Agric. Biol. Chem., 43 (10), 2137. 2142, 1979 2137

Isolation of Intact Chloroplasts from Spinach Leaf by Centrifugation

in Gradients of the Modified Silica "Percoll"*

Tetsuko TAKABE, Mikio NISHIMURA and Takashi AKAZAWA

Research Institute for Biochemical Regulation, School of Agriculture , Nagoya University, Chikusa, Nagoya 464, Japan

Received April 23, 1979

The isolation of the photosynthetically competent chloroplast preparations was under-

taken by means of the density gradient centrifugation on the modified silica sol "Percoll." A

clear separation of the intact chloroplast sustaining the high photosynthetic activities (light

dependent CO2 fixation ca. 130ƒÊmol/mg Chl• hr) was established. The contamination of

mitochondria and peroxisomes was estimated to be less than 3% by measuring the activities

of their marker enzymes. The chloroplasts were proved to be free from endoplasmic reticulum

and cytosol. The photosynthetic CO2 fixation of the isolated chloroplast preparations was

saturated by illumination of the light intensity of 20,000 Lux (12mW/cm2, 400•`750 nm).

The separation of intact chloroplasts sustain-ing the high photosynthetic activities (C02-fixation and 02 evolution) is crucial for elucidating the nature of photosynthetic reaction and its regulatory mechanisms. The procedures reported by Walker1,2) and Jensen and Bassham3) employing grinding of leaf tissues in specified buffers, followed by rapid differential centrifugation, have been widely used in recent years. The preparations thus obtained, however, contain broken chloroplasts, mitochondria and other particulate fractions4) and are obviously not suitable for studying those aspects of photosynthesis research, such as photorespiration, which are believed to involve the cooperative interaction of several organelles, e.g.,chloroplasts, peroxisomes and mitochondria.5) The sucrose density gradient

technique has been widely used for the isolation

of organelles, but, although sucrose gradients

have been useful in determining subcellular

enzyme localization,6•`8) chloroplasts obtained

generally retain only limited activity in light-

dependent CO2 fixation.6,8) Morgenthaler et

a1.9 ,10) have succeeded in isolating intact chloro

plasts from spinach leaf, which retain high

photosynthetic activities (80ƒÊmol CO, fixed/

mg Chl• hr) by isopycnic centrifugation in a

mixture of silica sol (Ludox AM) and PEG.

The chloroplasts also sustained a high activity

of protein biosynthesis and the method was

thus found useful for examining the biosynthe

sis of several proteins including RuP2 car

boxylase11,12) Cyt b-55913) and coupling factor

1 (CF1).14) It should be noted that the outer

membranes of such chloroplasts were seen to

be intact.

We have attempted to establish standard

separation method for intact chloroplasts by

the gradient centrifugation in the modified

silica sol coated with polyvinylpyrrolidone,

'P ercoll'.15,16)

MATERIALS AND METHODS

Preparation of crude chloroplasts. Freshly harvested leaf of spinach (Spinacia oleracea L. cv. Kyoho) was used throughout the experiment; 35g (wet wt.) of leaf

Abbreviations: BSA, bovine serum albumin; Chl,

chlorophyll; Cyt, cytochrome; G-R, grinding-resus-

pension; HEPES, N-2-hydroxyethylpiperazine-N•L-

ethane-sulfonic acid; MES, 2-(N-morpholino) ethane

sulfonic acid; PEG, polyethylene glycol; PEP, phospho-

enolpyruvate; PPBF, Percoll solution containing 5

polyethylene glycol, 1% bovine serum albumin and 1

Ficoll 400; RuP2, ribulose-1,5-bisphosphate.

* This paper No. 50 is in the series "Structure and

Function of Chloroplast Proteins." The research was

supported in part by the grant-in-aid from the Ministry

of Education of Japan (310413, 347099, 376039) and

the Nissan Science Foundation (Tokyo).

2138 T. TAKABE, M. NISHIMURA and T. AKAZAWA

tissues were diced into small segments (1 cm in width)

and immediately homogenized by blending for 2 sec

in 120ml of 50mM MES-NaOH buffer (pH 6.1) con

taining 0.33M sorbitol, 2mM Na2-EDTA, 1mM MnCl2,

1 MM MgCl2, 20mM NaCI, and 2mM isoascorbic acid

as described by Jensen and Bassham.3) The resulting

homogenate was briefly filtered through one layer of

Miracloth (Chicopee Mills Inc., Milltown, N. J.) and

the chloroplasts collected by centrifugation at 2,500•~g

for 70 sec at 4•Ž. The crude chloroplast preparation

was resuspended in 5ml of the grinding medium.

Gradient centrifugation in Percoll. Five g of PEG

6000 (Sigma, St. Louis), 1g of BSA (Sigma, St. Louis),

and 1g of Ficoll 400 (Pharmacia Fine Chemicals,

Uppsala) were dissolved in 100ml of Percoll (Pharmacia

Fine Chemicals, Uppsala). This mixture was referred

to as PPBF throughout the paper. The density of

PPBF was 1.142g/cm3. A linear gradient of 11 to

90% (v/v) PPBF was made by a Hitachi DGK-U

automatic density gradient maker. The gradient

solution contained in addition 0.33m sorbitol, 2mM

Na2-EDTA, 1mM MnC12, 1mM MgCI, 1mM Na-

pyrophosphate, 5mM isoascorbic acid, 5mM•Ž gluta

thione, and 50mM HEPES-NaOH buffer (pH 6.8),

which was referred to as G-R ('grinding-resuspension')

medium.9) Two ml of the crude chloroplast prepa

ration were loaded onto 35ml of a linear gradient

solution and centrifuged in a Beckman SW-27 swinging

bucket type rotor at 7,000rpm for 15 min; the maximal

centrifugal field was 8,820•~g. After centrifugation,

the chloroplast fractions in the 35 ml gradient were

harvested by aspiration into a centrifuge tube, diluted

3 fold with the G-R medium, and sedimented again

under the same conditions as described in the prepa

ration of crude chloroplast. The chloroplast prepa

ration thus obtained was resuspended in the G-R

medium and stored in an ice bath until used. Al-

ternatively, 0.9ml fractions were collected from 35-m1

centrifuge tubes, and aliquots were used for measure

ments of the enzyme activities.

Enzyme assays. (a) RuP2 carboxylase (EC 4.1.1. 39) activity was measured as described by Nishimura et al.17) (b) Cyt c oxidase (EC 1.9.3.1) activity was determined by measuring the decrease of the absorbance at 550nm due to the formation of reduced Cyt c according to the method of Smith.18) (c) Catalase (EC 1.11.16) was assayed by measuring the disappearance of H202 spectrophotometrically at 240nm following the method of Luck.19) (d) The assay method for NADPH-Cyt c reductase of Lord et al.21) was employed

after a slight modification. The reaction mixture contained in a total volume of 1ml; 20mM K-phosphate

(pH 8.0), 0.2mM NADPH, 0.02mM Cyt c, and 10mM KCN. The reaction was started by adding NADPH , and the reduction of Cyt c was monitored at 550n

m using a Gilford Model 240 recording spectrophotometer.

(e) NADH-nitrate oxidoreductase (EC 1.6.1.1) activity

was measured as Hageman and Hucklesby.21) The

reaction mixture contained in a total volume of 1ml;

25 mm K-phosphate buffer (pH 7.5), 0.4mM KNO3i

and 0.1mM NADH. The reaction was started by

adding NADH at 30•Ž. After 15min, the reaction

was stopped by adding 1% (w/v) sulfanylamide in

1.5N HCl. Then 1ml of 0.02% (w/v) N-(1-naphthyl)-

ethylenediamine-HCI was added and allowed to stand

for 10min. One ml of 20% cold trichloroacetic acid

was then added to the mixture. After the centrifugation

at 3,000•~g for 10 min, the absorption at 540nm was

measured. Control experiments were done without

NADH. (f) PEP carboxylase (EC 4.1.1.31) activity

was measured as described by Maruyama et al.22)

The reaction mixture contained in a total volume of

1 ml, 80mM Tris-HCI (pH 7.8), 10 mat NaH14C03

(0.2 mCi/mmol), 2mM PEP, 2mM MgC12, 5mM gluta-

thione, 0.2mM NADH, and 14 units of malate dehydro

genase. The reaction was started by adding PEP at

30•Ž. After 15 min, the reaction was stopped by

adding 50ƒÊl of acetic acid. The reaction mixture was

dried at 85•Ž for 60 min and radioactivity measured

in a Packard liquid scintillation spectrometer.

Analytical methods. Chl content was determined according to the method of Walker.28) Measurements of the density of Percoll solution were carried out using an ATAGO refractometer (Tokyo) . It was confirmed that the refractive index is directly proportional to the density of the Percoll solution.

Photosynthetic 14C02 fixation and 02 evolution.

Photosynthetic C02 fixation and 02 evolution were

measured basically following the procedures reported

by Cockburn et al.24) The reaction mixture (final

volume lml) contained: 50 trim HEPES-NaOH (pH 8 .0),

0.33 M sorbitol, 2mM Na2-EDTA, 1mM MnCl2, 1mM

MgCl2, 10mM NaH14CO3 (0 .5mCi/mmol) and chloro

plasts (1020 tog Chl). The reaction was started by

adding NaH14C03 at 25•Ž and incubation continued

for varied periods. The radioactivities fixed were

measured in a liquid scintillation spectrometer .

Photosynthetic 02 evolution was measured using

a Rank Bros. oxygen electrode. Illumination was

provided by a white tungsten lamp (200 W). Unless

otherwise indicated, light intensity was 30,000 Lux

(14mW/em2 of 400•`750nm) which is saturating the

photosynthetic reaction under the conditions presently

employed.

For the purpose of calculating the percentage con

tamination of the broken chloroplasts in the intact

chloroplast preparations, the method of Heber and

Santarius25) was employed.

Isolation of Spinach Chloroplasts in Percoll Gradients 2139

RESULTS AND DISCUSSION

After rapid disruption of spinach leaf tissues

in a blendor, the crude chloroplasts obtained

by centrifugation at 2500•~g were layered on

top of a gradient solution of 1•`90% (v/v)

PPBF solution. After an additional centri

fugation at 8820•~g for 15min, two Chl-

containing bands were separated, the lower

band containing intact chloroplasts and the

upper one broken chloroplasts. Figure 1

shows the separation profile of chloroplasts,

the density of intact and broken chloroplasts

being 1.14g/cm3 and 1.09g/cm3, respectively.

Without the addition of PEG 6000, we found

that the separation of two types of chloro

plasts is somewhat incomplete. Although the

density of each fraction was slightly larger than

that reported by Morgenthaler et al.9)using

FIG. 1. Localization of Enzyme Activities in the

Separated Fractions by Percoll Density Gradient

Centrifugation.

Fractions separated by Percoll gradient centrifugation were subjected to the following assays: A, Chl and density; B, RuP2 carboxylase; C, Cyt c oxidase and catalase. Experimental details for the Percoll density

gradient centrifugation and the enzyme activity measurements are described in the text. The enzyme activities in 0.9-ml fractions are expressed as /4mo1 substrates utilized or products formed/min.

the Ludox-PEG 6000 gradient solution, a clearer separation was achieved in the present study, density difference (0.005) of two fractions being slightly larger than the one reported by the former workers. It will be noted that RuP2 carboxylase, a chloroplast marker enzyme, is localized exclusively in the intact chloro

plasts, while Cyt c oxidase activity is detectable near to the broken chloroplast fraction. The density at which mitochondria are located is 1.07 (g/cm3), somewhat larger than the buoyant density of mitochondria reported for spinach isolated in Ludox HS.15) On the other hand, catalase, a peroxisomal marker enzyme, was localized on top of the gradient.

The degree of contamination of mitochondria and peroxisomes in both crude and

purified intact chloroplasts was tested by assaying the activities of the individual marker enzymes. As presented in Table I, the presence of catalase activities detectable in the crude preparation (13% of the total activity) has drastically reduced to 1.4% in the preparation of intact chloroplasts. Similarly the crude preparations of chloroplasts contained about 22% of the total Cyt c oxidase activities, but this contamination was reduced to 2.8 after recovery from the Percoll gradient. One third of the total NADPH-Cyt c reductase, a marker enzyme of the endoplasmic reticulum, was found in the crude preparation, but was barely detectable in the intact chloroplasts. Reardon et al.281 have found no nitrate reduc-tase activity, which is a cytoplasmic marker,

TABLE I. ENZYME CONTENTS IN INTACT

CHLOROPLASTS SEPARATED BY PERCOLL

GRADIENT CENTRIFUGATION

N.D.: not detectable.

2140 T. TAKABE, M. NISHIMURA and T. AKAZAWA

in the intact chloroplasts isolated by density

gradient centrifugation on Ludox AM. We also tested the contamination of cytosol by assaying nitrate reductase and PEP carboxylase activities, which are believed to be localized in the cytoplasm of C, mesophyll cells.27) These activities were not detectable in the prepa rations of intact chloroplasts. It has often been pointed out4) that the plastids separated by the sedimentation velocity or isopycnic centrifugation techniques contain as much 10% as contaminated mitochondria and micro-bodies. However, Miflin and Beevers28) re

ported that the plastid fraction separated by a relatively brief centrifugation on a semi-linear sucrose gradient contains less than 2 of the contaminated microbodies and mitochondria. The results given in Table I appear, therefore, to show that the isolated chloro

plasts from spinach leaf tissues are pure. The preparations were inspected by a phase-contrast microscope. Photograph of Fig. 2 shows the typical, refractile appearance of chloroplast having an intact envelope.

From the results of the measurements of

the Hill-reaction with ferricyanide as electron

FIG. 2. Intact Chloroplasts Seen Under Phase Con

trast Light Microscope (Bar is 10ƒÊm).

acceptor, the preparation thus obtained was found to contain approximately 90% of "Class I" chloroplasts .

The high photosynthetic activities of the

intact chloroplasts in comparison with those

of the crude preparations are shown in Fig. 3

(A; 0, evolution and B; CO, fixation). The

photosynthetic activities showed a linear in-

crease up to 10 min incubation, and the maxi

mal activity was calculated to be 130ƒÊmol

CO, fixed/mg Chl -hr.

FIG. 3. Photosynthetic 02 Evolution (A) and C02 Fixation (B) by Intact Chloroplasts .

Light-dependent C02 fixation and 02 evolution activities were simultaneously measured in the

same reaction vessel following the method of Cockburn et a1.24) Reaction was carried out in a

vessel of Rank Bros. oxygen electrode. l0-ƒÊl samples withdrawn by a microsyringe at the stated

reaction intervals were treated with 0.1 ml of acetic acid and applied to a liquid scintillation counter

for measuring the photosynthetic CO, fixation.

Isolation of Spinach Chloroplasts in Percoll Gradients 2141

Fin. 4. pH Dependence of Photosynthetic C02

Fixation by Intact Chloroplasts.

Assay conditions used were the same as that described

in Fig. 3, except that of varying pH values of Tricine

buffer solution in the reaction mixture. Incubation

time was 10 min.

FIG. 5. Activities of Photosynthetic CO, Fixation

as a Function of Light Intensities.

The basic experimental conditions were the same as

that employed in Fig. 3, except that the light intensities

were varied as stated. Incubation time was 10 min.

The optimal pH of the CO, fixation reaction was 8.0, nearly equal to that reported by other investigators3,24) (Fig. 4).

The photosynthetic activities (CO,-fixation)

were saturated by illumination of 20,000 Lux

(12mW/cm2 of 400•`750m) (Fig. 5). Spinach

chloroplasts can also be separated in a medium

whose density is intermediate between those of

broken and intact chloroplasts. Approxi-

mately 20ml of a solution containing 50%(v/v)

PPBF to bring its density to 1 .10g/cm3 was

placed on a cushion of 10ml of a solution

containing 90% (v/v) PPBF. After centri-

fugation at 8,820•~g for 10 min, the broken

chloroplasts formed a very sharp band on top

of the gradient. The intact chloroplasts also

formed a sharp band on the cushion. We find

that these chloroplasts are active and pure

nearly to the same extent as separated by a

standard centrifugation method described

above.

As the osmolality of Percoll is low (<20mOs/Kg H2O), the isolation procedure of the intact chloroplasts from spinach leaf by Percoll

gradient centrifugation is evidently feasible for analytical studies of photosynthesis and other reactions in vitro. At the same time we can expect that the method is potentially useful for isolating other organelles, e.g., mitochondria and peroxisomes, retaining their

physiological integrity. Hopefully we can ex-amine the intracellular interactions of these organelles in green cells more precisely than was previously possible.

Acknowledgment. The authors wish to record their

sincere thanks to Dr. C. A. Price for invaluable dis

cussions and guidance in connection with this investi

gation and his kind help for preparing the manuscript.

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