose 1,5-bisphosphate carboxylase in leaves of the c(4) plant pearl millet

The light-dependent development of the photosynthetic apparatus in the first leaf of the C₄ plant pearl millet (Pennisetum americanum) was monitored by immunologically determining the concentration of phospho-enolpyruvate carboxylase and ribulose 1,5-bisphosphate carboxylase. A competitive enzyme-linked immunosorbent assay procedure using antibodies to the monomeric subunit of phosphoenolpyruvate carboxylase and the large and small subunit of ribulose 1,5-bisphosphate carboxylase was used to quantitate the amounts of these polypeptides in the first leaf of etiolated seedlings and etiolated seedlings exposed to light for varying periods of time. Phosphoenolpyruvate carboxylase was present in etiolated tissue; however, light stimulated its synthesis nearly 23-fold. Maximum accumulation of phosphoenolpyruvate carboxylase occurred approximately 4 days after etiolated plants were placed in the light. Both the large subunit and the small subunit of ribulose 1,5-bisphosphate carboxylase were present in leaves of etiolated seedlings. Light also stimulated the synthesis of both of these polypeptides, but at different rates. In etiolated leaves there was approximately a 3-fold molar excess of the small subunit to large subunit. Exposure of the etiolated leaves to light resulted in the molar ratio of the large subunit to the small subunit increasing to approximately 0.72. These data indicate that the net synthesis of these two polypeptides is not coordinately regulated at all times.     

Active-site histidines in recombinant cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase examined by site-directed mutagenesis

The functions of His²⁹¹, His²⁹⁵ and His³²⁴ at the active-site of recombinant A. nidulans ribulose-1,5-bisphosphate carboxylase/ oxygenase have been explored by site-directed mutagenesis. Replacement of His²⁹¹ by K or R resulted in unassembled proteins, while its replacement by E, Q or N resulted in assembled but inactive proteins. These results are in accord with a metal ion-binding role of this residue in the activated ternary complex by analogy to x-ray crystallographic analyses of tobacco and spinach enzymes.His³²⁴ (H327 in spinach), which is located within bonding distance of the 5-phosphate of bound bi-substrate analog 2-carboxyarabinitol 1,5-bisphosphate in the crystal structures, has been substituted by A, K, R, Q and N. Again with the exception of the H324K and R variants, these changes resulted in detectable assembled protein. The mutant H324A protein exhibited no detectable carboxylase activity, whereas the H324Q and H324N changes resulted in purifiable holoenzyme with 2.0 and 0.1% of the recombinant wild-type specific carboxylase activity, respectively. These results are consistent with a phosphate binding role for this residue.The replacement of His²⁹⁵, which has been suggested to aid in phosphate binding, with Ala in the A. nidulans enzyme leads to a mutant with 5.8% of the recombinant wild-type carboxylase activity. All other mutations at this position resulted in unassembled proteins. Purified H295A and H324Q enzymes had elevated K_m(RuBP) values and unchanged CO₂/O₂ specificity factors compared to recombinant wild-type.Abbreviations CABP D-2-carboxyarabinitol 1,5 bisphosphate - IPTG isopropyl-b-d-thiogalactopyranoside - L large subunit of rubisco - PAGE polyacrylamide gel electrophoresis - rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-P₂, ribulose 1,5 bisphosphate - S small subunit of rubisco - SDS sodium dodecyl sulfate - X-gal 5-bromo-4-chloro-3-indolyl-b-d-galactoside