Planta 9 by Springer-Verlag 1978

Planta 142, 317-320 (1978)

Density Gradient and Differential Centrifugation Methods for Chloroplast Purification and Enzyme Localization in Leaf Tissue The Case of Citrate Synthase in Pisum sativum L.*

B.A. Elias** and Curtis V. Givan Department of Plant Biology,The University, Newcastle upon Tyne NE1 7RU, U.K.

Abstract. Intact chloroplasts, isolated by differentialcentrifugation and sucrose density-gradient methods, have been used to study the degree of apparent artifactual adsorption of citrate synthase (EC 4.1.3.7) to the organelles. Unfractionated homogenates layered directly on to sucrose density gradients gave elution profiles showing definite citrate synthase activity in the intact and broken plastid regions, along with the major mitochondrial peak. Nonreversible triose-phosphate dehydrogenase (EC 1.2.1.9), a cytosolic marker, showed no activity in any particulate region of the gradient. Crude chloroplast pellets and twice washed (resedimented and resuspended) chloroplasts layered on to the gradient gave progressively reduced citrate synthase activity in the plastid regions. In addition, the peak in the mitochondrial region of the gradient was virtually eliminated when washed chloroplasts were fractionated on the gradient. Differences in protein binding behavior on the chloroplasts may necessitate the inclusion of a washing step in chloroplast purification procedures. Moreover, repeated sedimentation and resuspension can also be a useful procedure to reduce mitochondrial contamination of chloroplast preparations. Key words: Centrifugation methods - Chloroplasts - Citrate synthase - Enzyme adsorption - P i s u m .

Introduction

Density-gradient techniques based on aqueous buffers now constitute the most popular method for purifying plant cell organelles, e.g. when determining the subcellular location of enzymes in plant tissues. Organelles purified by density-gradient procedures are * Work supported by the Rubber Research Institute of Malaysia ** Present Address." Labor ftir Pflanzenphysiologie, Institut fiir Allgemeine Botanik, ETH Ztirich, Sonneggstrasse 5, CH-8006 Ztirich, Switzerland

usually purer than those obtained by the older differential-centrifugation methods, where the organelle of interest is not further purified once it has been sedimented (cf. Leech, 1977). The principal advantage of the density-gradient method is that it can resolve populations of different organelles from one another, with little or no particulate cross-contamination (cf. Givan & Harwood, 1976; Leech, 1977; Yamazaki and Tolbert, 1970). While density-gradient methods definitely should continue to be employed in careful enzyme-localization studies, the gradient method a l o n e may not invariably provide a completely accurate picture. Marker enzymes arc obviously essential to confirm the purity of gradient fractions. But even using markers it is often difficult to rule out the possibility that an enzyme may associate with an organelle band owing to artifactual adsorption of a soluble enzyme originating from elsewhere. Nearly all tissue-homogenizing procedures break organelles, thereby causing solubilization of organellular enzymes. Also, unfractionated homogenates contain soluble cytosolic enzymes. Adsorption of soluble or solubilized enzymes to organelles may produce a potentially misleading picture in the gradient elution profile. In the case of nitrate reductase, Dalling et al. (1972) found that bovine serum albumen (BSA) prevented adsorption of the soluble enzyme to chloroplast membranes. BSA has, therefore, tended to become a routine ingredient in media used in enzyme-localization work (e.g. Miflin and Beevers, 1974; Elias and Givan, 1977). Leech (1977) has expressed concern lest BSA cause different types of organelles to stick together irreversibly ; however, Miflin and Beevers' work indicates that there need be no serious drawback of this kind. In this paper we examine the behavior of the enzymes citrate synthase and nonreversible, NADPlinked triose-P dehydrogenase on sucrose density gradients. We suggest that density-gradient procedures can and should be supplemented by differential centrifugation methods. In particular, repeated resuspen-

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318

B.A. Elias and C.V.Givan: Chloroplast Purification and Enzyme Localization

sion and resedimentation of chloroplast fractions is useful (a) to decrease mitochondrial contamination, and (b) to lessen the likelihood of invalid conclusions resulting from enzyme-adsorption artifacts. Materials and Methods

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Chloroplast Isolation and Purification Pisum sativum cv. Laxton Superb seedlings were grown as described earlier (Givan, 1975), and leafy shoots homogenized for a few seconds in a mechanical blender. The homogenizing buffer comprised D ( - ) sorbitol, 330raM; BSA, 0.1% (w/v); HEPES, 50 mM, pH 6.5. After filtration through Miracloth, the crude brei was either layered directly onto the density gradient or was centrifuged at 5000 g for 15 s (excluding acceleration time) with manual braking. The pellet ("crude chloroplasts") was then either layered directly onto the density gradient, or alternatively, resuspended in grinding medium (20 ml) and resedimented at 3000 g for 30 s (excluding acceleration time), resuspended and again resedimented as before. The resuspension and resedimentation procedure was termed "washing". The density gradient procedure was based on Miflin & Beevers (1974) and has been used previously by us (Elias and Givan, 1977). In this procedure, the centrifugation time is short (15 min), so that intact and broken (=envelope-free) chloroplasts approach equilibrium positions, whereas peroxisomes and mitochondria do not; after a 15 min spin, the latter two organelles thus band higher up the density gradient than either intact or broken chloroplasts.

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Enzyme and Chlorophyll Assays

Non-reversible glyceraldehyde-3-P dehydrogenase (NADP dependent) (EC 1.2.1.9) was assayed by the method of Kelly and Gibbs (1973). The assay mixture contained 2.2 ml 120 mM glycylglycineNaOH buffer, pH 7.7; NADP, 18 ~tmoles; triose-P isomerase (Sigma), 20 units, plus 0.5 ml eluted gradient fraction in a total volume of 3.0 ml. The assay was run at 30 ~ C, and the reaction was initiated by addition of 1 pmole dihydroxyacetone-P. Citrate synthase (EC 4.1.3.7) was assayed by the spectrophotometric method of Ochoa (1955). In this assay, citrate synthase was coupled to malate dehydrogenase, and the increase in absorbance at 340 nm followed. Chlorophyll was determined according to Arnon (1949).

Results and Discussion

Marker-enzyme elution profiles for density gradients of the type used here were-originally determined by Miflin and Beevers (1974), with particular reference to organelle-localized enzymes. The marker data of Miflin and Beevers have been essentially confirmed by us (Elias and Givan, 1977) and by Bryan et al. (1977). Intact chloroplasts, possessing envelopes and retaining stromal protein, move quickly to a near-equilibrium position well down in the gradient and are well separated from broken chloroplasts, peroxisomes and mitochondria. Separation between mitochondria and broken chloroplasts is less good, but some resolution is possible if the spin is halted m time to prevent mitochondria from passing into the region of broken (envelope-free) chloroplasts. In order to check on the extent of nonspecific

(i.e. generalized and indiscriminate) adsorption of soluble or solubilized enzymes to chloroplasts, we examined the elution profile of the cytosolic marker NADP-linked glyceraldehyde 3-P dehydrogenase (nonreversible) (Kelly and Gibbs, 1973). Figure 1 shows elution profiles for chlorophyll and for the nonreversible dehydrogenase, obtained when a Miracloth-filtered leaf homogenate was layered directly onto the sucrose gradient and centrifuged through it. The three chlorophyll peaks represent intact chloroplasts (A), broken (envelope-free) chloroplasts (B), and small chloroplast fragments (C) (Elias, 1977). Previous work has shown that particulate triose-P isomerase, a marker for intact chloroplasts, is found only in association with chlorophyll band A (Miflin and Beevers, 1974; Bryan et al., 1977; Elias and Givan, 1977). The cytosolic marker nonreversible glyceraldehyde-3-P dehydrogenase was entirely at the top of the gradient and was not associated with any chlorophyll peak. One can, therefore, reasonably conclude that Miflin and Beevers (1974) were correct in assuming that intact chloroplasts purified in this way did not suffer from gross, indiscriminate contamination by soluble or solubilized enzymes. Figure 2 shows elution profiles for chlorophyll and citrate synthase, when the Miracloth-filtered leaf homogenate was layered directly onto the sucrose gradient (without prior preparation of a plastid-enriched

B.A. Elias and C.V.Givan: Chloroplast Purification and Enzyme Localization

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Fig. 3. Density gradient elution profiles of crude chloroplastenriched pellet layered on sucrose density gradient, showing the distribution of citrate synthase activity and chlorophyll

pellet). The three characteristic chlorophyll peaks were evident, as in Figure 1. The elution profile for citrate synthase was complex. The major peak was clearly in the mitochondrial region of the gradient, where the marker fumarase also occurs (cf. Elias and Givan, 1977; Bryan et al., 1977), as do the mitochondrial enzymes NAD-specific isocitrate dehydrogenase (Elias and Givan, 1977) and cytochrome oxidase (Miflin and Beevers, 1974). In addition to the major mitochondrial peak of citrate synthase, one can see definite smaller peaks or " foothills" of activity associated with the broken (band B) chloroplasts and with intact (band A) chloroplasts. Assuming that there was no generalized, indiscriminate adsorption of soluble enzymes to intact chloroplasts, one could easily conclude that citrate synthase was present in chloroplasts as well as in mitochondria-particularly if use of BSA were regarded as a guarantee against the occurrence of adsorption artifacts. A considerable release and solubilization of citrate synthase from broken organelles is clearly indicated by the activity at the top of the gradient. Figure 3 shows elution-profile data obtained when a crude chloroplast pellet from a single differentialcentrifugation spin (see methods) was layered onto a density gradient. Compared to Figures 1 and 2, the elution profiles in Figure 3 differed both with respect to the patterns displayed by chlorophyll and citrate synthase. Only two chlorophyll peaks were present in the gradient profile depicted in Figure 3,

representing the intact and broken (envelope-free) organelles. Peak C (small fragments) was absent. The elution pattern for citrate synthase was also altered when a crude chloroplast pellet was prepared prior to gradient separation. Firstly, the total amount of citrate synthase relative to chlorophyll in the gradient was much reduced. Secondly, citrate synthase was now largely restricted to the mitochondrial region of the gradient, with much less of its total activity being found in the intact chloroplast region. We have shown elsewhere that the mitochondrial marker fumarase and the peroxisomal marker catalase are readily detected in the appropriate regions of the density gradient when a resuspended c r u d e chloroplast pellet has been spun through the gradient (Elias and Givan, 1977). Figure 4 shows an elution profile obtained when a crude chloroplast pellet was twice washed as described in the methods section (i.e. resuspended and resedimented). In this case there was no significant peak of citrate synthase activity anywhere in the gradient-neither in the chloroplast region n o r in the zone higher up where mitochondria band when present. The absence of citrate synthase activity in the mitochondrial region of the gradient supports our earlier suggestion that washing (resuspending and resedimenting) the crude chloroplast pellet removes much of the mitochondrial and peroxisomal contamination found in crude chloroplast pellets, as judged by the virtual absence of fumarase and catalase from the gradient fractions (Elias and Givan, 1977).

320

B.A. Elias and C.V.Givau: Chloroplast Purification and Enzyme Localization

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The evidence presented here suggests t h a t - c o n t r a r y to what could easily be concluded on the basis of a single density-gradient separation of the filtered crude homogenate-citrate synthase is not a chloroplastic enzyme in pea leaf cells. Citrate synthase is apparently confined to mitochondria, along with NAD-specific isocitrate dehydrogenase, fumarase, etc. Howevei', citrate synthase does readily adsorb to chloroplasts, so that it is necessary to wash the crude plastid fraction prior to gradient separation in order to remove adsorbed enzyme activity. As regards the relative merits of density gradients v e r s u s differential-centrifugation methods, we believe that it may be best to use the two methods in conjunction with one another, rather than using either procedure by itself. Density-gradient separations used without prior sedimentation and washing of the chloroplasts may, as with citrate synthase, lead to invalid conclusions owing to adsorption artifacts-even though BSA has been included in all media and generalized, indiscriminate enzyme adsorption minirnized. Density-gradient methods must certainly be used in critical enzyme localization work on chloroplasts; however, in our experience (cf. Leech, 1977 for an alternative view), repeated resuspension and recentrifugation of the crude chloroplast pellet can be quite effective in reducing contamination of chloroplasts by other organelles. In so far as enzyme-localization questions are concerned, we have usually found that chloroplast preparations purified by repeated resuspension and resedimentation gave results which could be subsequently confirmed on gradient-purified chloroplasts. Chloroplasts that have been washed by resuspension and resedimentation may als0 suffer less

adsorption of enzymes derived from non-chloroplastic sources. Moreover, chloroplasts obtained in this way have been less harshly treated than organelles spun through high osmolarities of sucrose. High concentrations of sucrose usually cause severe dehydration, with resulting deleterious effects on photosynthetic activity (cf. Plaut and Bravdo, 1973). Rocha and Ting (1970) have previously used differential centrifugation methods in conjunction with isopycnic gradients in an attempt to purify chloroplasts. While isopycnic procedures may no longer be the best choice for chloroplast purification (cf. Miflin and Beevers, 1974), the differential sedimentation procedure has a useful role to play in supplementing ratetype gradient techniques for chloroplast purification and should by no means be altogether discarded. The authors express their sincere thanks tO Prof. Dr. Ph. Matile for his critical review of the manuscript.

References

Arnon, D.I. : Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol. 24, 1 15 (1949) Bryan, J.K., Lissik, E.A., Matthews, B.F.: Changes in enzyme regulation during growth of maize. III. Intracellular localization of homoserine dehydrogenase in chloroplasts. Plant Physiol. 59, 673-679 (1977) Dalling, M.J., Tolbert, N.E., Hageman, R.H.: Intracellular location of nitrate reductase and nitrite reductase. I. Spinach and tobacco leaves. Biochim. Biophys. Acta 283, 505 512 (1972) Elias, B.A. : Tricarboxylic acid cycle enzyme localization studies in subcellular organelles of Pisum sativum L. Ph.D. Thesis, Univ. of Newcastle-upon-Tyne U.K. (1977) Elias, B.A., Givan, C.V.: Alpha-ketoglutarate supply for amino acid synthesis in higher plant chloroplasts. Intrachloroplastic localization of NADP-specific isocitrate dehydrogenase. Plant Physiol. 59, 738-740 (1977) Givan, C.V. : Light-dependent synthesis of glutamine in pea-chloroplast preparations. Planta 122, 281-291 (1975) Givan, C.V., Harwood, J.L.: Biosynthesis of small molecules in chloroplasts of higher plants. Biol. Rev. 51, 365-406 (1976) Kelly, G.J., Gibbs, M.: Nonreversible D-glyceraldehyde 3-phosphate dehydrogenase of plant tissues. Plant Physiol. 52, 111-118 (1973) Leech, R.M. : Subcellular fractionation techniques in enzyme distribution studies. In : Regulation of enzyme synthesis and activity in higher plants, pp. 289-327, Smith, H., ed. New York: Academic Press 1977 Miflin, B.J., Beevers, H.: Isolation of intact plastids from a range of plant tissues. Plant Physiol. 53, 870-874 (1974) Ochoa, S.: Crystalline condensing enzyme from pig heart. In: Methods enzymol. Vol. 1, pp. 685 694, Colowick S.P., Kaplan, N.O., eds. New York: Academic Press 1955 Plaut, Z., Bravdo, B.: Response of carbon dioxide fixation to water stress. Plant Physiol. 52, 28 32 (1973) Rocha, V., Ting, I.P. : Preparation of cellular plant organelles from spinach leaves. Arch. Biochem. Biophys. 140, 398-407 (1970) Yamazaki, R.K., Tolbert, N.E.: Enzymic characterization of leaf peroxisomes. J. Biol. Chem. 245, 5137 5144 (1970) Received 2 May; accepted 15 June 1978

Density gradient and differential centrifugation methods for chloroplast purification and enzyme localization in leaf tissue : The case of citrate synthase in Pisum sativum L.

Intact chloroplasts, isolated by differential-centrifugation and sucrose density-gradient methods, have been used to study the degree of apparent arti...
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