Cellular Fractionation of <i>Chlamydomonas reinhardii</i> with Emphasis on the Isolation of the Chloroplast

Klein, U.; Chen, Changguo; Gibbs, Martin; Platt‐Aloia, K. A.

doi:10.1104/pp.72.2.481

Cited by 77 publications

(82 citation statements)

References 20 publications

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“…These rates were sustained for over 10 min. Similar to higher plant chloroplasts (and chloroplasts isolated by Klein et al [6], and the Pressate [1]), the addition of 10 mm phosphate results in severe inhibition of this activity (trace C). The addition of 1 mM 3PGA and 1 mm OAA both stimulate O2 evolution in the presence of high phosphate (trace B).…”

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confidence: 62%

“…The chloroplast preparation described here results in more cytoplasmic as well as mitochondrial contamination of the chloroplast fraction than the preparation described by Klein et al (6). This preparation has the advantage of utilizing batch rather than synchronous cultures, which facilitates the use of mutant strains of Chlamydomonas.…”

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confidence: 86%

“…The large size of the chloroplast would seem to complicate isolation of the intact organelle of this organism. Recently, Klein et al (6) have reported a method of isolating functional chloroplasts from Chlamydomonas which utilizes differential solubilization of the plasma membrane and organellar membranes by detergents. The (-) (from R. P. Levine), and the phosphoribulokinase-deficient strain F60 (-) (9) were grown under fluorescent lights in Tris/acetate/phosphate medium on a rotary shaker as described in Gorman and Levine (5).…”

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confidence: 99%

“…Presumably, the presence of the cell wall in this preparation resulted in containment of cytoplasmic components. Protoplasts can be easily prepared from Chlamydomonas by utilizing a cell wall-digesting enzyme (autolysine) which is released during the mating of gametes (6,8). When protoplasts produced in this manner, from both Wt cells and those from the phosphoribulokinase-deficient mutant F60 (9), were used in the production of a modified Pressate, intact chloroplasts were released.…”

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confidence: 99%

“…The Pressate which resulted from disruption of the cells in the Yeda pressure bomb was fractionated on a two step Percoll density gradient. The intact chloroplasts, intermediate in density between the broken chloroplasts and protoplasts (6), were removed from the gradient and washed to remove the Percoll. The data in Table I Chloroplast preparations using Wt cells give similar enzyme and organellar activity profiles.…”

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Partial Purification of Intact Chloroplasts from Chlamydomonas reinhardtii

Belknap

1983

Plant Physiol.

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MATERIALS AND METHODSPartially purified intact chloroplasts were prepared from batch cultures of both wild type (Wt) and a mutant strain of Chlamydomonas reinhardtii. Protoplasts were generated from log phase cultures of Wt (137c) and the phosphoribulokinase-deficient mutant F60 by incubation of the cells in autolysine. These protoplasts were suspended in an osmoticum, cooled, and then subjected to a 40 pounds per square inch pressure shock using a Yeda pressure bomb. The resulting preparation was fractionated on a Percoll step gradient which separated the intact chloroplasts from both broken chloroplasts and protoplasts.The chloroplast preparation was not significantly contaminated with the cytoplasmic enzyme activity phosphoenolpyruvate carboxylase (>5%), and contained (100%) stromal enzyme activity ribulose-1,5-bisphosphate carboxylase. The (-) mating types were incubated separately in gamete inducing medium for 24 h (final cell concentrations of approximately 1.5 x 107 cells/ml). Gametes were then harvested and resuspended in sufficient deionized H20 to result in final cell concentrations of 2 x 108 cells/ml. These concentrated (+) and (-) gamete solutions were then mixed in equal volumes and incubated at room temperature for 45 min, with occasional gentle mixing, to allow mating to occur. At the end of the mating period, the mating solution was centrifuged at 17,000g (3°C) for 20 min to remove gametes and debris. The supernatant from this centrifugation, which contained the autolysine activity, was then separated into 9-ml lots and stored at -20°C. Autolysine prepared and stored in this fashion retains activity over a period of 6 months.Preparation of Protoplasts. Protoplasts were prepared from log phase cultures of Wt (+) and F60 in Tris/acetate/phosphate medium. Cell suspensions containing 5 mg Chl were harvested and resuspended in 9 ml of the autolysine solution, which had been thawed immediately prior to use. This suspension was placed on a rotary shaker in the dark (25°C) for 30 min to allow digestion of the cell walls to occur. At the end of the digestion period, the protoplasts were removed from the autolysine solution by centrifugation at 3,000g for 2 min. The supernatant from this centrifugation was discarded, and the pellet gently resuspended in 35 ml of 20 mm Hepes (pH 7.0, 25°C). This solution was then centrifuged at 3,000g for 1 min, and the supematant discarded. The protoplasts were washed with three more 35-ml volumes of the Hepes buffer in a similar fashion to ensure complete removal of the autolysine.

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confidence: 62%

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confidence: 86%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Partial Purification of Intact Chloroplasts from Chlamydomonas reinhardtii

Belknap

1983

Plant Physiol.

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Optimization of a subcellular metal fractionation method for fish liver: Homogenization, subcellular separation, and trial isolation of nuclear materials

Urien

Caron

Lerquet

et al. 2020

Limnology & Ocean Methods

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The subcellular compartmentalization of metals within aquatic organisms reflects their internal behavior after metal uptake and can provide important information about their potential toxicity. Commonly, the fractionation protocol used to determine subcellular metal partitioning in aquatic organisms consists of mechanically homogenizing the tissue, separating subcellular components into fractions by differential centrifugation and heat-denaturation steps, and determining the amount of metals associated with each fraction. However, the accurate separation of subcellular cell components is challenging and the nature and purity of the operationally defined subcellular fractions are rarely assessed. In the absence of this type of validation, however, the interpretation of subcellular metal fractionation results could be compromised. The aim of the present study was to adjust a subcellular fractionation protocol for the liver of field-collected fish and to test the adjusted protocol using fraction-specific enzyme markers. Overall, our results illustrate the need to optimize fractionation procedures when studying a new species or organ. In the course of this study, the categorization of some fractions was revised in accordance with the enzymatic results obtained, in order to yield a more credible subcellular fraction distribution scheme. In addition, trial assays aimed at isolating nuclear materials from the cellular debris were conducted, using DNA as a marker for nuclear material. Tested protocols failed to isolate the nuclei and results suggested that nuclei were probably trapped by disrupted cellular membranes. Recommendations on how to improve future subcellular fractionation studies on freshwater fish are discussed.

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From Current Algae Products to Future Biorefinery Practices: A Review

Eppink

Olivieri

Reith

et al. 2017

Advances in Biochemical Engineering/Biotechnology

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Microalgae are considered to be one of the most promising next generation bio-based/food feedstocks with a unique lipid composition, high protein content, and an almost unlimited amount of other bio-active molecules. High-value components such as the soluble proteins, (poly) unsaturated fatty acids, pigments, and carbohydrates can be used as an important ingredient for several markets, such as the food/feed/chemical/cosmetics and health industries. Although cultivation costs have decreased significantly in the last few decades, large microalgae production processes become economically viable if all complex compounds are optimally valorized in their functional state. To isolate these functional compounds from the biomass, cost-effective, mild, and energy-efficient biorefinery techniques need to be developed and applied. In this review we describe current microalgae biorefinery strategies and the derived products, followed by new technological developments and an outlook toward future products and the biorefinery philosophy.

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

Cited by 77 publications

References 20 publications

Partial Purification of Intact Chloroplasts from Chlamydomonas reinhardtii

Partial Purification of Intact Chloroplasts from Chlamydomonas reinhardtii

Optimization of a subcellular metal fractionation method for fish liver: Homogenization, subcellular separation, and trial isolation of nuclear materials

From Current Algae Products to Future Biorefinery Practices: A Review

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