Isolation of protoplasts by means of a ?species-specific? autolysine inChlamydomonas

Schlösser, U. G.; Sachs, H.; Robinson, David G.

doi:10.1007/bf01280359

Cited by 67 publications

(19 citation statements)

References 25 publications

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“…The pellet was washed with 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) (pH 7.0) and subsequently incubated in 40 ml autolysin (Sfiltemeyer et al 1988). Autolysin was prepared according to Schl6sser et al (1976). After the cell walls were completely removed (15-30 min), protoplasts were collected (1000" 9; 10 min) and washed with Hepes buffer (pH 7.0).…”

Section: Introductionmentioning

confidence: 99%

Evidence for the contribution of pseudocyclic photophosphorylation to the energy requirement of the mechanism for concentrating inorganic carbon in Chlamydomonas

Sültemeyer

Biehler

Fock

1993

Planta

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Abstract. Mass-spectrometric measurements of 1602 and 802 were made to compare the rates of light-dependent 02 evolution and uptake by Chlamydomonas reinhardtiiDang. grown in air (0.035% CO2; low-C~ cells) or CO2-enriched air (5% CO2; high-Ci cells) at pH 5.5 and 8.0.While at pH 5.5, no differences were observed in the isotopic Oz-gas exchange of high-and low-Ci cells, at pH 8.0 the rates of true 02 evolution and uptake were considerably higher in low-C i than in high-C~ cells. The enhanced rates of 02 uptake and evolution by low-Ci cells were completely inducible within 6 h after transferring high-C~ cells to ambient air. At pH 8.0, 02 uptake in the light was inhibited by 2 gM 3-(3,4-dichlorophenyl)-l,1 dimethylurea in both types of alga, but this effect was more pronounced in low-C~ than in high-C~ cells. When the cells were grown at pH 5.5 the activities of the superoxide-radical-degrading enzymes, superoxide dismutase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase, were similar regardless of the CO2 concentration provided during growth. At pH 8.0, however, the activities of these enzymes were 4 to 20 times higher in low-C~ than in high-Ci cells. When high-Ci cells were allowed to acclimate to ambient air for 6 h at pH 8.0, the activities of superoxide dismutase, ascorbate peroxidase and monodehydroascorbate dehydrogenase increased to more than 50 % of the level observed with low-C i cells. These results are consistent with an enhanced operation of 02 photoreduction which could provide energy to the * To whom correspondence should be addressed; FAX: 49 (631) 205 29 98Abbreviations." AP = ascorbate peroxidase; Chl = chlorophyll; C i = inorganic carbon (HCO~-+ CO2); DAR = dehydroascorbate reductase; DCMU = 3-(Y,4'-dichlorophenyl)-1,1-dimethylurea; E o = rate of true 02 evolution; GR = glutathione reductase; high-C i cells = cells grown at 5 % CO2; low-C i cells = cells grown at 0.035 % CO 2 ; MDAR = monodehydroascorbate reductase; NET = rate of net 02 evolution; PPFD=photosynthetic photon flux density; SOD = superoxide dismutase; Uo= rate of true O z uptake inorganic-carbon-concentrating mechanism via pseudocyclic photophosphorylation.

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Section: Introductionmentioning

confidence: 99%

Evidence for the contribution of pseudocyclic photophosphorylation to the energy requirement of the mechanism for concentrating inorganic carbon in Chlamydomonas

Sültemeyer

Biehler

Fock

1993

Planta

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“…The autolysine was obtained as described earlier (20). Protoplasts could be produced with an integrity of 100% because the autolysine specifically lyses only the algal cell wall (35). Chloroplasts were isolated from protoplasts by lysis with digitonin and purification by differential and Percoll cushion centrifugation (for details see ref.…”

Section: Methodsmentioning

confidence: 99%

Localization of Nitrogen-Assimilating Enzymes in the Chloroplast of Chlamydomonas reinhardtii

Fischer¹,

Klein²

1988

Plant Physiol.

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The specific activities of nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase, and glutamate dehydrogenase were determined in intact protoplasts and intact chloroplasts from Chlamydomonas reinhardtii. After correction for contamination, the data were used to calculate the portion of each enzyme in the algal chloroplast. The chloroplast of C. reinhardtii contained all enzyme activities for nitrogen assimilation, except nitrate reductase, which could not be detected in this organelle. Glutamate synthase (NADH-and ferredoxin-dependent) and glutamate dehydrogenase were located exclusively in the chloroplast, while for nitrite reductase and glutamine synthetase an extraplastidic activity of about 20 and 60%, respectively, was measured. Cells grown on ammonium, instead of nitrate as nitrogen source, had a higher total cellular activity of the NADH-dependent glutamate synthase (+95%) and glutamate dehydrogenase (+33%) but less activity of glutamine synthetase (-10%). No activity of nitrate reductase could be detected in ammonium-grown cells. The distribution of nitrogen-assimilating enzymes among the chloroplast and the rest of the cell did not differ significantly between nitrate-grown and ammonium-grown cells. Only the plastidic portion of the glutamine synthetase increased to about 80% in cells grown on ammonium (compared to about 40% in cells grown on nitrate).

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“…Autolysine was prepared according to the following method which is essentially that described by Schlosser et aL (24) with a few modifications: 1,000-ml cultures of C. reinhardii 11-32/b and 11-32/c were inoculated from 200-ml precultures. The starting cell density was about 2,000 cells/ml.…”

Section: Methodsmentioning

confidence: 99%

Cellular Fractionation of Chlamydomonas reinhardii with Emphasis on the Isolation of the Chloroplast

Klein

Chen²,

Gibbs³

et al. 1983

Plant Physiol.

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A method for cellular fractionation of Chiamydomonas reinhardii, SAG 11-32/b, and isolation of intact chloroplasts from synchronized cells of the alga is described. The procedure for ceil fractionation comprises essentially four steps: (1) protoplast production with autolysine; (2) lysis of the protoplasts with digitonin; (3) aggregation of broken protoplasts; and (4) separation of organelles by differential centrifugations.Replacing the differential centrifugations (step 4) by Percoll cushion centrifugations yields intact chloroplasts. Starting with 100 milliliters of an algal culture containing 3000 micrograms chlorophyll, intact chloroplasts with 100 to 200 micrograms of chlorophyll can be isolated. Envelope integrity is about 90% (ferricyanide assay). Examination of the chloroplasts by electron microscopy and marker enzyme activities indicated some mitochondrial and cytoplasmic contamination.The biochemistry and physiology of unicellular green algae have been studied intensively because these algae can be maintained easily under laboratory conditions. Cultures can be synchronized (21) and some strains grow heterotrophically as well as autotrophically and mixotrophically. Many similarities with the metabolism of higher plants have been established. The pathway of photosynthetic CO2 reduction was primarily elucidated with unicellular green algae. Enzymes of the glyoxylate cycle and glycolate metabolism, previously found in higher plants, could also be measured in algae, proving the general occurrence of these pathways in plants. Although it was possible to relate these pathways and cycles to cellular organelles in higher plants, very few investigations are reported on the compartmentation of metabolism in unicellular green algae. The first detailed study of localization of enzymes in a unicellular alga of the Chlamydomonas type was made by Kombrink and Wober (16) who were able to demonstrate the activity of starch-metabolizing enzymes in chloroplasts of Dunaliella marina. In their investigation, they used a new method involving DEAE-dextran for cell lysis.One reason for the difficulties in cell fractionation of unicellular

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Isolation of protoplasts by means of a ?species-specific? autolysine inChlamydomonas

Cited by 67 publications

References 25 publications

Evidence for the contribution of pseudocyclic photophosphorylation to the energy requirement of the mechanism for concentrating inorganic carbon in Chlamydomonas

Evidence for the contribution of pseudocyclic photophosphorylation to the energy requirement of the mechanism for concentrating inorganic carbon in Chlamydomonas

Localization of Nitrogen-Assimilating Enzymes in the Chloroplast of Chlamydomonas reinhardtii

Cellular Fractionation of Chlamydomonas reinhardii with Emphasis on the Isolation of the Chloroplast

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