The glycolate pathway and photosynthetic competence in euglena

The development of glycolate pathway enzymes has been determined in relation to photosynthetic competence during the regreening of Euglena cultures. Phosphoglycolate phosphatase and glycolate dehydrogenase rapidly reached maximal levels of activity but the complete development of ribulose 1,5-diphosphate carboxylase and concomitant photosynthetic carbon dioxide fixation were not attained until 72 hours of illumination. Specific inhibitors of protein synthesis showed that the formation of ribulose 1,5-diphosphate carboxylase in both division-synchronized and regreening cultures was prevented by both cycloheximide and d-threo-chloramphenicol, whereas phosphoglycolate phosphatase formation was only inhibited by d-threo-chloramphenicol but not by l-threo-chloramphenicol or cycloheximide. Since cycloheximide prevented ribulose diphosphate carboxylase synthesis and photosynthetic carbon dioxide fixation without affecting phosphoglycolate phosphatase synthesis during regreening, it was concluded that photosynthetic competence was not necessary for the development of the glycolate pathway enzymes. The inhibition of phosphoglycolate phosphatase synthesis by d-threo-chloramphenicol but not by l-threo-chloramphenicol or cycloheximide shows that the enzyme was synthesized exclusively on chloroplast ribosomes, whereas protein synthesis on both chloroplast and cytoplasmic ribosomes was required for the formation of ribulose 1,5-diphosphate carboxylase. Although light is required for the development of both Calvin cycle and glycolate pathway enzymes during regreening it is concluded that the two pathways are not coordinately regulated.     

Evidence for in vivo Light-induced Synthesis of Ribulose-1,5-diP Carboxylase and Phosphoribulokinase in Greening Barley Leaves

When actinomycin D, puromycin, streptomycin, chloramphenicol, and cycloheximide, known inhibitors of protein synthesis, were applied to leaves of intact seedlings or detached leaves of barley prior to their greening, the same general response resulted: the light-induced increase in activity of ribulose 1,5-diphosphate carboxylase was prevented while that of phosphoribulokinase was only partially suppressed; synthesis of chlorophyll was arrested. This is taken as preliminary evidence that de novo synthesis of protein may be responsible for the observed increase in ribulose-1,5-diphosphate carboxylase activity during greening. However, other factors may be involved with the light-induced stimulation of phosphoribulokinase.

Carbohydrate metabolites and substrates of the enzymes failed to induce the formation of ribulose-1,5-diphosphate carboxylase and phosphoribulokinase in the dark. No evidence was found for the presence of inhibitors in etiolated seedlings or activators in illuminated leaves of barley. Carboxylase activity almost equal to that of the illuminated water control was stimulated by MgCl₂ in the dark; MgCl₂ had no effect on the activity of the kinase.

     

Evidence for lack of turnover of ribulose 1,5-diphosphate carboxylase in barley leaves

Turnover of ribulose 1,5-diphosphate carboxylase in barley leaves (Hordeum vulgare L.) was followed over time in light and dark. The enzyme was degraded in prolonged darkness and was resynthesized after the plants were returned to light. Labeling with ¹⁴C showed that simultaneous synthesis and degradation (turnover) did not occur in light. In contrast, the remaining soluble protein was turned over rapidly in light. Although ribulose 1,5-diP carboxylase can be both degraded and synthesized, these processes seem not to occur simultaneously, but can be induced independently by changing environmental conditions.     

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.     

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.     

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.     

Ribulose 1,5-bisphosphate and activation of the carboxylase in the chloroplast

Ribulose 1,5-bisphosphate in the chloroplast has been suggested to regulate the activity of the ribulose bisphosphate carboxylase/oxygenase. To generate high levels of ribulose bisphosphate, isolated and intact spinach chloroplasts were illuminated in the absence of CO₂. Under these conditions, chloroplasts generate internally up to 300 nanomoles ribulose 1,5-bisphosphate per milligram chlorophyll if O₂ is also absent. This is equivalent to 12 millimolar ribulose bisphosphate, while the enzyme, ribulose bisphosphate carboxylase, offers up to 3.0 millimolar binding sites for the bisphosphate in the chloroplast stroma. During illumination, the ribulose bisphosphate carboxylase is deactivated, due mostly to the absence of CO₂ required for activation. The rate of deactivation of the ribulose bisphosphate carboxylase was not affected by the chloroplast ribulose bisphosphate levels. Upon addition of CO₂, the carboxylase in the chloroplast was completely reactivated. Of interest, addition of 3-phosphoglycerate stopped deactivation of the carboxylase in the chloroplast while ribulose bisphosphate accumulated. With intact chloroplasts in light, no correlation between deactivation of the carboxylase and ribulose bisphosphate levels could be shown.     

Derepression of the synthesis of D-ribulose 1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum.

The synthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase in Rhodospirillum rubrum was greatly influenced by the conditions of culture. When grown photolithotrophically in an atmosphere containing low levels of CO2 (1.5 to 2%), enzyme synthesis was derepressed, with the result that the enzyme comprised up to 50% of the soluble protein of the cells as determined by immunological quantitation. This response was not observed when R. rubrum was grown photolithotrophically in an atmosphere of 5% CO2 in hydrogen. Similarly, the derepression of ribulose 1,5-bisphosphate carboxylase/oxygenase was observed in photoheterotrophically (butyrate)-grown cultures only after the HCO3- supply was nearly exhausted. The increase in enzyme activity observed in derepressed cultures was not paralleled by an increase in the in vivo CO2 fixation rate. Apparently, R. rubrum derepresses the synthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase when exposed to low CO2 concentrations to scavenge the limited CO2 available to such cultures.     

Localization and properties of ribulose diphosphate carboxylase from castor bean endosperm

A substantial portion of the ribulose 1,5-diphosphate carboxylase activity in the endosperm of germinating castor beans (Ricinus communis var. Hale) is recovered in the proplastid fraction. The partially purified enzyme shows homology with the enzyme from spinach (Spinacia oleracea) leaves, as evidenced by its reaction against antibodies to the native spinach enzyme and to its catalytic subunit. The enzyme from the endosperm of castor beans has a molecular weight of about 500,000 and, with the exception of a higher affinity for ribulose 1,5-diphosphate, has similar kinetic properties to the spinach enzyme. The castor bean carboxylase is inhibited by oxygen and also displays ribulose 1,5-diphosphate oxygenase activity with an optimum at pH 7.5.     

Inhibition of ribulose 1,5-diphosphate carboxylase by 6-phosphogluconate

6-Phosphogluconate is a much more effective inhibitor of the photosynthetic carboxylation enzyme, ribulose-1, 5-diphosphate carboxylase, than other sugar phosphates and sugar acids of the reductive and oxidative pentose phosphate cycles. The inhibition appears to be noncompetitive with ribulose 1,5-diphosphate. Since 6-phosphogluconate is unique to the oxidative cycle and inhibits at concentrations comparable to those found in vivo, it is proposed that its inhibition of the carboxylase may be a regulatory factor. If so, it would operate during darkness as a different control factor from those factors postulated to activate the carboxylase during photosynthesis.     

Enzyme levels in relation to obligate phototrophy in chlamydobotrys

During the transition from photoheterotrophic growth on acetate to phototrophic growth on carbon dioxide, there is a decrease in isocitrate lyase and increase in ribulose-1,5-diphosphate carboxylase activity in Chlamydobotrys stellata cultures. The increase in ribulose-1,5-diphosphate carboxylase activity is the result of protein synthesis, there being a close correlation between increase in enzyme activity and protein precipitated by antibody to ribulose-1,5-diphosphate carboxylase. The purified ribulose-1,5-diphosphate carboxylase was similar to the constitutive enzyme from other green algae having a molecular weight of 530,000 and composed of two types of subunit of molecular weight 53,000 and 14,000.     

Ribulose diphosphate carboxylase synthesis in euglena: increased enzyme activity after transferring regreening cells to darkness

The transfer of dark-grown cultures of Euglena gracilis Klebs strain Z regreening in the light back into darkness resulted in a dramatic increase in ribulose diphosphate carboxylase activity. On a culture volume basis activity increased 4-fold over a 24-hour dark period, although on a protein basis activity declined because of rapid cell division. Mixed assays with light- and dark-growing cell extracts provided no evidence for the removal of an inhibitor of ribulose diphosphate carboxylase upon transferring regreening cells back to darkness. Although ribulose diphosphate carboxylase activity increased over a 24-hour dark period, there was no concomitant increase in the potential of the cells for photosynthetic carbon dioxide fixation.     

Development of ribulose-1,5-diphosphate carboxylase in castor bean cotyledons

Light was not essential for the development of ribulose-1,5-diphosphate carboxylase protein or catalytic activity in the photosynthetic cotyledons of germinating castor beans (Ricinus communis). Cotyledons developing in the dark showed higher activity than those in the light. Returning cotyledons developing in the light to darkness resulted in a significant increase in ribulose-1,5-diphosphate carboxylase activity compared to cotyledons in continuous light.     

Activation and inhibition of ribulose 1,5-diphosphate carboxylase by 6-phosphogluconate

Ribulose 1,5-diphosphate carboxylase, when activated by preincubation with 1 mm bicarbonate and 10 mm MgCl₂ in the absence of ribulose 1,5-diphosphate, remains activated for 20 minutes or longer after reaction is initiated by addition of ribulose diphosphate. If as little as 50 μm 6-phosphogluconate is added during this preincubation period, 5 minutes before the start of the reaction, a further 188% activation is observed. However, addition of 6-phosphogluconate at the same time or later than addition of ribulose diphosphate, or at any time with 50 mm bicarbonate, gives inhibition of the enzyme activity. Possible relevance of these effects in vivo regulatory effects is discussed.     

pH Dependence of the Km(CO(2)) of Ribulose 1,5-Diphosphate Carboxylase

The Km(CO(2)) values of ribulose 1,5-diphosphate carboxylase in freshly ruptured spinach (Spinacia oleracea L.) chloroplasts and in the purified form isolated from spinach leaves were found to be pH dependent. Raising the pH of the assay solution produced a substantial decrease in the Km(CO(2)) of both enzyme systems. In freshly ruptured chloroplasts at pH 7.2 the Km(CO(2)) was 25 mum, at pH 8 it decreased to 19 mum, and at pH 8.8 a further decrease to 7 mum was found. With the purified enzyme at pH 7.2 the Km(CO(2)) was 147 mum, while the corresponding Km values for pH 8 and 8.8 were 34 and 15 mum CO(2), respectively. The latter figure approximates the physiological Km(CO(2)) of 10 mum estimated for photosynthesizing leaves and intact chloroplasts. The maximum velocity for both enzyme systems at optimum substrate levels was at pH 8, but the highest calculated rate of CO(2) uptake at atmospheric CO(2) levels occurred at pH 8.8. These results support the proposal that the light-induced efflux of protons out of the chloroplast stroma may be a major factor involved with the reported in vivo light activation of ribulose 1,5-diphosphate carboxylase.     

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.     

Influence of Inorganic Phosphate on Photosynthesis of Wheat Chloroplasts: II. RIBULOSE BISPHOSPHATE CARBOXYLASE ACTIVITY

Isolated wheat chloroplasts were pre-incubated in the dark inthe presence of various concentrations of inorganic phosphatewith or without carbon dioxide, oxaloacetate, glycerate, and3-phosphoglycerate. The effect of subsequent illumination onphotosynthetic oxygen evolution, ribulose bisphosphate carboxylaseactivity, ATP content, and ribulose bisphosphate content wasinvestigated. Inorganic phosphate had little effect on ribulosebisphosphate carboxylase activity in darkness or during theinitial phase of illumination, but it prevented the declinein activity that occurred during later stages of illumination,when photoreduction of CO₂ was decreasing in rate. Additionof inorganic phosphate to chloroplasts illuminated without phosphaterestored the ribulose bisphosphate carboxylase activity, increasedthe ATP, and decreased the ribulose bisphosphate in the organelles.The responses to CO₂, oxaloacetate, glycerate, and 3-phosphoglyceratesuggest that the decreased activity of ribulose bisphosphatecarboxylase during photosynthesis results from ATP consumption. Purified ribulose bisphosphate carboxylase was activated byinorganic phosphate, but this activation did not occur in thepresence of ATP. ATP inhibited ribulose bisphosphate carboxylasewhen it was present in combination with various photosyntheticmetabolites. Inactivation of ribulose bisphosphate carboxylase in chloroplasts,illuminated in the absence of inorganic phosphate, is not dueto lack of activation by inorganic phosphate or ATP. It mayresult from decreased stromal pH. Key words: Ribulose bisphosphate carboxylase, Chloroplasts, Wheat, Phosphate, ATP     

Purification, some properties and quaternary structure of the D-ribulose 1,5-diphosphate carboxylase of Alcaligenes eutrophus.

D-Ribulose 1,5-diphosphate carboxylase has been purified from autotrophically grown cells of the facultative chemolithotrophic hydrogen bacterium Alcaligenes eutrophus. The enzyme was homogeneous by the criteria of polyacrylamide gel electrophoresis. The molecular weight of the enzyme was 505000 determined by gel filtration and sucrose density gradient centrifugation, and a sedimentation coefficient of 18.2 S was obtained. It was demonstrated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis that the enzyme consists of two types of subunits of molecular weight 52000 and 13000. Electron microscopy on the intact and the partially dissociated enzyme lead to the construction of a model for the quaternary structure of the enzyme which is composed of 8 large and 8 small subunits. The most probable symmetry of the enzyme molecule is 4:2:2. Michaelis constant (Km) values for ribulose 1,5-diphosphate, Mg2+, and CO2 were 0.59 mM, 0.33 mM, and 0.066 mM measured under air. Oxygen was a competitive inhibitor with respect to CO2 suggesting that the enzyme also exhibits an oxygenase activity. The oxygenolytic cleavage of ribulose 1,5-diphosphate was shown and a 1:1 stoichiometry between oxygen consumption and 3-phosphoglycerate formation observed.     

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.     

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.