d-Ribulose 1,5-diphosphate carboxylase from the blue-green alga Aphanocapsa 6308

d-Ribulose 1,5-diphosphate carboxylase from extracts of the unicellular blue-green alga Aphanocapsa 6308 has been purified by ammonium sulphate precipitation and linear sucrose density gradient centrifugation. The molecular weight was estimated to be 525 000 and the enzyme consisted of two types of sub-unit of molecular weights 51 000 and 15 000. The small sub-units were not detected after purification involving acid precipitation but were observed if the acid precipitation step was omitted. The Michaelis constants for Mg²⁺ and CO₂, when tested under air, were 0.35 mM and 0.071 mM respectively. Oxygen acted as a competitive inhibitor with respect to CO₂, suggesting that the enzyme also acts as an oxygenase. This was confirmed by measuring ribulose diphosphate-dependent O₂ uptake. A 1:1 stoichiometry between ribulose diphosphate utilization and O₂ consumption was observed. 6-Phosphogluconate inhibited carboxylase activity both at high (20 mM) and low (1 mM) bicarbonate concentrations. The data are compared with the properties of ribulose diphosphate carboxylase from other autotrophic prokaryotes and from chloroplasts.Abbreviations RuDP d-Ribulose 1,5-diphosphate - EDTA ethylene diamine tetraacetic acid - GSH reduced glutathione - SDS sodium dodecyl sulphate - 6PGluc 6-phosphogluconate - STB supplemented Tris buffer     

Purification,some properties and quaternary structure of thed-ribulose 1,5-diphosphate carboxylase ofAlcaligenes eutrophus

d-Ribulose 1,5-diphosphate carboxylase has been purified from autotrophically grown cells of the facultative chemolithotrophic hydrogen bacteriumAlcaligenes 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 (K _m ) values for ribulose 1,5-diphosphate, Mg^2-, 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.Abbreviations DTE dithioerythritol - EDTA ethylenediamine tetraacetate - RuDP d-ribulose 1,5-diphosphate     

Carboxydismutase activity in plants with and without β-carboxylation photosynthesis

Summary A comparison was made of the activity of carboxydismutase (ribulose-1,5-diphosphate carboxylase) between higher plant species which possess the -carboxylation (C₄-dicarboxylic acid) pathway for photosynthesis and species which lack this pathway. Contrary to earlier findings no marked difference in the level of this enzyme was found between the two groups of species. Chloroplast-containing vascular-bundle-sheath cells which seem to be present only in plants with -carboxylation apparently contain relatively high carboxydismutase activity.C.I.W.-D.P.B. Publ. No. 453.     

Carbonic anhydrase activity of Prochloron sp. isolated from an ascidian host

The prokaryotic algal symbiont of ascidians, Prochloron sp., was found to exhibit carbonic anhydrase activity which is largely associated with the cell surface. This extracellular carbonic anhydrase activity was inhibited, while the intracellular activity was not affected, by chloride or bromide. Acetazolamide and ethoxyzolamide inhibited carbonic anhydrase activity with I₅₀ values of 7×10^-4 and 3×10^-4M, respectively. These I₅₀ values are similar to those observed for intracellular carbonic anhydrases of Synechococcus sp. PCC7942, Chlamydomonas reinhardii and spinach.Abbreviations AZA acetazolamide - CA carbonic anhydrase - chl chlorophyll - EZA ethozyzolamide - I₅₀ concentration of an inhibitor required to cause 50% inhibition - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - U unit     

The effect of SO 3 -- on the activity of ribulose-1,5-diphosphate carboxylase in isolated spinach chloroplasts

Summary SO ₃ ^-- inhibits the activity of ribulose-1,5-diphosphate carboxylase in isolated spinach chloroplasts. It shows a non-competitive inhibition pattern with respect to ribulose-1,5-diphosphate and Mg⁺⁺ but a competitive one with respect to HCO ₃ ^- . The K _i -values are 14 mM SO ₃ ^-- and 9.5 mM SO ₃ ^- respectively for the non-competitive inhibition but only 3.0 mM SO ₃ ^-- in the case of competitive inhibition with HCO ₃ ^-- as a substrate. Thus it is concluded that the competitive inhibition type will predominate at low SO ₃ ^-- and low internal CO₂ concentrations.The abbreviations used RuDph ribulose-1,5-diphosphate - DTT dithiothreitol - EDTA ethylenediaminetetraacetate     

CHLOROPLAST STRUCTURE AND FUNCTION IN ac-20, A MUTANT STRAIN OF CHLAMYDOMONAS REINHARDI : I. CO2 Fixation and Ribulose-1,5-Diphosphate Carboxylase Synthesis

A mutant strain of the green alga Chlamydomonas reinhardi, ac-20, is described in which both the rate of CO₂ fixation by whole cells and the rate of carboxylation of ribulose-1,5-diphosphate in cell-free extracts are reduced, particularly when sodium acetate is present in the growth medium. Of the enzymes of the reductive pentose phosphate cycle tested, only ribulose-1,5-diphosphate carboxylase activity is reduced in the mutant strain, and it appears that the low carboxylase activity limits the strain's rate of photosynthetic carbon metabolism. Evidence is presented to show that the fluctuation in the level of the enzyme activity in the presence or absence of acetate results from the fluctuation in the level of some factor(s) limiting the rate of synthesis of the protein.     

Chlorophyll Accumulation, CO2 Fixation Enzymes, and Hill Activity, in Greening Roots of Lens culinaris

The carboxylation of ribulose-1,5-diphosphate was demonstrated in vitro with extracts of ctiolated seedling roots. The presence of ribulose-1,5-diphosphate carboxylase was characterized in the subcellular fraction enriched in amyloplasts. Synthesis of chlorophyll, development of CO² fixation capacities and of Hill activity upon illumination have been studied with roots of Lens culinaris seedlings. The marked increases in CO² fixation with ribulose-1,5-diphosphate as the substrate and in Hill activity that occur after a lag phase seem to be related to cytological changes during the greening of roots.     

Carbon dioxide fixation in Pyrobotrys stellata

The green alga Pyrobotrys stellata Korshik., an obligate phototroph, is unable to utilise carbon dioxide for growth, although assimilation of acetate is dependent on the photosynthetic process. The incorporation of ¹⁴CO₂ from ¹⁴C-bicarbonate into the cells of P. stellata is only 3% of that in Chlorella pyrenoidosa Chick. The activity of the key enzyme of the Calvin cycle, ribulose-1-5-diphosphate carboxylase, is very low in P. stellata, being only 7% of that in C. pyrenoidosa. The determination of the products of ¹⁴CO₂ fixation in intact cells confirms that ribulose-1-5-diphosphate activity is very low in P. stellata, since little carbon-14 is found in 1–3 diphosphoglyceric acid, the product of carboxylation of ribulose-1-5-diphosphate. It is concluded that the inability of P. stellata to utilize carbon dioxide for growth in the light is probably the result of the low ribulose-1-5-diphosphate carboxylase activity in the organism.     

THE PATH OF CARBON IN PHOTOSYNTHESIS BY MARINE PHYTOPLANKTON12

Using cultures of a number of different marine algae (diatoms Skeletonema costatum (Grev.) Cleve and Phaeodactylum tricornutum Bohlin, chrysophyte Isochrysis galbana Parke, green flagellate Dunaliella tertiolecta Butcher, dinoflagellate Gonyaulax tamarensis Lebour) the short-term, pattern of ¹⁴CO₂ assimilation has been investigated. In all except D. tertiolecta the labelling of amino acids and intermediates of the tricarboxylic acid (Krebs) cycle was significantly heavier than that of sugar phosphates. Over periods of 30–120 s labelling in amino acids and Krebs cycle intermediates accounted for 41–95% of the ¹⁴C fixed (depending on the alga). Over shorter times (< 10 s) the pattern in the 2 diatoms showed significant labelling of C₄ acids (and related com-pounds) and little labelling of sugar phosphates. The reverse wits seen with D. tertiolecta. Also, in the 2 diatoms and in G. tamarensis significant inhibition of photosynthesis by oxygen could only be achieved with 100% oxygen; atmospheric levels having little effect. Parallel measurements of 2 carboxylating enzymes showed that ribulose-1,5-diphosphate carboxylase (RuDPCase) was significantly greater than phospho (enol)pyruvate carboxylase (PEPCase) activity only in the green flagellate. It is suggested that photosynthesis in marine diatoms depends on an active PEPCase utilizing bicarbonate as a substrate and that a less active RuDPCase utilizes CO₂. In D. tertiolecta the pattern more closely resembles that of a “Calvin (C₃)” plant. The dinoflagellate and the chrysophyte appeared to show a mixed C₃ and C₄ photosynthesis.     

PURIFICATION OF RIBULOSE 1,5-BISPHOSPHATE CARBOXYLASE AND CARBON ISOTOPE FRACTIONATION BY WHOLE CELLS AND CARBOXYLASE FROM CYLINDROTHECA SP. (BACILLARIOPHYCEAE.)1,2,3

Ribulose 1,5-biphosphate carboxylase has been purified to homogeneity from extracts of Cylindrotheca sp. (strain N-1), a marine, pennate diatom. The carboxylase has a molecular weight and structural composition similar to the enzyme from higher plants. When assayed in the presence of 1 mM NaHCO₃ the enzyme was stimulated nearly 40% by 1 mM aspartate and over 20% by 1 mM malate, and was inhibited to over 60% by 1 mM phosphoenolpyruvate. Similar experiments, using spinach carboxylase, failed to show activation by these metabolites. When assayed in the presence of 20 mM NaHCO₃, 6-phosphogluconate (1 mM) inhibited activity of ribulose bisphosphate carboxylase from Cylindrotheca by 60%, and higher concentrations of maiate (10 mM) inhibited activity by 25% Carbon isotope fractionation by ribulose bisphosphate carboxylase was -32.6% (ppt) when measured under N₂ using homogeneous enzyme, whereas maximum carbon isotope fractionation by the whole alga grown in 1% -C0₂-in air averaged - 16.8%. Carbon isotope fractionation by the whole alga varied with the density of the culture and was maximum at a low cell density (1.7 ± 10⁶ cellslml). At higher densities, the fractionation decreased by 4.0%. Carbon isotope fractionation has been used previously to determine the pathway of carbon metabolism in other organisms; the results of this investigation seem to indicate that this strain uses both the reductive pentose phosphate pathway and the C₄ carbon pathway for primary CO₂ fixation.     

Experimental Studies on the Physiology of the Phycobiont and Mycobiont Ramalina ecklonii By

The lichenized fungus and alga of the fruticose lichen Ramalini ecklonii were isolated into pure cultures. The ascospores of the fungus failed to germinate in less than five weeks incubation in spite of the use of a variety of cultural conditions. The fungus showed a considerable increase in growth on malt extract agar. Both organisms showed a marked tolerance for high concentrations of glucose although growth was quantitatively reduced. The fungus was able to use a variety of carbon and nitrogen sources as well as an extract of algal cells. Cultivation in the absence of biotin and thiamine failed to yield significant amounts of growth. The alga yielded 27 mg of dry weight after three weeks in a synthetic medium under low light intensities. The alga could be grown in satisfactory amounts on CO₂ and inorganic salts with moderate light intensities. Experiments using ¹⁴CO₂ showed the fungus able to incorporate the extra-cellular and intra-cellular products of algal metabolism. The rate of incorporation of extra-cellular products was inhibited by high concentrations of biotin and thiamine. The alga assimilated ^l4CO₂ which was retained by the cells over a period of 14 days, at which time 78 per cent of the activity was insoluble in 80 per cent ethanol. An extract of the fungus labelled with ¹⁴C glucose was partially taken up by the alga and 50 per cent of the label was insoluble in 80 per cent after three days incubation in the light. No lichen acids were found in either the fungal cultures or the algal cultures although large amounts (e.g. 2 liters) of material were extracted and chromatographed. Usnic acid was produced by the intact lichen thallus.     

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.     

Properties of Ribulose-1,5-Diphosphate Carboxylase (Carboxydismutase) From Chinese Cabbage and Photosynthetic Microorganisms

Ribulose-1,5-diphosphate carboxylase (carboxydismutase) was prepared from Chinese Cabbage [Brassica petsai (Parl)] and the K(m) values and molecular weight were determined. These parameters were found to be in good agreement with values reported for this enzyme from other higher plants. Investigation of carboxydismutase activity from the photosynthetic micro-organisms Chlamydomonas reinhardi (IU 89+), Plectonema boryanum (IU 594), and Chromatium strain D showed striking similarity to the higher plant enzyme, when the sedimentation coefficients were compared.     

Regulation of glucose-6-phosphate dehydrogenase in spinach chloroplasts by ribulose 1,5-diphosphate and NADPH/NADP+ ratios

The activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from spinach chloroplasts is strongly regulated by the ratio of NADPH/NADP⁺, with the extent of this regulation controlled by the concentration of ribulose 1,5-diphosphate. Other metabolites of the reductive pentose phosphate cycle are far less effective in mediating the regulation of the enzyme activity by NADPH/NADP⁺ ratio. With a ratio of NADPH/NADP⁺ of 2, and a concentration of ribulose 1,5-diphosphate of 0.6 mM, the activity of the enzyme is completely inhibited.This level of ribulose 1,5-diphosphate is well within the concentration range which has been reported for unicellular green algae photosynthesizing in vivo. Ratios of NADPH/NADP⁺ of 2.0 have been measured for isolated spinach chloroplasts in the light and under physiological conditions.Since ribulose 1,5-diphosphate is a metabolite unique to the reductive pentose phosphate cycle and inhibits glucose-6-phosphate dehydrogenase in the presence of NADPH/NADP⁺ ratios found in chloroplasts in the light, it is proposed that regulation of the oxidative pentose phosphate cycle is accomplished in vivo by the levels of ribulose 1,5-diphosphate, NADPH, and NADP⁺.It already has been shown that several key reactions of the reductive pentose phosphate cycle in chloroplasts are regulated by levels of NADPH/NADP⁺ or other electron-carrying cofactors, and at least one key-regulated step, the carboxylation reaction is strongly affected by 6-phosphogluconate, the metabolite unique to the oxidative pentose phosphate cycle. Thus there is an interesting inverse regulation system in chloroplasts, in which reduced/oxidized coenzymes provide a general regulatory mechanism. The reductive cycle is activated at high NADPH/NADP⁺ ratios where the oxidative cycle is inhibited, and ribulose 1,5-diphosphate and 6-phosphogluconate provide further control of the cycles, each regulating the cycle in which it is not a metabolite.     

Characterization of a symbiotic,heterocystous, N2-fixing cyanobacterium fromCycas coralloid roots

A symbiotic, heterocystous, N₂-fixing blue-green alga, isolated from the coralloid roots of a xerophytic plant,Cycas revoluta, grew best in liquid medium supplemented with 4 mM NO ₃ ^– . Morphologically, the isolated alga was identical to that of the natural endophyte but the cell size had decreased markedly. The alga was heterotrophic. Intact coralloid roots had nearly 4 to 5 times more nitrogenase activity compared with natural- and laboratory-grown agla but nitrate reductase was inducible in both the forms. Plasmid(s) were found in both algal forms.     

Some properties of fructose 1,6-diphosphatase of rat liver and their relation to the control gluconeogenesis

1. Fructose 1,6-diphosphatase has been purified tenfold from rat liver. The final preparation was not contaminated by either glucose 6-phosphatase or phosphofructokinase. The properties of the enzyme have been investigated in an attempt to define factors that could be of revelance to metabolic control of fructose 1,6-diphosphatase activity. 2. The metal ions Fe²⁺, Fe³⁺ and Zn²⁺ inhibited the activity of fructose 1,6-diphosphatase even in the presence of an excess of mercaptoethanol; other metal ions tested had no effect. The inhibition produced by Zn²⁺ was reversed by EDTA, but that produced by either Fe²⁺ or Fe³⁺ was not reversible. 4. The enzyme has a very low K_m for fructose 1,6-diphosphate (2·0μm). Concentrations of fructose 1,6-diphosphate above 75μm inhibited the activity; however, even at very high fructose 1,6-diphosphate concentrations only 70% inhibition was obtained. 5. The activity was also inhibited by low concentrations of AMP, which lowered V_max. and increased K_m for fructose 1,6-diphosphate. Evidence is presented that suggests that AMP can be defined as an allosteric inhibitor of fructose 1,6-diphosphatase. 6. The inhibitions by both fructose 1,6-diphosphate and AMP were extremely specific. Also, the degree of inhibition was not affected by the presence of intermediates of glycolysis, of the tricarboxylic acid cycle, of amino acid metabolism or of fatty acid metabolism. 7. It is suggested that the intracellular concentrations of AMP and fructose 1,6-diphosphate could be of significance in controlling the activity of fructose 1,6-diphosphatase in the liver cell. The possible relationship between these intermediates and the control of gluconeogenesis is discussed.     

Ribulose diphosphate oxygenase. V. Presence in ribulose diphosphate carboxylase from Rhodospirillum rubrum

Ribulose-1,5-diphosphate carboxylase was purified fifteenfold from Rhodospirillum rubrum grown autotrophically under H₂ and CO₂. There was RuDP oxygenase activity associated with the carboxylase. The oxygenase had maximal activity at pH 9.4. Although these bacterial RuDP oxygenase and carboxylase activities were cold labile, activity could not be restored by treatment at 50° in the presence of Mg⁺⁺ and a sulfhydryl reagent, in contrast to results with the enzyme from eukaryotes.     

EFFECTS OF HYDROXAMATE SIDEROPHORES (STRONG Fe (III) CHELATORS) ON THE GROWTH OF ALGAE1

Varying concentrations of Fe were tested with three hydroxamate siderophores to demonstrate the interactions affecting growth of Chlamydomonas reinhardtii and Chlorella vulgaris. Schizokinen was purified from the excretions of the blue-green alga Anabaena sp. grown in low-Fe medium. Chlamydomonas reinhardtii was inhibited by schizokinen when in molar excess of the Fe concentration; the inhibition was overcome with excess Fe. The growth of C. vulgaris was not affected by this chelator. Results with desferrioxamine were similar. A weaker chelator, rhodorulic acid, did not inhibit the growth of either alga. Low concentrations of the chelators may stimulate algal growth when Fe precipitates are hydrolyzed. Since different algae respond differently to the presence of the chelators, the observed interactions could be important in determining competitive relationships when Fe is limiting. If an alga can excrete a strong chelating agent, as does Anabaena, algae lacking the ability to compete with the chelator may not grow.     

PURIFICATION AND CHARACTERIZATION OF GLYCOLATE OXIDASE FROM THE BROWN ALGA SPATOGLOSSUM PACIFICISM (PHAEOPHYTA)1

Glycolate oxidase was purified to apparent homogeneity from the brown alga Spatoglossum pacificum Yendo. The 1326-fold purified glycolate oxidase enzyme exhibited a specific activity of 22. 4 micromoles glyoxylate formed ·min^?1·mg protein^?1. The molecular weight of the native enzyme was estimated to be 230,000 by gel filtration. The subunit molecular weight of the enzyme was determined to be 49,000 by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, suggesting that the native enzyme is a tetramer. There were two absorption peaks at 345 and 445 nm, indicating that glycolate oxidase is a flavoprotein. This enzyme had a high isoelectric point (pI 9.6) and a high pH optimum (pH 8.3). The K_m values for glycolate and l -lactate were 0.49 and 5.5 mM, respectively. This enzyme also had a broad specificity for other straight-chain α-hydroxy acids but not for β-hydroxyacids. Cyanide, azide, N-ethylmaleimide, and p-chloromercuribenzoic acid did not affect the enzyme, whereas 2-pyridylhydroxymethanesulfonic acid strongly inhibited it. These properties of glycolate oxidase from the brown alga S. pacificum are similar to the properties of the glycolate oxidasesfrom higher plants. Polyclonal antibodies raised against the polypeptide fragment of Spatoglossum glycolate oxidase could recognize glycolate oxidase from Spinacia oleracea L., although the cross-reactivity was weak. The N-terminal sequence of two internal polypeptide fragments of the enzyme from S. pacificum showed a high degree of similarity to that of glycolate oxidase from higher plants. These results suggest that glycolate oxidase from higher plants and brown algae share the same ancestral protein.