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[40] Isolation of highly purified intact chloroplasts and of multiorganelle complexes containing chloroplasts
[40]
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419
Acknowledgments This work was supported by the Bank of Sweden Tercentenary Research Foundation. D. J. Morr6 was the 1989-1990 Tage Erlander professor of the Swedish Natural Science Research Council.
[40] Isolation o f H i g h l y P u r i f i e d I n t a c t C h l o r o p l a s t s a n d o f MultiorganeUe Complexes Containing Chloroplasts By CHRISTER LARSSON
Introduction For studies on major chloroplast reactions, such as those of the Calvin cycle, intact chloroplasts prepared solely by differential centrifugation are preferred because of the rapidity and simplicity of that purification process. However, for studies on compartmentation of metabolism between the chloroplast and other cellular compartments more highly purified chloroplasts are needed, since contamination (by cytoplasm, mitochondria, peroxisomes, etc.) would otherwise obscure the results. Such highly purified chloroplasts can be prepared by phase partitioning. ~As a by-product a fraction containing "multiorganelle complexes" is obtained. These complexes consist of one or two chloroplasts surrounded by cytoplasm, mitochondria, and peroxisomes all enclosed by some plasma membrane. Multiorganelle complexes are formed during homogenization of leaf tissue, and they are one of the contaminants removed during the purification process. The multiorganelle complexes can, however, be used to advantage as a reference point representing "highly contaminated chloroplasts" with a metabolic capacity close to that of the intact cell.l
Procedure
A phase system (of, e.g., 50 g), to provide the fresh upper and lower phases indicated below, is prepared in a separatory funnel by weighing and pipetting from stock solutions of polymers [20% (w/w) dextran (Dx) T500 (Pharmacia LKB, Uppsala, Sweden), 40% (w/w) poly(ethylene glycol) (PEG) 3350 (Union Carbide, New York, NY)] and salts, z'3 After I C. Larsson and P.-,~. Albertsson, Biochim. Biophys. Acta 357, 412 (1974). 2 D. E. Brooks and R. Norris-Jones, this volume [2]. H. Walter and C. Larsson, this volume [4].
METHODS IN ENZYMOLOGY, VOL. 228
Copyright © 1994by Academic Press, Inc. All rights of reproduction in any form reserved.
420
PARTITIONING OF PARTICULATES
[40]
FIG. I. Electron micrographs of (a) intact chloroplasts, (b) broken chloroplasts (thylakoids), and (c) multiorganelle complexes, all obtained by phase partitioning. Note that the multiorganelle complexes are surrounded by only a thin layer of cytoplasm and are, therefore, similar to free intact chloroplasts (a) in size and density. Observe also that the different
[40]
ISOLATION OF INTACT CHLOROPLASTS
421
equilibration at 4° , the phase system is well shaken and left standing for phase settling to occur. Upper and lower phases are collected and stored separately in the cold or are frozen.
Intact Chloroplasts A well-drained chloroplast pellet, obtained from 50 g of spinach leaves by differential centrifugation,4 is suspended in 1 ml of 0.3 M sorbitol, 5 mM potassium phosphate, pH 7.8. The chloroplast suspension (1.0 ml) is added to a phase mixture (7.0 g) to yield a phase system (8.0 g) composed of 5.8% (w/w) Dx T500, 5.8% (w/w) PEG 3350, 0.3 M sorbitol, and 5 mM potassium phosphate, pH 7.8, at a temperature of 4°. The phase system is mixed by 10-20 inversions of the tube and is either permitted to stand at unit gravity for about l0 rain or subjected to low-speed centrifugation (swing-out buckets) to decrease the time required for phase settling. If centrifugation is used care must be taken that material partitioning into the upper phase not be sedimented onto the interface. The upper phase, containing mainly thylakoids (Fig. lb) but also multiorganelle complexes and other contaminants, is removed (Pasteur pipette) and the lower phase plus interface is reextracted twice with fresh upper phase. The final lower phase plus interface is diluted 10-fold with a suitable medium, and the intact chloroplasts (Fig. la) are sedimented by centrifugation (500 g for 5 rain, preferably using swing-out buckets) and resuspended.
Multiorganelle Complexes A batch preparation of multiorganeUe complexes (Fig. lc) is obtained in a similar way. In contrast to that of multiorganelle complexes, the partitioning of thylakoids is very sensitive to changes in the interfacial potential. Thus, if 5 mM KCI is included in the phase system (by pipetting from 2 M KC1) the chloroplast thylakoids will collect at the R. G. Jensen and J. A. Bassham, Proc, Natl. Acad. Sci. U.S.A. 56, 1095 (1966).
particulates in a, b, and c expose different membrane surfaces to the phase system, which provides the basis for their separation by phase partitioning, ce, Chloroplast envelope, outer membrane; tin, thylakoid membrane; pm, plasma membrane; m, mitochondrion; p, peroxisome. Bars, I/zm. [From P.-/~. Albertsson, B. Andersson, C. Larsson, and H.-E. /kkeflund, Methods Biochem. Anal. 28, 115 (1982). Copyright © 1982 John Wiley & Sons, Inc. Reprinted by permission of John Wiley & Sons, Inc.]
422
PARTITIONING OF PARTICULATES
[40]
[40]
ISOLATION OF INTACT CHLOROPLASTS
423
interface together with the intact chloroplasts, while the multiorganelle complexes will remain in the upper phase (compare Refs. 3 and 5). The first upper phase from the isolation of intact chloroplasts (above) can be used as a source for multiorganelle complexes. However, because the amounts of multiorganelle complexes in chloroplast preparations are low (0.5-5% of total chlorophyll) it is usually necessary to scale up the process. The best yields are obtained from cotyledons. L6 A chloroplast preparation from 200-300 g of leaves is partitioned in a phase system of 24-36 g using a separatory funnel. After phase settling for 15-20 rain, the lower phase plus interface is discarded and the upper phase is reextracted twice with fresh lower phase (also containing 5 mM KC1). The final upper phase is diluted at least once with a suitable medium, and the multiorganelle complexes are collected by centrifugation at 500 g for 5 min. Properties of Fractions Intact chloroplasts purified by phase partitioning incorporate ~4CO2 almost exclusively into glycolate, starch, and intermediates of the Calvin cycle and starch synthesis (Fig. 2A). By contrast, multiorganelle complexes give a much broader spectrum of ~4C-labeled products including sucrose, malate, glycerate, aspartate, alanine, glycine, serine, and other amino acids (Fig. 2B). Intact chloroplasts can be separated from broken chloroplasts (thylakoids, Fig. lb) by differential4 or by Percoll gradient centrifugation.7 However, intact chloroplasts and multiorganelle complexes, which are readily separated by phase partitioning because of differences in their surface properties (Fig. 1), are separated poorly by methods which depend on 5 C. Larsson, M. Sommarin, and S. Widell, this volume [44]. 6 C. Larsson, C. CoUin, and P.-A. Albertsson, Biochim. Biophys. Acta 245, 425 (1971). 7 K. W. Joy and W. R. Mills, this series, Vol. 148, p, 179.
Flo. 2. Radioautographs of 14C-labeled products after 6 min of photosynthetic 14CO2 fixation by (A) intact chloroplasts and (B) multiorganelle complexes, purified from spinach leaves by phase partitioning. Products were separated by thin-layer electrophoresis (horizontal) and chromatography (vertical) [P. Schiirmann, J. Chromatogr. 39, 507 (1969)]. ALA, Alanine; ASP, aspartate; DHAP, dihydroxyacetone phosphate; GLC, glucose; GLL, glycolate; GLR, glycerate; GLU, glutamate GLY, glycine; MAL, malate; PEP, phosphoenolpyruvate; 3-PGA, 3-phosphoglycerate; PGL, phosphoglycolate; SBP, sugar bisphosphates; SER, serine; SMP, sugar monophosphates; SUCR, sucrose; THR, threonine. [Modified from C. Larsson and P.-/~. Albertsson, Biochim. Biophys. Acta 357, 412 (1974)].
424
PARTITIONING OF PARTICULATES
[41]
size and density. Because multiorganelle complexes have a metabolic capacity close to that of the intact cell (Fig. 2B), they may constitute a serious problem in studies on chloroplast metabolism. This is well illustrated by Bickel et al. 8 in a study on the biosynthesis of prenylquinones. After incubation of a standard chloroplast preparation with [~4C]tyrosine they found about 3% of total label in plastoquinone and a-tocopherol. If instead chloroplasts purified by phase partitioning were used, only 0.01% of the ~4C was in prenylquinones. Bickel et al. 8 were also able to show (by reconstitution experiments) that peroxisomes were necessary for the conversion of tyrosine to homogentisate, a precursor for the prenylquinones. Thus, the high activity of prenylquinone synthesis observed with the unpurified chloroplasts was due to contamination by peroxisomes. It should be noted that the ~4CO2 fixation rate of the chloroplasts is not changed by the purification process. Chloroplasts purified by phase partitioning are therefore well suited for studies on compartmentation of metabolic pathways. Acknowledgements Work in the author's laboratory was supported by the Swedish Natural Science Research Council. g H. Bickel, B. Buchholz, and G. Schultz, in "Advances in the Biochemistryand Physiology of Plant Lipids" (L. ,~. Appelqvist and C. Liljenberg, eds.), p. 369. Elsevier, Amsterdam, 1979.
[41] P u r i f i c a t i o n a n d C h a r a c t e r i z a t i o n o f P l a n t M i t o c h o n d r i a and Submitochondrial Particles B y PER
GARDESTROM,PATRICE X. PETIT, and IAN M. MOLLER
Introduction To study plant respiration it is important to isolate functionally intact mitochondria from different tissues. Functionally intact plant mitochondria can be obtained by differential centrifugation, yielding a crude fraction.~ For many types of studies it is also necessary to have pure mitochondria. Different methods have been used to remove contaminating material,
i R. Douce, J. Bourguignon, R. Brouquisse, and M. Neuburger, this series, Vol. 148, p. 403.
METHODS IN ENZYMOLOGY,VOL. 228
Copyright © 1994by AcademicPress, Inc. All fights of reproductionin any formreserved.
I S O L A T I O N OF I N T A C T C H L O R O P L A S T S
419
Acknowledgments This work was supported by the Bank of Sweden Tercentenary Research Foundation. D. J. Morr6 was the 1989-1990 Tage Erlander professor of the Swedish Natural Science Research Council.
[40] Isolation o f H i g h l y P u r i f i e d I n t a c t C h l o r o p l a s t s a n d o f MultiorganeUe Complexes Containing Chloroplasts By CHRISTER LARSSON
Introduction For studies on major chloroplast reactions, such as those of the Calvin cycle, intact chloroplasts prepared solely by differential centrifugation are preferred because of the rapidity and simplicity of that purification process. However, for studies on compartmentation of metabolism between the chloroplast and other cellular compartments more highly purified chloroplasts are needed, since contamination (by cytoplasm, mitochondria, peroxisomes, etc.) would otherwise obscure the results. Such highly purified chloroplasts can be prepared by phase partitioning. ~As a by-product a fraction containing "multiorganelle complexes" is obtained. These complexes consist of one or two chloroplasts surrounded by cytoplasm, mitochondria, and peroxisomes all enclosed by some plasma membrane. Multiorganelle complexes are formed during homogenization of leaf tissue, and they are one of the contaminants removed during the purification process. The multiorganelle complexes can, however, be used to advantage as a reference point representing "highly contaminated chloroplasts" with a metabolic capacity close to that of the intact cell.l
Procedure
A phase system (of, e.g., 50 g), to provide the fresh upper and lower phases indicated below, is prepared in a separatory funnel by weighing and pipetting from stock solutions of polymers [20% (w/w) dextran (Dx) T500 (Pharmacia LKB, Uppsala, Sweden), 40% (w/w) poly(ethylene glycol) (PEG) 3350 (Union Carbide, New York, NY)] and salts, z'3 After I C. Larsson and P.-,~. Albertsson, Biochim. Biophys. Acta 357, 412 (1974). 2 D. E. Brooks and R. Norris-Jones, this volume [2]. H. Walter and C. Larsson, this volume [4].
METHODS IN ENZYMOLOGY, VOL. 228
Copyright © 1994by Academic Press, Inc. All rights of reproduction in any form reserved.
420
PARTITIONING OF PARTICULATES
[40]
FIG. I. Electron micrographs of (a) intact chloroplasts, (b) broken chloroplasts (thylakoids), and (c) multiorganelle complexes, all obtained by phase partitioning. Note that the multiorganelle complexes are surrounded by only a thin layer of cytoplasm and are, therefore, similar to free intact chloroplasts (a) in size and density. Observe also that the different
[40]
ISOLATION OF INTACT CHLOROPLASTS
421
equilibration at 4° , the phase system is well shaken and left standing for phase settling to occur. Upper and lower phases are collected and stored separately in the cold or are frozen.
Intact Chloroplasts A well-drained chloroplast pellet, obtained from 50 g of spinach leaves by differential centrifugation,4 is suspended in 1 ml of 0.3 M sorbitol, 5 mM potassium phosphate, pH 7.8. The chloroplast suspension (1.0 ml) is added to a phase mixture (7.0 g) to yield a phase system (8.0 g) composed of 5.8% (w/w) Dx T500, 5.8% (w/w) PEG 3350, 0.3 M sorbitol, and 5 mM potassium phosphate, pH 7.8, at a temperature of 4°. The phase system is mixed by 10-20 inversions of the tube and is either permitted to stand at unit gravity for about l0 rain or subjected to low-speed centrifugation (swing-out buckets) to decrease the time required for phase settling. If centrifugation is used care must be taken that material partitioning into the upper phase not be sedimented onto the interface. The upper phase, containing mainly thylakoids (Fig. lb) but also multiorganelle complexes and other contaminants, is removed (Pasteur pipette) and the lower phase plus interface is reextracted twice with fresh upper phase. The final lower phase plus interface is diluted 10-fold with a suitable medium, and the intact chloroplasts (Fig. la) are sedimented by centrifugation (500 g for 5 rain, preferably using swing-out buckets) and resuspended.
Multiorganelle Complexes A batch preparation of multiorganeUe complexes (Fig. lc) is obtained in a similar way. In contrast to that of multiorganelle complexes, the partitioning of thylakoids is very sensitive to changes in the interfacial potential. Thus, if 5 mM KCI is included in the phase system (by pipetting from 2 M KC1) the chloroplast thylakoids will collect at the R. G. Jensen and J. A. Bassham, Proc, Natl. Acad. Sci. U.S.A. 56, 1095 (1966).
particulates in a, b, and c expose different membrane surfaces to the phase system, which provides the basis for their separation by phase partitioning, ce, Chloroplast envelope, outer membrane; tin, thylakoid membrane; pm, plasma membrane; m, mitochondrion; p, peroxisome. Bars, I/zm. [From P.-/~. Albertsson, B. Andersson, C. Larsson, and H.-E. /kkeflund, Methods Biochem. Anal. 28, 115 (1982). Copyright © 1982 John Wiley & Sons, Inc. Reprinted by permission of John Wiley & Sons, Inc.]
422
PARTITIONING OF PARTICULATES
[40]
[40]
ISOLATION OF INTACT CHLOROPLASTS
423
interface together with the intact chloroplasts, while the multiorganelle complexes will remain in the upper phase (compare Refs. 3 and 5). The first upper phase from the isolation of intact chloroplasts (above) can be used as a source for multiorganelle complexes. However, because the amounts of multiorganelle complexes in chloroplast preparations are low (0.5-5% of total chlorophyll) it is usually necessary to scale up the process. The best yields are obtained from cotyledons. L6 A chloroplast preparation from 200-300 g of leaves is partitioned in a phase system of 24-36 g using a separatory funnel. After phase settling for 15-20 rain, the lower phase plus interface is discarded and the upper phase is reextracted twice with fresh lower phase (also containing 5 mM KC1). The final upper phase is diluted at least once with a suitable medium, and the multiorganelle complexes are collected by centrifugation at 500 g for 5 min. Properties of Fractions Intact chloroplasts purified by phase partitioning incorporate ~4CO2 almost exclusively into glycolate, starch, and intermediates of the Calvin cycle and starch synthesis (Fig. 2A). By contrast, multiorganelle complexes give a much broader spectrum of ~4C-labeled products including sucrose, malate, glycerate, aspartate, alanine, glycine, serine, and other amino acids (Fig. 2B). Intact chloroplasts can be separated from broken chloroplasts (thylakoids, Fig. lb) by differential4 or by Percoll gradient centrifugation.7 However, intact chloroplasts and multiorganelle complexes, which are readily separated by phase partitioning because of differences in their surface properties (Fig. 1), are separated poorly by methods which depend on 5 C. Larsson, M. Sommarin, and S. Widell, this volume [44]. 6 C. Larsson, C. CoUin, and P.-A. Albertsson, Biochim. Biophys. Acta 245, 425 (1971). 7 K. W. Joy and W. R. Mills, this series, Vol. 148, p, 179.
Flo. 2. Radioautographs of 14C-labeled products after 6 min of photosynthetic 14CO2 fixation by (A) intact chloroplasts and (B) multiorganelle complexes, purified from spinach leaves by phase partitioning. Products were separated by thin-layer electrophoresis (horizontal) and chromatography (vertical) [P. Schiirmann, J. Chromatogr. 39, 507 (1969)]. ALA, Alanine; ASP, aspartate; DHAP, dihydroxyacetone phosphate; GLC, glucose; GLL, glycolate; GLR, glycerate; GLU, glutamate GLY, glycine; MAL, malate; PEP, phosphoenolpyruvate; 3-PGA, 3-phosphoglycerate; PGL, phosphoglycolate; SBP, sugar bisphosphates; SER, serine; SMP, sugar monophosphates; SUCR, sucrose; THR, threonine. [Modified from C. Larsson and P.-/~. Albertsson, Biochim. Biophys. Acta 357, 412 (1974)].
424
PARTITIONING OF PARTICULATES
[41]
size and density. Because multiorganelle complexes have a metabolic capacity close to that of the intact cell (Fig. 2B), they may constitute a serious problem in studies on chloroplast metabolism. This is well illustrated by Bickel et al. 8 in a study on the biosynthesis of prenylquinones. After incubation of a standard chloroplast preparation with [~4C]tyrosine they found about 3% of total label in plastoquinone and a-tocopherol. If instead chloroplasts purified by phase partitioning were used, only 0.01% of the ~4C was in prenylquinones. Bickel et al. 8 were also able to show (by reconstitution experiments) that peroxisomes were necessary for the conversion of tyrosine to homogentisate, a precursor for the prenylquinones. Thus, the high activity of prenylquinone synthesis observed with the unpurified chloroplasts was due to contamination by peroxisomes. It should be noted that the ~4CO2 fixation rate of the chloroplasts is not changed by the purification process. Chloroplasts purified by phase partitioning are therefore well suited for studies on compartmentation of metabolic pathways. Acknowledgements Work in the author's laboratory was supported by the Swedish Natural Science Research Council. g H. Bickel, B. Buchholz, and G. Schultz, in "Advances in the Biochemistryand Physiology of Plant Lipids" (L. ,~. Appelqvist and C. Liljenberg, eds.), p. 369. Elsevier, Amsterdam, 1979.
[41] P u r i f i c a t i o n a n d C h a r a c t e r i z a t i o n o f P l a n t M i t o c h o n d r i a and Submitochondrial Particles B y PER
GARDESTROM,PATRICE X. PETIT, and IAN M. MOLLER
Introduction To study plant respiration it is important to isolate functionally intact mitochondria from different tissues. Functionally intact plant mitochondria can be obtained by differential centrifugation, yielding a crude fraction.~ For many types of studies it is also necessary to have pure mitochondria. Different methods have been used to remove contaminating material,
i R. Douce, J. Bourguignon, R. Brouquisse, and M. Neuburger, this series, Vol. 148, p. 403.
METHODS IN ENZYMOLOGY,VOL. 228
Copyright © 1994by AcademicPress, Inc. All fights of reproductionin any formreserved.