Isolation, characterization, and crystallization of ribulosebisphosphate carboxylase from autotrophically grown Rhodospirillum rubrum

Schloss, John V.; Phares, E.F.; Long, Mary V.; Norton, I.L.; Stringer, Claude D.; Hartman, Fred C.

doi:10.1128/jb.137.1.490-501.1979

Cited by 78 publications

(30 citation statements)

References 73 publications

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“…Furthermore, this increased activity is paralleled by an increase in the amount of Rbu-P2 carboxylase/oxygenase actually synthesized by the organism, with this enzyme produced at a level which represents up to 50% of the soluble protein under conditions of carbon dioxide deprivation. We further show that the adaptation occurs within the first transfer of heterotrophically grown cells to photolithotrophic growth conditions and not after 10 to 14 weeks of growth and transfer as previously reported (12). Such a response to nutrient (carbon dioxide) limitation is by no means unprecedented since various degrees of derepression of Rbu-P2 carboxylase/oxygenase synthesis have been observed in the hydrogen-oxidizing bacterium Alcaligenes eutrophus (7), the obligate chemolithotroph Thiobacillus neapolitanus (3), the cyanobacterium Anacystis nidulans (8), and the facultative autotroph Pseudomonas oxalaticus OXI (6).…”

Section: Discussionsupporting

confidence: 84%

“…Recently, it was reported that levels of Rbu-P2 carboxylase/oxygenase representing up to 40o of the soluble protein of R. rubrum may be obtained upon repeated transfer (10 to 14 weeks) of cells grown under a C02/H2 atmosphere (12). Thus, the levels of Rbu-P2 carboxylase/oxygenase obtained by these investigators in R. rubrum are similar to the levels found in the chloroplasts of eucaryotic cells.…”

supporting

confidence: 55%

“…Schloss et al, with a 50-liter culture, obtained about 50 U/g (dry weight) of cells in one experiment with a 3.75% inoculum and less than 10 U/g (dry weight) of cells with a 10% inoculum after transfehlng malate-grown cells to the 2% Cm2-H2 atmosphere (12). Certainly, we have not observed the rather lengthy adaptation pedaodof 14 weeks previously reported (12). In a separate experiment, we varied the bubbling rate of a culture grown in a 2% C02-H2 atmosphere.…”

Section: Resultsmentioning

confidence: 99%

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

Sarles¹,

Tabita²

1983

J Bacteriol

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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%6 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.

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Section: Discussionsupporting

confidence: 84%

supporting

confidence: 55%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

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

Sarles¹,

Tabita²

1983

J Bacteriol

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“…Storage of extracts at -80°C was adequate for preserving activity over a few weeks, but for long term, the less convenient preservation in liquid N2 is required. Symbiodinium Rubisco is much more unstable than the Form II enzyme of R. rubrum, which is readily purified and, after purification, retains 50-70% of its activity after storage for 1 month at 4°C (Schloss et al 1979). The thermal sensitivity of carbon fixation in plants is generally the consequence of a lowered activation state of the Form I Rubisco caused by thermal instability of the AAA + motor protein, Rubisco activase (Portis 2003), which is, in turn, partially protected against elevated temperature by the GroEL-type chloroplast heat shock protein cpn60b (Salvucci 2008).…”

Section: Stability Of Extracted Rubiscomentioning

confidence: 99%

The determination of activity of the enzyme Rubisco in cell extracts of the dinoflagellate alga Symbiodinium sp. by manganese chemiluminescence and its response to short‐term thermal stress of the alga

Lilley

Ralph

Larkum

2010

Plant Cell & Environment

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The dinoflagellate alga Symbiodinium sp., living in symbiosis with corals, clams and other invertebrates, is a primary producer in coral reefs and other marine ecosystems. The function of the carbon-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) in dinoflagellates is difficult to study because its activity is rapidly lost after extraction from the cell. We report procedures for the extraction of Rubisco from Symbiodinium cells and for stable storage. We describe a continuous assay for Rubisco activity in these crude cell extracts using the Mn 2+ chemiluminescence of Rubisco oxygenase. Chemiluminescence time courses exhibited initial transients resembling bacterial Form II Rubisco, followed by several minutes of linearly decreasing activity. The initial activity was determined from extrapolation of this linear section of the time course. The activity of fast-frozen cell extracts was stable at -80°C and, after thawing and storage on ice, remained stable for up to 1 h before declining non-linearly. Crude cell extracts bound [ 14 C] 2-carboxy-D-arabitinol 1,5-bisphosphate to a high molecular mass fraction separable by gel filtration chromatography. After pre-treatment of Symbiodinium cell cultures in darkness at temperatures above 30°C, the extracted Rubisco activities decreased, with almost complete loss of activity above 36°C. The implications for the sensitivity to elevated temperature of Symbiodinium photosynthesis are assessed.

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“…In addition, the results of this study specifically indicate that the T. intermedius enzyme is not the unique evolutionary octameric form of RuBPCase that was originally proposed (19,20,22), thus leaving R. rubrum as the only bacterial species with an independently confirmed 0-type enzyme (cf. 9,13,25,28).…”

Section: Results and Discussion Purification Of Rubpcase From T Intementioning

confidence: 99%

Presence of two subunit types in ribulose 1,5-bisphosphate carboxylase from Thiobacillus intermedius

Bowman¹,

Chollet²

1980

J Bacteriol

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Ribulose bisphosphate carboxylase (EC 4.1.1.39) has been purified to homogeneity from glutamate-CO2-thiosulfate-grown Thiobacillus intermedius by pelleting the protein from the 93,000 x g supernatant fluid followed by ammonium sulfate fractionation and sedimentation into a discontinuous sucrose density gradient. The molecular weight of the native protein approximated that of the higher plant enzyme (550,000) based on its relative electrophoretic mobility in polyacrylamide disc gels compared with that of standards of known molecular weight, including crystalline tobacco ribulose bisphosphate carboxylase. Sodium dodecyl sulfate electrophoresis in 12% polyacrylamide disc gels and Sephadex G-100 chromatography in the presence of sodium dodecyl sulfate indicated that the purified Thiobacillus protein, like the tobacco enzyme, consisted of two types of nonidentical subunits. The molecular weights of the large and snall subunits were estimated to be about 55,000 and 13,000, respectively, by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The carboxylase activity of the protein purified from spinach leaves and T. intermedius responded similarly to the effectors reduced nicotinamide adenine dinucleotide phosphate and 6phosphogluconate. Contrary to a previous report (K. Purohit, B. A. McFadden, and A. L. Cohen, J. Bacteriol. 127:505-515, 1976), these results indicate that ribulose bisphosphate carboxylase purified from Thiobacillus intermedius closely resembles the higher plant enzyme with respect to quaternary structure, molecular weight, and regulatory properties.

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Isolation, characterization, and crystallization of ribulosebisphosphate carboxylase from autotrophically grown Rhodospirillum rubrum

Cited by 78 publications

References 73 publications

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

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

The determination of activity of the enzyme Rubisco in cell extracts of the dinoflagellate alga Symbiodinium sp. by manganese chemiluminescence and its response to short‐term thermal stress of the alga

Presence of two subunit types in ribulose 1,5-bisphosphate carboxylase from Thiobacillus intermedius

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