Purification and Some Properties of Chlorella fusca Ribulose 1,5-Diphosphate Carboxylase

Ribulose 1,5-diphosphate carboxylase has been purified from extracts of autotrophically grown Chlorella fusca by ammonium sulfate precipitation and centrifugation on a linear sucrose density gradient. The enzyme was homogeneous by the criterion of polyacrylamide gel electrophoresis. The molecular weight of the enzyme was 530,000, and it was composed of two types of subunit of molecular weight 53,000 and 14,000. Ribulose 1,5-diphosphate, CO(2), and Mg(2+) had Michaelis constant values of 15 mum, 0.3 mm, and 0.37 mm, respectively. At high bicarbonate concentration (17 mm and 50 mm), 6-phosphogluconate inhibited the enzyme, the inhibition being noncompetitive with respect to ribulose 1,5-diphosphate (Ki 0.065 mm), whereas at low bicarbonate concentration (1 mm), 6-phosphogluconate activated the enzyme. Oxygen was a competitive inhibitor with respect to CO(2), suggesting the enzyme also functions as an oxygenase. This was confirmed by direct assay, a 1: 1 stoichiometry between ribulose 1,5-diphosphate consumed and O(2) uptake being observed.     

Light-induced de Novo Synthesis of Ribulose 1,5-Diphosphate Carboxylase in Greening Leaves of Barley

An antibody specific for ribulose 1,5-diphosphate carboxylase was used to isolate the enzyme from greening barley (Hordeum vulgare L.) leaves. The increase in enzymatic activity during greening was due to de novo synthesis of the enzyme. Increases in enzymatic activity were accompanied by corresponding increases in enzyme protein and by incorporation of radioactive leucine, all of which were inhibited by low concentrations of cycloheximide. ¹⁴C-Labeled amino acids were incorporated into the enzyme by covalent peptide bonding.     

Ribulose Diphosphate Carboxylase Synthesis in Euglena: III. Serological Relationships of the Intact Enzyme and its Subunits

Ribulose 1,5-diphosphate carboxylase was isolated from Euglena gracilis Klebs strain Z Pringsheim, Chlorella fusca var. vacuolata, and Chlamydobotrys stellata, and the subunits from each enzyme were separated and purified by gel filtration on Sephadex G-200 in the presence of sodium dodecyl sulfate. Rabbit antibody was elicited against purified Euglena ribulose 1,5-diphosphate carboxylase whole enzyme and the isolated large and small subunits. Euglena ribulose 1,5-diphosphate carboxylase showed partial immunological identity on Ouchterlony gels with the Chlorella and Chlamydobotrys carboxylases. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates between antibody to the Euglena large subunit and the isolated large subunits of the Chlorella and Chlamydobotrys enzymes showed this was due to determinants on the large subunit. There was no serological affinity between the small subunits of the Euglena, Chlorella, and Chlamydobotrys carboxylases, and NH₂-terminal amino acid analyses provided further evidence of variability in the structure of the small subunits.     

Effect of Nitrogen Fertilizer on Photosynthesis and Ribulose 1,5-Diphosphate Carboxylase Activity in Spring Wheat in the Field

Wheat was grown in the field with different levels of nitrogenousfertilizer, and the rate of photosynthesis and the activityof ribulose 1,5-diphosphate carboxylase in the flag leaves determined.Additional nitrogen increased the dry-weight and leaf area ofthe plants, but did not increase grain yield; the rate of photosynthesisof the flag leaves was unchanged but the activity of ribulose1,5-diphosphate carboxylase increased. The significance of theseobservations to the loss of potential yield of wheat and therelationship between, photosynthesis and carboxylase activityis considered.     

Xylulose 1,5-Bisphosphate Synthesized by Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase during Catalysis Binds to Decarbamylated Enzyme

Xylulose 1,5-bisphosphate (XuBP) is synthesized from ribulose 1,5-bisphosphate (RuBP) at carbamylated catalytic sites on ribulose 1,5-bisphosphate carboxylase (Rubisco) with significant amounts of XuBP being formed at pH less than 8.0. XuBP has been separated by high performance liquid chromatography and identified by pulsed amperometry from compounds bound to Rubisco during catalysis with the purified enzyme and from celery (Apium graveolens var Utah) leaf extracts. XuBP does not bind tightly to carbamylated sites, but does bind tightly to decarbamylated sites. Upon incubation of fully activated Rubisco with 5 micromolar XuBP, loss of activator CO₂ occurred before XuBP bound to the enzyme catalytic sites, even in the presence of excess CO₂ and Mg²⁺. Binding of XuBP to decarbamylated Rubisco sites was highly pH dependent. At pH 7.0 and 7.5 with 10 millimolar MgCl₂ and 10 millimolar KHCO₃, the apparent dissociation constant for XuBP, K_d, was 0.03 micromolar, whereas at pH 8.0 and 8.5, the apparent K_d was 0.35 and 2.0 micromolar, respectively. This increase in K_d with pH was a result of a decrease in the association rate constant and an increase in the dissociation rate constant of XuBP bound to decarbamylated sites on Rubisco. The K_d of 2-carboxyarabinitol 1-phosphate binding to carbamylated sites was only slightly pH dependent.     

Loss of Ribulose 1,5-Diphosphate Carboxylase and Increase in Proteolytic Activity during Senescence of Detached Primary Barley Leaves

Symptoms typical of senescence occurred in green detached primary barley (Hordeum vulgare L.) leaves placed in darkness and in light. Chlorophyll, total soluble protein, ribulose 1,5-diphosphate carboxylase protein and activity each progressively decreased in darkness and to a lesser extent in light. In all treatments most of the total soluble protein lost was accounted for by a decrease in ribulose 1,5-diphosphate carboxylase protein, suggesting that the chloroplast was a major site of degradation early in senescence.     

Isotope Discrimination by Ribulose 1,5-Diphosphate Carboxylase: No Effect of Temperature or HCO(3) Concentration

Carbon 13 isotope discrimination by ribulose 1,5-diphosphate carboxylase from soybean (Glycine max [Merr.] cv. Amsoy) was studied as a function of temperature, bicarbonate concentration, and pH. None of these factors affected the degree of discrimination against ¹³C. The average δ¹³C was −28.3%, a value close to that found for whole C₃ plants. The zero temperature response observed here with ribulose 1,5-diphosphate carboxylase corroborates data from whole plants. The lack of effect of bicarbonate concentration on discrimination is consistent with both current theories of alternate forms of carboxylase.     

In Vitro Protein Synthesis by Plastids of Phaseolus vulgaris: V. Incorporation of C-Leucine into a Protein Fraction Containing Ribulose 1,5-Diphosphate Carboxylase

A crude chloroplast preparation of primary leaves of Phaseolus vulgaris was allowed to incorporate ¹⁴C-leucine into protein. A chloroplast extract was prepared and purified for ribulose 1,5-diphosphate carboxylase by ammonium sulfate precipitation, chromatography on Sephadex G-200, and chromatography on Sepharose 4B. The distribution of radioactive protein and enzyme in fractions eluted from Sepharose 4B was nearly the same. The radioactivity in the product was in peptide linkage, since it was digested to a trichloroacetic acid-soluble product by Pronase. Whole cells in the plastid preparation were not involved in the incorporation of amino acid into the fraction containing ribulose 1,5-diphosphate carboxylase, since incorporation still occurred after removal of cells. The incorporation into the fraction containing ribulose 1,5-diphosphate carboxylase occurs on ribosomes of plastids, since this incorporation is inhibited by chloramphenicol. These plastid preparations may be incorporating amino acid into ribulose 1,5-diphosphate carboxylase, but the results are not conclusive on this point.     

Binding of Ribulose 1,5-Bisphosphate to the Non-Catalytic Sites of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase and Its Metabolic Implications

This is the first report showing that ribulose bisphosphatecarboxylase/oxygenase has the non-catalytic sites to bind ribulosebisphosphate (RuBP). A plot of the binding number against theRuBP concentration in the equilibrium binding assay gave a bumpycurve with an intermediate plateau at 0.3 to 0.5 mM RuBP. Thebinding was saturated with 1.5 mM RuBP. The concentrations offree and binding forms of RuBP and the functioning forms ofthe enzyme in chloroplasts could be predicted using the kineticdata of the binding. (Received October 5, 1993; Accepted November 22, 1993)     

Effects of Photosynthetic Intermediates on the Activation State of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from Euglena gracilis Z

The half-saturating concentration of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from Euglena gracilis Z for CO₂ in its activation by CO₂ in the presence of a saturating concentration of MgCl₂ (KJ was measured by analyzing the partial reversible inactivation of the fully activated enzyme in the medium with dilute CO₂. The K_d of the Euglena enzyme was 12.5 μm. The K,_d values were 6.3/im for the enzyme from soybean, 10.8 fiM from maize, 23.3 jiM from Scenedesmus obliquus, and 20.8 μm from Anabaena 7120. The activated state of Euglena RuBisCO was stabilized by 6-phosphogluconate, fructose 1,6-bisphosphate, and 3-phosphoglycerate in the medium containing low concentrations of CO₂. Both fructose 6-phosphate and ATP stimulated inactivation in the medium. NADPH not only stabilized the activated state of the enzyme, but also enhanced the enzyme activity over the full activity measured in the absence of NADPH. NADP⁺ did not nullify the effects of NADPH on the activation at all. The physiological significance of the effects of these photosynthetic metabolites on the activated state of Euglena RuBisCO is discussed.     

Relative Contribution of Ribulose 1,5-Diphosphate Carboxylase and Phosphoenolpyruvate Carboxylase to CO2 Fixation Activity in Roots of Dark-grown or Light-grown Lens culinaris Seedlings

The pathway of carbon assimilation in greening roots was compared to the pathway in leaves of Lens culinaris seedlings by means of labelling distribution analysis among the products of ¹⁴CO₂ fixation in vivo, and in vitro with ribulose 1,5-diphosphate as the substrate. In green leaves, CO₂ fixation via ribulose 1,5-diphosphate carboxylase predominated largely while, in green roots, this carboxylase activity and the phosphoenolpyruvate carboxylase contributed almost equally to the whole in vivo CO₂ fixation. A participation of the activities of both carboxylases according to the double carboxylation pathway in the synthesis of dicarboxylic acids (malate and aspartate) was demonstrated in vitro after 48 h of greening in roots but seemed to be absent in in vivo experiments.     

Factors Affecting the Synthesis and Degradation of Ribulose-1,5-Diphosphate Carboxylase in Hydrogenomonas facilis and Hydrogenomonas eutropha

Hydrogenomonas facilis and H. eutropha cultured in fructose medium retained high levels of ribulose-1,5-diphosphate carboxylase only when the following conditions were fulfilled: low aeration, FeCl(3) addition to fructose medium, and cell harvest at or prior to mid-exponential phase of growth. Repression of carboxylase synthesis was demonstrated under conditions of high oxygen tension during growth of H. eutropha on fructose. Upon depletion of fructose in the growth medium, carboxylase activity fell abruptly in both organisms. The decline could not be attributed to a repressive mechanism. Rapid inactivation of carboxylase was promoted by transfer of mid-exponential-phase H. eutropha to a basal salts medium lacking fructose. During severe fructose starvation, N(2), H(2), 80% H(2) to 20% air, 2,4-dinitrophenol, actinomycin D, streptomycin, bicarbonate, and magnesium ion deficiency spared carboxylase. Nitrogen starvation or chloramphenicol afforded no protection during severe starvation. In vitro inactivation was also demonstrated in crude cell-free extracts from nonstarved, fructose-grown H. eutropha. Substrate bicarbonate protected against this loss. Inactivation of the carboxylase could not be demonstrated either by starvation of autotrophically grown cells or in autotrophic extracts. Autotrophic extracts mixed with heterotrophic extracts lost their carboxylase activity, but mixing with heterotrophic extracts that had been heated to 50 C resulted in no loss of activity. Mechanisms are proposed to accommodate these observations.     

Chlorophyll Formation in Euglena gracilis var. bacillaris: Effect of Enzyme Inhibitors

SYNOPSIS. The synthesis of chlorophylls in non-proliferating dark-grown etiolated cells of Euglena gracilis var. bacillaris is markedly inhibited by the enzyme inhibitors dinitrophenol (DNP), p -chloromercuribenzoate (pCMB) and sodium fluoride. The pCMB inhibition of greening is reversed to a great extent by glutathione but not by cysteine. The inhibitory effect of fluoride is reversed by both succinate as well as Mg⁺⁺ during illumination. Fluoride also inhibits the photosynthetic growth of the euglenas; this inhibition is competitively reversed by succinate.     

Kinetics of Accumulation of Ribulose-1,5-bisphosphate Carboxylase during Greening in Euglena gracilis: Photoregulation

The preparation of a rabbit antibody to ribulose-1,5-bisphosphate carboxylase (RuBPCase) from Euglena gracilis and its use to quantitate RuBPCase in dark- and light-grown cells and during light-induced chloroplast development (greening) are described. Light-grown Euglena have at least 36 times more RuBPCase than dark-grown Euglena. Light is required for both the initiation and continued increase in net synthesis of RuBPCase over the dark level: brief illumination 12 hours before exposure to continuous light eliminates the lags in the accumulation and increase in activity of RuBPCase (as well as in chlorophyll accumulation); net synthesis is blocked in greening cells returned to the dark or exposed to 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Streptomycin or cycloheximide prevents RuBPCase accumulation when added at the beginning of greening but only partially blocks accumulation when added after 25 hours of greening. After 24 hours of greening, the activity of RuBPCase per milligram chlorophyll continues to increase slowly while concentration of the enzyme per milligram chlorophyll remains constant. This increased activity may be due to activation of the enzyme as well as to net synthesis.     

Linearity and Functioning Forms in the Carboxylase Reaction of Spinach Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase

The reaction of spinach RuBisCO activated with CO₂ and Mg²⁺proceeded in two phases, an initial burst for a few minutesand the subsequent linear phase, in the presence of saturatingconcentrations of CO₂, ribulose 1,5-bisphosphate (RuBP), andMg²⁺. The percentage of the activity in the linear phase tothat in the initial burst was 55% with RuBisCO prepared withpolyethylene glycol, and very close to the value with the enzymereleased immediately from isolated chloro-plasts. RuBisCO preparedwith ammonium sulfate had a much larger decrease of the activityin the linear phase. The Euglena enzyme had a linear courseof reaction with time for up to 20 minutes. The K_m for CO₂ of spinach RuBisCO activated beforehand was 20µM in the initial burst, and 28 µM in the linearphase. In the carboxylase reaction initiated with inactive enzyme,the activity was initially negligible, but in 5 minutes increasedto the level observed in the linear phase of the activated enzyme.The K_m for CO₂ in the linear phase of the pre-inactivated enzymewas 70 µM. The concentration of RuBP was the immediate cause of the two-phasiccourse of the carboxylase reaction of spinach RuBisCO. The curvatureof the time course was not observed below 35 µM RuBP.The enzyme required over 88 µM RuBP for the conventionaltwo-phasic course. Further increase of the concentration ofRuBP increased the extent of the curvature, but did not startthe curvature sooner after the start of the reaction. Even ifspinach RuBisCO was in the linear phase, dilution of RuBP orits consumption by the enzymatic reaction to less than 30 µMcaused the enzyme to show the resumed biphasic reaction courseafter addition of a high concentration of RuBP. ¹This paper is the twenty-fourth in a series on PhotosyntheticCarbon Metabolism in Euglena gracilis. (Received September 19, 1988; Accepted November 25, 1988)     

Light Regulatilon of the Synthesis of Ribulose 1,5-Bisphosphate Carboxylase and Fructose 1, 6-Bisphosphatase

The activity of ribulose 1,5-bisphosphate carboxylase (RuBPCase, E. C. 4. 1. 1. 395, fructose 1,6-bisphosphatase (FBPase, E. C. 3. 1. 3. 11) and sedoheptulose 1,7-bisphosphatase (SBPase, E. C. 3. 1. 3. 37) was assayed in the etiolated cotyledons of Brassica juncea after red light or far- red light stimulation. There seemed to be a light-sensitive phase in the course of germination as indicated by the response of leaves to light. During this phase red light stimulated the synthesis of RuBPCase and FBPase, but not SBPase. This effect of red light could be reversed by farred light. Therefore, the initiation of the synthesis of the two enzymes was mediated by phytochrome. The amount of enzyme synthesized was not concerned with the number of light quanta. Phytochrome is only involved in the initiation of the synthesis of certain enzymes, but whether the synthesis will proceed continuosely ro not depends on many other factors, e. g. the availability of substrate and energy.     

Kinetics of Accumulation of Ribulose-1,5-bisphosphate Carboxylase during Greening in Euglena gracilis: Nutritional Regulation

The accumulation of ribulose-1,5-bisphosphate carboxylase (RuBPCase) in resting Euglena gracilis strain Z during greening is photo-regulated (Freyssinet, Eichholz, Buetow 1984 Plant Physiol 75: 850-857. Greening resting cells are not photosynthetically competent for about the first 24 hours in the light. Therefore, substrates for a net synthesis of the enzyme must come from endogenous constituents. During this time, degradation of endogenous paramylum (carbohydrate) reserves provides the main source of substrates. By about 24 hours of greening, resting cells are photosynthetically competent and RuBPCase accumulation becomes highly sensitive to 3-(3,4 dichlorophenyl)-1,1-dimethylurea. Therefore, from about 24 hours of greening onward, substrates (and/or energy) for RuBPCase synthesis are provided by photosynthesis. Ethanol, a nutritional substrate ordinarily used constitutively by Euglena for growth, inhibits RuBPCase accumulation when added to the resting medium in the light. The alcohol exerts this negative regulatory effect by limiting the availability of substrates needed for a net synthesis of the enzyme.     

Protein Degradation in Lemna with Particular Reference to Ribulose Bisphosphate Carboxylase: II. The Effect of Nutrient Starvation

The concept of ribulose bisphosphate carboxylase as a storage protein is not supported in the case of Lemna minor, where the enzyme appears to be particularly stable under conditions of nitrogen starvation. Total nutrient starvation in light and in the dark induced the degradation of this enzyme, but not at an enhanced rate compared with other leaf proteins and, surprisingly, darkness inhibited the degradation of chlorophyll which occurs with total nutrient starvation in the light. The data suggest that Lemna is not programmed to senesce in response to nutrient starvation. Differences in the pattern of protein degradation, which occurred under the stress conditions employed, are not consistent with a simple model of protein degradation in which the degradative system is assumed to be located in the vacuole. The data is best explained by a dual system in which cytosolic proteins are degraded by a vacuolar/lysosomal system and chloroplast proteins are degraded within the chloroplast. Whatever the system of degradation, our data do not support the proposed correlation between the rate of protein degradation and either protein charge or size.