preparation of cellular plant organelles from spinach leaves

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 140, 398-407 (1970)

Preparation of Cellular Plant Organelles From Spinach Leaves’

VICTOR ROCHA AND IRWIN P. TING

Department of Life Sciences, University of California, Riverside, California 92501

Received April 20,197O; accepted July 7,197O

Broken chloroplasts, intact chloroplasts, mitochondria, and microbodies were isolated and purified from spinach leaf tissue using a sequential sedimentation rate- equilibrium density (S-p) centrifugation technique. Organelle pellets (10009 and 3000g) were prepared in 0.5 M sucrose, 1 mM EDTA, 1 mM 2-mercaptoethanol, and 0.1% bovine serum albumin all in 0.05 M Tris buffer, pII 7.5. The organelle pellets were separately layered on linear sucrose-density gradients of 40-XOy, (w/v) and centrifuged for 3 hr at 25,000 rpm (& = 74 X lo9 rad2.sec-1) in an ultracentrifuge. The 1OOOg pellet resolved into broken chloroplasts (withont outer envelope and stroma), intact chloroplasts (with outer envelope andstroma) plus mitochondria, and microbodies; the 30009 pellet resolved into broken chloroplasts, mit,ochondria, and microbodies. Electron micrographs and marker enzymes revealed a high degree of purity. Markers used were glycolate oxidase and slow particulate malic dehydrogenase for microbodies, cytochrome c oxidase and fast particulate malic dehydrogenase for mitochondria, NADP glyceraldehyde K-phosphate dehydrogenase and chlorophyll for intact chloroplasts, and chlorophyll for broken chloroplasts. Est,i- mates of purity using the enzyme markers suggested that the intact chloroplast fraction was about 13cj, contaminated by mitochondria and 3.6c/; by microbodies. The mitochondrial fraction was contaminated about 12% with microbodies and was essentially free from chlorophyll. The 30009 microbody fraction was essentially pure. The estimated isopycnic densities for the organelles were: broken chloroplasts, 1.17; intact chloroplasts, 1.21; mitochondria, 1.21; and microbodies, 1.2.; g/cm3. When estimated by centrifugation in a 2%50cjb (w/v) sucrose gradient, the sediment,ation-rate orders were: intact chloroplasts > broken chloroplasts > microbodies > mitochondria.

Studies concerned with in vivo metabolic events frequently require the isolation and purification of intact cellular organelles. We have been interested in the intracellular localization of various enzymes of COZ metabolism and previously reported the existence of several isoenzyme systems distributed in different subcellular organelles (1, 2). Also, we reported multiple forms of malic dehydrogenase in spinach leaves, and that one was associated with the chloroplast fraction (3). Recently, Tolbert et al (4, 5) using improved organelle-separation techniques presented data indicating that spinach chloroplasts do not have NAD malic dehydrogenase activity, but that iso-

1 Supported in part by the University of Cali- fornia Intramural Research Fund and by NSF Grant GB-6735.

enzymes were present in both mitochondrin and microbody fractions. Past reports of malic dehydrogenase activity (and certainly other enzymes) localized in spinach chloroplasts may be due to contamination by microbodies and/or mitochondria.

Because of the importance of knowing the exact intracellular localization of enzymic activity, and because of our interest in the demonstration of the localization of isoenzymes in different cellular compartments, we have investigated further the sucrose- density gradient centrifugation method for the preparation and purification of intact cellular plant organelles. In this paper, we report a simple and effective method for the isolation of chloroplasts, mitochondria, and microbodies (peroxisomes) in high purity.

398

PREPARATION OF CELLULAR PLANT ORGANELLES 399

MATERIALS AND METHODS

Heagenk. OAA,2 NAD, NADH, NADPH, 3- PGA, L-cysteine, cytochrome c, peroxidase (horse- radish), DEAE-cellulose, PMS, and glycolate were obtained from Calbiochem (Los Angeles, Cali- fornia). ATP, o-dianisidine diHC1, FMN, C&H, and NBT were obtained from Sigma Chemical Corp. (St. Louis, Missouri). Hydrolyzed starch used for starch gel electrophoresis was purchased from Connaught Medical Research Laboratories (Toronto, Canada). Reagents for acryIamide gel clectrophoresis were obtained from Eastman Chemical Corp. All ot,her chemicals used were of reagent grade.

Enqrr~R assags. All spectrophot,ometric assays were conducted at, 25”with a double-beamspectro- photomet,er and aut,omatic recorder. Malic dehydrogenase was assayed spect,rophotometrically in the direct ion of oxaloacetate reduction (6). The standard assay contained 0.004 rnbr N,4DH, 0..5 mM OAA, enzyme, and 0.03 M Tris (pH 7.4) in a total voIume of 3.0 ml. The oxidation of NADH was determined at 340 rnF. Initial velocities were used 10 calculate activities.

Cytochrome c oxidase was assayed spect,ro- phot,omet,ricalIy by following the oxidation of reduced cytochrome (’ at, 5.50 rnp (7). The reaction mixt,ure cont,ained 0.623 mM reduced cytochrome c, enzyme (treated with Brij 23) and 0.05 M Trig (pH 7.4) in a total volume of 1.25 ml. Cytochrome c was reduced chemically with dithionite. The ac- t,ivitg of glycolate oxidase was determined using the redox dye, o-dianisidine. The standard assay cont,ained 1.0 mM FMN, 1.3 ml1 o-dianisidine diIIC1, 1 rnM glycolate, 6.04 ml peroxidase (1 mg/ ml), enzyme (t,reated with Brij .58), and 0.05 nt Tris (pH 8.0) in a t)otal volume of 1.0 ml. Initial velocities were determined by following the increase in absorption of oxidized o-dianisidine at 44.5 rnp. NADPglyceraldehyde :&phosphate dehydrogenase was assayed by COUplirlg with phosphoglyceric Acid kinase (8). The reaction mixture cont,ained 0.87 rn~ cysteine, O.:i rnM GSH, 6.67 rnhf 3-PGA, 0.5 1llM ATP, 6.67 mM MgSOI , 0.094 mM NAI>PH, enzyme (t,reated with Bri,j 5X), and 0.03 M Tris (pH 7.4) in a total vollime of 3.0 ml. Chlorophyll was determined by the method of Arnon (!I).

Gel elecfrophmesis. St.arch-gel electrophoresis

2 Abbreviations used are : OAA, oxaloacetatc; NAl), nicotinamide a.denine dinucleotide; NADH, reduced nicotinamide adenine dinucleotide; NAl)PH, reduced nicotinamide adenine dinucleo- t’ide phosphate; 3-P(;A, 3-phosphoglyceric acid; PMR, phenazine methosulphate; ATP, adenosine Qiphosphate; FMN, tlavin mononucleotide; GSH, reduced glutat.hione; NBT, nitro blue tetrazolinm; MDH, malic dehydrogenase.

was conducted according t,o Fine and Costello (10) as described earlier (11) using a phosphate- citrate buffer at pH 7.0. Flat-bed acrylamide-gel elect,rophoresis was conducted according to Akroyd (12).

Estimation of oryanelle serIimenta&n rates.

Relative organelle sedimentation rates were estimated by centrifugation in a linear sucrose-density gradient [23-50~0 (w/vjj. Rat,es for broken chloroplasts, mitochondria, and microbodies were est.i- mated by centrifugntion at 1OOOg for lti-min intervals up to 60 min. Intact chloroplast,s, because of t,he high sedimentation rat,es, were centrifuged at 5OOg. Sedimentation rates, S, were calculated from the following st-andard expression (13) : dr/dt = Sm2r. No corrections were made for viscosity, gradient, density, Or osmotic effects.

Isolation and prlr<jcaiion of cellular particles. Fresh spinach was purchased at local markets, washed thoroughly with distilled water, and de- ribbed. The washed tissue was st.ored overnight in the cold prior t,o use. 100 g of the prepared spinach leaf t,issue was thoroughly minced with a food chopper ilk l.iO ml of grinding medium (0.5 lg sucrose, 1 m&l EDTA, 1 rnbr 2-mercaptoethanol, 0.15; bovine serrun albumin) all in 0.05 M Tris, pH 7.5 (14). After mincing, the spinach preparation was ground in a blender for 2-3 set at, low speed. The initial mince and shcJrt, low-speed grinding is absolutely necessary for the isolation of intact chloroplasts in high yield. The homogenate was gently filtered through eight, layers of cheese- clot’h. The filtrate containing organelles was ten- t,rifuged at, 2Xg for 90 see t,o remove debris and intact cells. The supernatant fraction was centrifuged at 1OOOg for .5 min in an angle rotor to obtain the 1OOOg pellet (%(I-1000 g). The lat,ter supernatant. fluid was recentrifuged in the angle rotor for 15 min at 3OOOg. Pellet’s were gently suspeJJded in 405~; sucrose (w/v) by agitation with a rubber policeman. All operations were conduct.ed iJJ a cold room or over ice (O-4’). Linear slicrosc-density gradients of 40-X0’>; (w/v) (34 ml) were prepnrcd in 50 ml cellulose nitrate centrifuge tubes with the use of a double-chamber, gravity-flow apparatus. The sucrose was prepared in 0.05 M Tris, pH 7.;5, and 1 mM EDTA. Linearity of t.he gradient. wits confirmed by refractometry. Atso, deusities of certain portions in the gradient. were estimated with hydrometers. Prior to use, the gradients were chilled to about 0’.

The lOOO- and 3OOOg resuspended pellets were layered over separate linear gradients by pipet ting down the side of the centrifuge tubes. Gradient,s were centrifuged for 3 hr in a Beckman SW-27 swinging bucket rotor at 25,000 rpm (ant = 74 X 10g rad2.sec-J) in a Spinco Model L2-50 preparative ultracentrifJJge. Centrifugation at lower speeds in

400 I:OCHA ANI) ‘rIN(:

swinging bucket rotors resulted in a percentage of the particles coming t,o their equilibrium positiolls, but a high degree of contamination of low-density components by high-densit.y components.

After centrifugatioll 10 equilibrium, the gradients were fractionated under pressure through a hole in the bot,tom of the tube into approximat,el2; i10 equal frnct ions of abolit 1.2 ml.

Prior l,o assay, each fraction was treated by sonicat.ion or with dct.ergents to liberate proteins.

Elcc!tvn mi~ros~op~~. Samples were prefixed with 2.6(,; glntaraldehyde in 0.1 Y phosphate buffer, pH 7.0, for I hr. After fixation, the microbody fraction was centrifuged at Zi,OOO rpm in an SW-27 swinging-huckei rowr in a Gpinco Model

1240 preparative ultracelilrifuge for 30 min. Sam- plcs were postfixed with l”(, bllflered osmium (0.1 M phosphate buffer, pH 7.0) overnight. After fixation the samples were dehydrated wit.h an acctollc seqrlence and embedded 111 Maraglas. Thin sec- tions were cut with an LRR-microtome and post- stained with lead (*itrate alld Ilranyl acetate. Sam- ples were observed wit,h au Hitachi HU-11 electron microscope.

Separation of ,malic deh!ytlrogenase isoenzymes. The protein of an homogenate isolated by grinding 100 g of leaves was precipitst,ed with arn- moniumsulfate (0-O.Rsaturation). The precipitate was resuspended irl 0.00.5 M phosphate buffer, pH 7.0, and dialyzed overnight against the same buffer. The sample was layered 011 a I X 10 DF:Al+cellnlose Rlliowexchallge column (1.5 X 15 cm of 0.61 meq/liter) equilibrated with 0.00.5 M phosphate bluffer, pH 7.0. After washing the column with 30 ml of 0.OO.i M phosphate bufler (pH 7.0), a linear phosphate gradient was applied to t,he column as described by Mat~dell arid Hershey (15). Fractions were collected (3.0 ml) and assayed for malic dehydrogenase. The peak frxc*- t.ions were analyzed by starch-gel electrophoresis.

11 EHULTS

SeparatioN of cellular orqanelles. When the lOOOy organelle pellet was purified by ce&ifugation 011 a linear 40-80 % (w/v) sucrose-density gradient, two visible chlorophyll-containing bands were resolved (Fig. lA, bands 1 and 2). The 300$ pellet centrifuged under identical condtttons resolved into ~1 single chlorophyll-containing band (Fig. lB, band 1) corresponding to the uppermost (less dense) green band of the 1OOOg pellet. In the position of the second chlorophyll band of the 1OOOg pellet, the 3OOOg pellet demonstrated a slightly yellow opaque band (lcig. lB, band 2). A third

A B

FIN. I. Photographs of spinach Icaf orgallelles separated by litlear sllcrose-delisity gradient cell- trifrlgation. Left, IOOt~g pellet showing broken chlorop1ast.s Wld illtact chloroplasts. Right, 3OOOg pellet showing broken chloroplasts, mito- rhondria, and microbodies. See text for details of method.

more dense band appeared as an ivory opaque region (Icig. 18, band 3).

Electron microscopy of the visible bands indicated that the upper chlorophgll-con- tairting band (p -I 1.17) of both the 1OOOy rind 3000!/ pellets (P’ig. IA and B, band 1) NXS composed primarily of broken chloroplasts (Fig. 2B). These plastids appear as stacks of grana lamcllae often held together by interconnecting lamellae (stroma lamal- he). The plnstids appear to lack both outer envelopes and stroma (stripped chloroplasts). Thr second chlorophyll band in a density region of about 1.21 (Fig. IA, band 2) appeared to be intact chloroplasts with intact outer envelopes (Fig. 2C). These chloroplnsts appear ns stacli!: of grann lamellae embedded in stroma. Elcctron- dense osmophilic globules aw visible wi-ithin. An occasional mitochondrion was evidwt. The second band of the 3000!/ pellet in the density region of 1.21 (E‘ig. lB, band 2) appears to be composed of intact mitochondria (Fig. %D). The mitochondria are double-membmnrt structurts with charac- teristic invaginations of the inner membranes. An occ:bsiorl:tl intact, chloroplwt NXY

FIG. 2. ISlectroll micrographs of an intact spinach leaf cell (A) (l(j,200 X ); hroketl chloroplast fraction (13) (i,2 JO X); intact chloroplast fraction (C) (8.300 X); mitochol~drial frac- t.ion (1)) (12,100 X); and microbody frart,ion (E) (16,500 X). 13 and C were from gradiwts similar to the 1OOOy pellet cwltrifugation shown in Fig. 1 and B, I), alld E wcrc from gm- dierrt,s similar to the 3OOOg pellet s11ow11 in Fig. 1. Actual samples were taken from fractions containing t.he mssim~lrn activity of e~~dog:e~~ous marker r~~zy~nes and/or chlorophyll. Reference markers represent 1 P.

-1-01

402 ROCHA AND TING

visible and probably accounts for the slightly yellow appearance of the band. In addition, there is some microbody contamination of the mitochondria. The dense band present in density position of about 1.25 (Fig. lB, band 3) appears to be a relatively pure microbody fraction (Fig. 2E). These bodies have no distinguishing ultrastructure other than the granular appearance. J$a,ny of the structures appear to be in a state of disor- ganization. No other organelles were observed.

If chlorophyll, cytochrome c oxidase, and glycolate oxidase are used as markers for chloroplasts, mitochondria, and microbodies respectively, an estimation of purity and intactness can be made (Table I). An analy- sis of the fractions throughout the 1OOOg gradient indicated two chlorophyll-containing bands (Fig. XA). Of the total chlorophyll in the gradient, approximately 50% occurs in the whole chloroplast fraction (in Fig. 3A, 54% chlorophyll in broken chloroplasts and 4F% in the intact chloroplasts). There was essentially no chlorophyll at the top of the gradient. Using the chlorophyll in the original leaf homogenate as a base, the final recovery of intact chloroplssts is about 0.15%.

The intact chloroplasts in the 1OOOg density gradient are contaminated with mitochondria. Using cytochrome c oxidase as a marker, me estimate that 64% of the mitochondrial membranes in the gradient are in the intact chloroplast region. Consid- ering both the 1OOOg and 3000g gradients, about 13 % of cytochrome c oxidase is in the intact chloroplast region of the 1OOOg gradient. Hence, the mitochondrial contamination is estimated to be 13 %. h!laking the same calculation for glycolate oxidase, we estimate that microbody contamination of intact chloroplasts is about 3.5%. Presum- ably, the cytochrome c oxidase activity in the intact chloroplast is associated with intact mitochondria. The ratio of malic dehydrogenase activity to cytochrome c oxidase in the intact chloroplast region was about 15.7 while in the broken chloroplast region it was 7.5. Because intact mitochondria in any region of the gradient should have a constant ratio of malic dehydrogenase

TABLE I

ESTIMATION OF PE:RCENT ORGANELLE CROSS CONTAMINATIONS

contaminating organelle Organelle

preparation Microbodies ,$!$k, $5: !Ec

Microbodies < l’i;, < 1% <l% Mitochon- 12’;; <1:;I <1y;

dria Intact 3 p . ’ ,/o 13“’ /O - 5-10ygb

chloroplasts

Broken Cl%;, 6% <l% - chloroplasts

a Based on organelle marker enzymes. b Apparently represents “intermediate class”

chloroplasts (broken outer membrane but still contain stroma) ; estimated from electron micrographs.

to cytochrome c oxidase, it is assumed that the cytochrome c oxidase activity in the broken chloroplast region represents mitochondrial membranes. The electron micrographs did not reveal intact mitochondria in the broken chloroplast region. This is further substantiated by the ratio of malic dehydrogenase to cytochrome c oxidase in the 3000g gradient. In the mitochondrial region, the ratio is 17.2 while in the broken chloroplast region it is 5.0; essentially the same as the 1OOOg gradient.

The microbody fraction appears to be relatively pure. Essentially no cytochrome c oxidase or triose phosphate dehydrogenase are in the microbody region. The contamination of other fractions by microbodies can be estimated from the glycolate oxidase activity. In the 1OOOg gradient, approximately 37% of the glycolate oxidase activity is at the top of the gradient. The 37% should be an estimate of organelle breakage. Within the gradient, 14.5 % of the glycolate oxidase activity was present in the intact chloroplast region while 85.5% was in the microbody region. Considering the 3000g gradient, 32 %I of the total glycolate oxidase was at the top. Considering just the gradient, 32% of the activity was in the mitochondrial region. Considering both the lOOO- and 3000g

PREPARATION OF CELLULAR PLANT GRC AN ELLlB 403

Fraction Number

_ 1.4

5 _ 1.2

4 -1.0

t -O.E\,

36 E

ci g

-0.6 i

E 2 0

-0.4

I - 0.2

1

- I.300

12 -1.275

IO -1.250

T 06 -1.225 \o

E E

2 x .Z

c z 06 -1.200;;

04 -1175

32 -1.150

1 1.30

_ 0.5 _ .I2 - 1.275

-0.4 _.I -1.25

I7 5

z-.06 -1.225;

- 0.3g I ,o

= a a. D .Z

‘0 +- z

: _ .06 -1.20 z

-0.25

Fraction Number

FIG. 3. Sucrose-density gradient elution profiles. Upper, IOOOg pellet; lower 3000g pellet, (see Fig. 1). Upper (1OOOg): Fraction 27 = broken chloroplasts; fraction 18 = intact chloroplasts and an occasional mitochondrion; fraction 10 = microbodies. Lower (3OOOg): fraction 23 = broken chloroplasts; fraction 16 = mitochondria; fraction 8 = microbodies. G.O. = glycolate oxidase, MDH = malic dehydrogenase, cyto c = cytochrome c oxidase, Chl = chlorophyll, TDH = NADP-glyceraldehyde-3-phosphate dehydrogenase.

404 ROCHA AND TING

gradients, 12 % of the microbody marker enzyme is in the 3000g mitochondrial fractions. Hence, we estimate the contamination to be about 12%. There is little contamination of microbodies by mitochondria, but some microbody contamination of the mitochondrial fraction. The electron micrographs suggest this same distribution.

Estimation of’ orgarbelle density and relative sedimentatiorr velocity. In a 40-80 %, (w/v) linear sucrose-density gradient, the density of broken chloroplasts was estimated to be 1.17 (Table II). The density of intact chloroplasts and mitochondria was estimated to be about 1.21, and the density of microbodies was estimated to be about 1.25.

Relative sedimentation velocities were estimated by centrifuging the organelles in a Z-50 70 linear, sucrose-density gradient for periods up to 1 hr (Fig. 4). The order of sedimentation n-as : intact chloroplasts > broken chloroplasts > microbodies > mitochondria. The calculations for mitochondrin and microbodies were based on four points, the calculation for broken ehloroplasts was based on two points, and for intact chloroplasts on a single point. Uncorrected relative sedimentation rates under these conditions were: intact chloroplasts = 161,000; broken chloroplasts = 30,500; microbodies = 9,000; mitochondria = 3,000.

Our estimates of the average diameters of particles from electron micrographs indicated that intact chloroplasts, mitochondria, and microbodies were on the order of 4.3, 0.9, and OM p respectively. Estimates from

TABLE II

Es,rtM.\rroN OF SPIN.\CH T,ka~ ORG.\NI~:~,I,K LIEN-

SI’I’Y IN A 40-80’;,i, (N/V) LINIMl sVCKOSI+

1)P:NSI'l'Y (:R.\DIENT" ~_~--. -

Organelle Density (g/a+) .-~ .-

Broken chloroplasts 1.167 zt O.OO.T* (7)< Intact chloroplasts 1.211 * 0.003 (5) Mitochondria I .208 + 0.004 (4) Microbodies 1.248 z!z 0.004 (5)

” The densities are estimated from the medium organelle posit.ion within the gradient.

* 1)ensit.y + SI) in grams per cubic centimeter. ’ Nttmber of experiments used in calculation.

100 -

broken chloroplasts

o 60 0 z 2 - 40 i

I/ d microbodies

‘“c

0 IO 20 30 40 50 60 Time (minutes1

FIG. 4. Estimat,ion of apparent sedimentat,ion ‘. veloctty m a 2:)~.5Oc/u (w/v) linear sucrose-density

gradient. Cent,rifugations are corrected to 1OOOg. Four identical gradients were prepared and re- moved at IS-mitt intervals. Estimated sedimentation coefficients (S) are: intact chloroplasts = 161,000; broken chloroplast,s = 30,500; microbodies = 9,000; mit,ochondria = 3,000.

the preparations isolated from the gradient were 3.8, 1.3, and 0.35 p for chloroplasts, mitoehondria, and microbodies. Hence, the preparation and gradient centrifugation tend to shrink chloroplasts and microbodies whereas mitochondria enlarge.

Intracellular localization of malic dehydrogenase isoenzymes. Four malic dehydrogenase peaks were obtained by elution of a protein homogenate from a DEAE-cellulose column with a 0.02-0.2 JI phosphate gradient (Fig. 5A). The four MDH isoenzymes appear to be localized such that peak I is unique to the microbody fraction while peak II is associated with the mitochondrial fraction (Fig. 5B and C). Peaks III and IV would not sediment at 100,000~1 for 2 hr and are, therefore, defined as soluble. Starch-gel electrophoresis confirmed that the column peaks represent four different electrophoretic forms of malic dehydrogenase (Fig. 6). In addition, the electrophoretic mobilities of the isolated isoenzymes correspond exactly with the zymogram of the total homogenate (Fig. 6). Density-gradient fractions intermediate between the microbody and mitochondria maxima have malic dehydrogenase starch-gel patterns which reflect the degree of organelle contamination (Fig. 7). Con-

PREPAI~ATION OF C:ICLLUI,AR PLAST OI~(;ANl5I,LI~:S 40.5

Phosphate & 0.02+0.2M Phosphate oroditnt 4.81 I -

4.4 A 4.0

3.6

3.2

2.8

2.4

e 1.6 - yo I L 5 1.4 -

2 1.2 -

f 1.0 - I 0.6-

0.4 -

0.2 -

2.0

1.6 C

1.6 II 1.4

I.2 fl

IO 20 30 40 50 60 70 60 Fraction number (3ml)

FIG. 5. Elution of malic dehydrogenase isoenzymes from a l)EAE-cellulose anion-exchange column. A. Peak I = isoenzyme associated with the microbody fraction; Peak II = isoenzyme associated with mitochondrial fraclion; Peaks III and IV = isoenaymes not. sedimentating at 100,Ot~Og. B. Elrltion profile of MI)H activit.y ex- t.racted from isolated microbodies (see Fig. 3). C. fCl\ltioll profile of MI)H activity extracted from isolated mitocholldria (see Fig. 3).

sequently, in addition to glycolate oxidase, slow-migrating particulate malic dehydrogenase appears to be an excellent marker protein for microbodies while fast-migrating particulate malic dehydrogenase is a good marker for mitochondria.

Acrylamide-gel electrophoresis of the total spinach leaf homogenate results in excellent separation of the two particulate malic dehydrogenase isoenzymes and the predom- inant soluble form. Poor resolution of the

minor soluble isoenzyme was obtained (Fig. S). Because of the mpidity of separation (about 90 mm), acrglamide gels might be preferred over starch gels for spinach IMDH isoenaymes.

I)ISCUSSION

A combination of sedimentation velocity (S) and isopycnic density (p) centrifugation on linear sucrose gradients from 40-80s (w/v) (p = 1.150-1.297) is a suitable method to prepare broken chloroplasts (lacking outer envelopes), intact chloroplssts (with outer envelopes), mitochondria, and microbodies from spinach leaves. A pellet obtained by grinding nsshed leaf tissue in a sucrose-Tris buffer containing IZDTA, mercaptoethanol, and bovine serum albumin, and centrifugation at 1000~ for 5 min results in m enriched intact chloroplast fraction containing mitochondria and traces of microbodies. Separation of the latter organelle fraction by centrifugation to equilibrium on

the linear sucrose-density gradient results in a pure broken chloroplast fraction, an intact chloroplast frwtion with mitochondria and a purified microbody fraction. A similar sucrose-gradient centrifugation of a 3000~~ organelle pellet results in a pure broken chloroplnst fraction, a relatively pure mitochondrial fraction contaminated with an occasional intact chloroplast and microbodies, and a pure microbody fraction free from mitochondrin and chloroplasts. Pre- viously, Leech (16) separated intact chloroplasts from other cellular particles using a two-phase discontinuous sucrose gradient, and Still and Price (17) separated intact chloroplasts from broken chloroplaxts using a sedimentation-rate technique in a zonal rotor. A sedimentation-density (S-p) double- centrifugation technique using lOOO- and 3000~~ pellets as reported here allows for the preparation of broken chloroplasts, intact chloroplasts, mitochondria, and microbodies.

The equilibrium density of the microbodies was calculated to be 1.27 gjcm3. This estimate agrees quite well with the reports of others for microbodies from other tissues (18). Our estimate of the equilibrium density of spinach mitochondria was 1 .Zl corresponding to cst.imates of others (14, IS). Intact chloroplwts appear t’o hove nearly

406 ROCHA ANI) TING

FIG. 6. Malic dehydrogenase starch-gel electrophoresis of the anion-exchange column chromatographic profiles shown in Fig. 5. a = total homogenate prior to column chromatography; b = fraction from Peak I of Fig. 3A; c = fraction from Peak II of Fig. 5A; d = fraction from Peak III of Fig. 5A; e = fraction from Peak IV of Fig. .?A; f = fraction from Peak I of Fig. 5B; g = fraction from Peak II of Fig. 3C.

FIG. 7. Malic dehydrogenase starch-gel electrophoresis of sucrose-density gradient fractions obtained from the 30009 pellet shown in Fig. 3. Num- bers 8,11,14,16, and 18 correspond to the fractions in Fig. 3. H = total homogenate.

the same density of 1.21 whereas broken chloroplasts are considerably lighter at

FIG. 8. Photograph of acrylamide gel electro-

about 1.17 g/cm3. As a particle approaches phoresis of the total homogenate obtained from spinach leaves. The photo should be compared

its isopycnic point, its sedimentation velocity with column a of Fig. 6.

within the gradient approaches zero (13). For this reason, our particles centrifuged at have reached equilibrium. Probably, how- maximum speed in a Spinco SW-27 rotor for ever, the error is small and the estimated p 3 hr (& = 74 X 10s rad2. se@) may not values are reasonably correct.

PREPARATIOX OF CXLLULAR PLANT OI:GANl’Ll,l~;S 407

Our estimation of the sedimentation velocities indicated that the order of sedimentation to reach equilibrium would be: intact chloroplasts > broken chloroplasts > microbodies > mitochondria. The sedimentation coefficients, however, depend on several factors. The S values will vary with the initial grinding and preparation medium since the elastic organelles should assume some density depending on the medium. Furthermore, the sedimentation velocity will change as the osmotic potential of the gradient changes and water and/or perhaps sucrose are transferred across the organelle limiting membrane; and also the scdimenta- tion rate will vary as the viscosity of the medium changes with sucrose density. The sedimentation velocity, therefore, should be a function of the method of preparation and nature of the gradient. Hence, our estima- tions of S should be taken as relative values for our conditions.

In the spinach leaf tissue which we are using there are four malic dehydrogenase isoenzymes separable by DEAE-cellulose column chromatography and starch-gel or acrylamide-gel electrophoresis. Two of the isoenzymes, both slow-moving on electrophoretic gels, are apparently particulate- bound and two (fast-moving) are soluble. The data seem to be relatively conclusive that the slow-moving malic dehydrogenase in spinach leaves is uniquely associated with microbodies (5)) and that the fast particulate malic dehydrogenase is uniquely associated with mitochondria. The fast-migrating soluble forms are apparently either readily solubilized by our extraction techniques, or are not associated with cellular particles. We agree with Tolbcrt et al. (4) that in spinach leaves the available evidence for a unique mnlic dehydrogenase isoenxyme associated with chloroplasts is not valid, and that, spinach chloroplasts probably do not have SAD-malic dehydrogenase. It is quite clear that malic dehydrogenase isoenzymes are excellent markers and are an efficient means of estimating the purity of organelles.

In summary, in combmation with proper grinding and initial extraction, a sequential sedimentation rate-equilibrium density (S-p) t.echnique for the separation and purification of spinach leaf organelles appears to be satisfactory to prepare broken chloroplxsts

(i.e., without outer envelopes and st,romzl), intact chloroplasts (i.e., witli outer envelopes, stroma, and soluble-stromal enzymes), intact mitochondria, and intact microbodies (peroxisomes). JIalic dehydrogenase isoenzymes sltould be useful to c&mate purity of the organellcs.

The aut.hors thank Miss Stephanie Lindsey an11 Dr. W. W. Thomson for aid ill cleptron microscol)y. One of 1,s (VX) a~kllr)wlctlgrs a fellowship from EOP Student, Aid Fund #~O,OOO.

2. h~UIiF:RJI 1 $. K., .\ NI) 1’1 NU, 1. P., /+bchi?,/.. Bioph,f/s. Acln 167, 2:<0 (L!Xs)

3. Rocr-ra, V., MTTKEILJI, S. Ii., .cu) TING, I. I’., Biochem. Biophys. Res. Cotnmm. 31, 890 (1968).

4. TOLBER,~, N. I:., OICSEIL, A., Y.Mu.~KI, It. K., HAGEMAN, R,. II., 21s~ KIS~KI, T., Plant Physiol. 44, 135 (1969).

5. YAM.IZ.LKI, R. K., ANI) TOLUKRT, N. I<;., Bio- chinz. Kioph?/s. A da, 178, II (1969).

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preparation of cellular plant organelles from spinach leaves

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