THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 263, No. 21, lasue of July 25, pp. 10289-10234,1988 Printed in U. S. A.

Purification and Characterization of Ferritins from Maize, Pea, and Soyabean Seeds DISTRIBUTION IN VARIOUS PEA ORGANS*

(Received for publication, December 21, 1987)

Jean-Pierre Laulhere, Anne-Marie Lescure, and Jean-Francois BriatS: From the Laboratoire de Biologie Moleculaire Vegetale, Centre National de la Recherche Scientifique UA 11 78, Universite Joseph Fourier, F-38041 Grenoble Cedex, France-

Ferritins from maize, pea, and soyabean seeds were purified. They contain two polypeptides of 28 and 26.5 kDa. The molecular weight of native pea seed ferritin has been estimated to be 540,000.

Pea and maize seed ferritins were compared by re- verse phase high performance liquid chromatography, amino acid composition, and two-dimensional gel elec- trophoresis. They are very similar, although four iso- forms of the 28-kDa polypeptide from the pea were observed in contrast to a unique polypeptide in maize. No isoforms of the 26.5-kDa polypeptide were de- tected.

Rabbit antibodies were produced in response to pea seed ferritin. It was shown by Western blot analysis that ferritins of the three plants analyzed share im- munological determinants. However, horse spleen fer- ritin was not recognized by the phytoferritin antibod- ies. Antibodies were also used to demonstrate that fer- ritins are not uniformly distributed in different pea organs from 30-day-old iron-unloaded plants. The pro- tein was more abundant in flowers than in fruits and roots, and was not detected in leaves.

Iron is essential for all living cells. Like other elements, iron functions both as a structural component and as a cofactor for enzymatic reactions (1).

In plants, iron deficiency causes chlorosis, and it has been known for over a century that addition of iron causes recovery of iron chlorotic plants (2). This observation can be explained by the fact that iron is essential for the synthesis of chloro- phyll as well as for the process of photosynthesis, iron being present in the active center of many electron carriers in photosystems I and 11. On the other hand, iron can be toxic to cells because ferrous ions with oxygen give rise to radicals such as OH. and 0; (3).

Ferritins, a class of proteins present in all living organisms, are known to sequester and thus detoxify iron that is taken up by cells but not utilized for metabolic requirements. On the other hand, under conditions of iron need, ferritin iron can be released for cellular utilization. Therefore, ferritins are key proteins acting as a buffer for iron, protecting cells from a harmful overdose of free iron, and tuning properly their immediate need for iron (for review see Refs. 1 and 4).

Animal ferritins have been extensively studied, and a wealth of information is now available concerning their structure,

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed.

synthesis, and function (4). However, as mentioned in a recent review (4), very little is known about plant ferritins. They have been purified from a few dicotyledons (5-7) and one monocotyledon (8). As in mammals, isoforms may exist (9). Plant ferritins are nuclear-encoded and plastid-located (10). The control of iron induction of their synthesis in bean leaves may occur at the transcriptional level (1 1).

We have initiated a study of the structure, synthesis, and function of phytoferritins. In this paper we describe a powerful method for the isolation of phytoferritins from seeds, and we characterize the native protein, its subunit(s), and isoforms. Antibodies raised against pea seed ferritins are also charac- terized and used to examine the distribution of ferritins within various organs of the pea plant.

EXPERIMENTAL PROCEDURES

Materials-Dried peas (Pisum sativum, var. Douce Provence) were obtained from Vilmorin, France, dried soyabeans (Glycine max, var. Mungo) were obtained from Sperli, Germany, and dried maizes (Zea mays, var. B73) were obtained from INRA, France.

Pea plants were grown on vermiculite for 30 days with 15 h of light (50,000 lux) and 9 h of darkness.

DEAE-cellulose was from Whatman (United Kingdom), Bio-Gel (1.5 M) from Bio-Rad, AcA 22 from Pharmacia (Sweden), and the reverse phase HPLC’ column was a 300-A pore C3 silica column (Ultrapore RPSC) from Beckman. Horse spleen ferritin was from Boehringer Mannheim. All other products used were of analytical grade.

Isolation of Plant Ferritins-Two different methods were used. In both procedures, dried seeds were allowed to swell in water for 2 days at 4 ‘C with constant air bubbling.

Pea seed ferritins were purified according to the method described by Crichton et al. (5). The DEAE-cellulose pooled fractions were then passed through a Bio-Gel 1.5 M column (1.5 X 50 cm) and eluted with 50 mM Tris-HC1, pH 7.5, 500 mM NaC1. The 2-ml fractions were analyzed for protein and iron concentration.

The DEAE-cellulose pooled fractions were further purified by reverse phase HPLC using a discontinuous acetonitrile gradient (10- 70%). Fractions with absorbance at 214 nm were pooled and lyophy- lized.

The second procedure for ferritins from pea, soyabean, and maize seeds was as follows. Volumes given are for 150 g of dry seeds. Swollen seeds were ground in a Waring Blendor prior to the addition of 2 volumes of extraction buffer (50 mM potassium phosphate, pH 7.0, 1% polyvinylpyrrolidone). This suspension was then homogenized for 30 s using a Polytron (PCU-2, Kinematica, Switzerland) a t maximum speed. After centrifugation at 10,000 rpm for 10 min, in a Sorvall GSA rotor, MgClz was added to the supernatant to a final concentra- tion of 50 mM to precipitate all debris and ribosomes. A second low speed centrifugation (10,000 rpm for 15 min) gave rise to a superna- tant which was filtered through a 500-pm nylon filter (Blutex GG, Tripette and Renaud, Paris). 20 min after the MgClz addition, triso- dium citrate and RNase A were added to final concentrations of 70

The abbreviations used are: HPLC, high performance liquid chro- matography; SDS, sodium dodecyl sulfate.

10289

10290 Characterization of Ferritins from Plants mM and 10 pg/ml, respectively, to complex the magnesium and to prevent phytoferritin precipitation. After 2 h at 4 “C this supernatant was centrifuged through 2 ml of 5 mM potassium phosphate, pH 7.0, 0.01% Triton X-100, and 50% glycerol. Pellets were resuspended in a final volume of 5 ml of 7 mM potassium phosphate, pH 7.0,0.006% Triton X-100, and 30% glycerol. This suspension was stored overnight at 4 “c prior to centrifugation at 10,000 rpm for 15 min in a Sorvall SS34 rotor. The brown pellet was resuspended in 1 ml of 10 mM potassium phosphate, pH 7.0, prior to a 3-min centrifugation in an Eppendorf tube. After washing the pellet with 200 pl of phosphate buffer, the 1.2 ml of supernatant containing ferritins was loaded on a AcA 22 gel filtration column (18 X 2.5 cm) previously equilibrated in 10 mM potassium phosphate, pH 7.0. Fractions of 2 ml were collected and used to determine protein and iron concentrations. After verification of the purity of the ferritins using polyacrylamide- SDS gel electrophoresis the fractions were pooled, aliquoted, and stored at -20 “C.

Protein and Iron Determination-Protein extractions from differ- ent pea organs were carried out according to Nechustai and Nelson (12).

of Bradford (13). Protein concentrations were determined according to the method

phenanthroline (0.02%) at 510 nm, pH 6 (50 mM acetic acid/NaOH Iron concentration was measured by absorbance of Fe2+/ortho-

buffer), using an excess of dithionite crystals as the reducer. Phytoferritins were selectively stained in polyacrylamide gels by

soaking in a 2% (w/v) K(Fe (CN)6) solution in 50% ethanol, 7% acetic acid.

Protein Gel Electrophoresis-Electrophoresis of proteins under de- naturing conditions was done in 12.5% polyacrylamide-SDS gel ac- cording to Laemmli (14).

Two-dimensional gel electrophoresis was performed according to O’Farrell (15) with pH gradient ranging from 4.6 to 7.2 in the first dimension.

Determination of the molecular weight of the native pea seed ferritin was done using a 5-10% polyacrylamide gradient gel run at 50 volts for 63 h at room temperature. The buffer system of the gel was TB (89 mM Tris borate, 89 mM boric acid). Gels were stained with Coomassie Blue R-250.

Amino Acid Composition-20 pg of seed ferritins purified by reverse phase HPLC were lyophilized and hydrolyzed in 50% propionic acid, 50% HCl, 0.014 M P-mercaptoethanol, and 0.1% phenol for 16 h at 110 “C. After evaporation of the samples, pellets were resuspended in 0.2 M sodium citrate, pH 2.2, and the amino acid composition deter- mined using an LKB Alpha analyzer calibrated with 10 ng of each amino acid.

Production of Antibodies Raised against Pea Seed Ferritins-100 pg of pea seed ferritins, purified by reverse phase HPLC, were injected into a rabbit on four occasions at 2-week intervals. The titer of the pea seed ferritin antibodies was determined after the second and third injection, prior to final bleeding which occurred 2 weeks after the fourth injection.

Western Blots-Protein samples were run in duplicate on a single gel. One half was stained and the other half was transferred to nitrocellulose BA83 paper (Whatman). Electroblotting was done ac- cording to Towbin et al. (16). Probing of the blots with pea seed ferritin antibodies was done according to Hahn and Stiegler (17) using anti-rabbit IgG sheep immunoglobulins coupled with peroxidase (Biosys).

RESULTS

Purification of Phytoferritins from Seeds-Ferritins were isolated from sorghum roots (8), and from soyabean (7), bean (6), pea, and lentil (5) seeds. For these four legumes, only one polypeptide was observed on SDS-polyacrylamide gel, and the molecular mass of this ferritin subunit was variable: 28 kDa for soyabean (7), 26.5 kDa for bean (6), 21.4 kDa for lentil, and 20.3 kDa for pea (5) ferritins.

This discrepancy provoked further investigation. The pea seed ferritin was purified by two different procedures, and the soyabean and maize seed ferritins were partially purified.

The procedure described by Crichton et al. (5), using alter- nate low speed and high speed centrifugations and DEAE- cellulose chromatography, was used first to purify ferritin from pea seeds. Proteins eluted from the DEAE column at

0.15 M NaCl coincided with a peak of iron detected with orthophenanthroline (Fig. lA). These protein fractions con- tained two polypeptides of 28 and 26.5 kDa (Fig. 1B).

Gel filtration in 0.5 M NaCl on a Bio-Rad 1.5 M column of the pooled DEAE-cellulose fractions containing ferritins gave one peak of protein corresponding to an iron peak. The gel filtration fractions contained the two polypeptides of 28 and 26.5 kDa (not shown). This result is consistent with the possibility that two different subunits build up phytoferritins, as do the H and L subunits of animal ferritins (Ref. 18, see “Discussion”).

Another procedure, described under “Experimental Proce- dures,” was also used to purify the ferritins from pea, soya- bean, and maize seeds. This method which is fast (no dialysis step) allows the purification of 50-80 mg of pure ferritin (Fig. 2, lune 2) from 1 kg of dry pea seeds. The yield obtained is 10-fold higher than that obtained with the method of Crichton et al. (5). This procedure makes it possible to purify, almost to homogeneity, ferritins of soyabean and maize seeds (Fig. 2, lane 3 and 4 ) . It is important to notice that SDS-polyacryl- amide gel electrophoresis of ferritins isolated by this second procedure also reveals two polypeptides in the range of 28 and 26.5 kDa for the three different plants we have considered (Fig. 2). The iron-to-protein ratio has been determined for the three plant seed ferritins analyzed and is reported in Table I. Based on the molecular mass of the native protein (see below), an estimate of the number of iron atoms/molecule of ferritin is also given in Table I.

Reverse phase HPLC of purified pea seed ferritins gave a major peak eluting at 48% acetonitrile (Fig. 3A). This is consistent with the strong hydrophobicity of ferritins. SDS- polyacrylamide gel electrophoresis of this protein fraction reveals two polypeptides of 28 and 26.5 kDa (Fig. 3B) . The same result was obtained with maize seed ferritin (not shown).

Determination of the Molecular Mass of the Ferritin Oligo- mer of Pea Seeds-An electrophoresis procedure with a 5- 10% polyacrylamide gradient gel calibrated with horse spleen ferritin was used to determine the molecular mass of the pea seed ferritin oligomer (Fig. 4). Ferritins are identified in this kind of gel by staining the iron with ferricyanide (not shown). Coomassie staining of the proteins (Fig. 4) coincides with iron staining. The mass of pea seed ferritin was estimated to be 540 kDa. This value is higher than the value of 463.7 kDa calculated from neutron scattering experiments by Crichton et al. (5) but confirms that phytoferritins have a higher molecular mass than mammalian ferritins (5).

characterization of Pea and Maize Seed Ferritins Using Two-dimensional Gel Electrophoresis-Ferritins purified by reverse phase HPLC from pea and maize seeds were analyzed by two-dimensional gel with a pH gradient in the first dimen- sion ranging from 4.6 to 7.2.

The 28-kDa polypeptide of the pea seed ferritin gives at least four isoforms with PI ranging from 5.94 to 6.17 (Fig. 5A). The equivalent polypeptide of maize seed ferritin does not exhibit isoforms and has a PI of 5.7 (Fig. 5B).

The 26,500-Da polypeptide of pea seed ferritin has a PI of 5.54, and no isoform has been detected (Fig. 5A). This obser- vation also applies to the equivalent polypeptide of maize seed ferritin, the PI of which is 5.65 (Fig. 5B).

Amino Acid Composition of Pea and Maize Seed Ferritins- The amino acid compositions of the pea and maize seed ferritins that have been purified by reverse phase HPLC are very similar (Table 11). The percentage of nonpolar amino acids for pea and maize is 40.9 and 40.2, respectively. Basic amino acids represent 15.1% of the pea seed ferritin and 15.7% of the maize protein. Acidic amino acids represent

Characterization of Ferritins from Plants 10291

B 1 2 3 4 5 6

0,4 t 0.3 .

- 9

02 . ozE

0.1

Fracllons Number

FIG. 1. DEAE-cellulose chromatography of pea seed ferritin. A, elution profile showing the superimpo- sition of the protein and the iron peak. B, polypeptides present in the different fractions eluted from the DEAE- cellulose column. Lane 1, 10 p1 of sample buffer without protein; lane 2, 10 pl of mixed fractions 23, 24, and 25; lane 3, 10 pl of mixed fractions 20, 21, and 22; lane 4, 4 pg of marker; lane 5, 10 p1 of mixed fractions 18, 19, and 20; lane 6, 10 pl of mixed fractions 16, 17, and 18. Analysis was performed on a 12.5% polyacrylamide-SDS gel.

1 2 3 5

94 - 87 0

30 - " I -

20 - "- FIG. 2. Subunit composition of ferritins isolated from pea

(lane 2), soyabean (lane 3) and maize (lane 4 ) seeds. Phytofer- ritins were isolated using the second procedure described under "Ex- perimental Procedures." Lane I contains 4 pg of marker. 5 pg of each phytoferritin fraction were loaded on a 12.5% polyacrylamide-SDS gel (lanes 2-4).

TABLE I Iron content of plant ferritins

Ironfprotein Iron atomsfmolecule"

nmollclg Pea 2.99 1800 Soyabean 4.56 2500 Maize 1.65 990

This ratio has been calculated using 540,000 as molecular weight for plant ferritins.

26.4% of the pea seed ferritin and 28% of the maize protein. Immunological Relationship of Ferritins from Different

Sources-Rabbit antibodies raised against pea seed ferritin

04 57 13 30

20 14

purified by reverse phase HPLC (Fig. 3) were obtained. West- ern blot analysis of purified ferritins from pea, soyabean, and maize seeds, and of horse spleen (Fig. 6A) , shows that anti- bodies raised against the pea seed ferritin recognize not only the two polypeptides of its own antigen (Fig. 6B, lane 2 ) but also the two polypeptides of the soyabean (Fig. 6B, lane 3 ) and maize (Fig. 6B, lane 4 ) seed ferritins. Horse spleen ferritin is not recognized by antibodies raised against phytoferritins (Fig. 6B, lane 5 ) .

Therefore, seed ferritins of two dicotyledons (pea and soy- abean) and one monocotyledon (maize) share immunological similarities but are not immunologically related to animal ferritins. This result is consistent with the very recent work by Sczekman and Joshi (7 ) showing that antibodies raised against ferritins from soyabean seeds do not cross-react with horse spleen and rat liver ferritins.

Distribution of Ferritins in Various Organs of the Pea Plant-To estimate the relative abundance of ferritin in dif- ferent pea plant organs, Western blot analysis was performed using total protein extracts of roots, leaves, flowers, and fruits from 30-day-old normal pea plants (i.e. not iron-loaded) and rabbit antibodies raised against pea seed ferritins. The poly- peptide patterns of these different organs are shown in Fig. 7A.

As can be seen in Fig. 7B, the relative amount of ferritin is variable in the different organs analyzed. No ferritins are detected in normal green leaves (Fig. 7B, lane 3) . The seed ferritin antibodies, diluted a t 1/500, are able to detect 5 ng of pure pea seed ferritin (not shown) when 15 pg of the leaf extract was loaded on the gel. Therefore, if ferritins are present in normal green leaves they represent less than 0.3 pg/mg proteins. The maximum quantity of ferritin is detected in flowers (Fig. 7B, lane 4) . Less ferritin is present in fruits (Fig. 7B, lane 5 ) . The amount detected in roots (Fig. 7B, lane 2 ) is lower than in fruits. The iron concentration of each

10292 Characterization of Ferritins from Plants

B 1 2

FIG. 3. Analysis of pea seed fer- ritins using reverse phase HPLC. 30 pg of pure ferritin was pelleted by cen- trifugation a t 100,000 rpm for 1 h in a Beckman TLA 100-2 rotor and resus- pended in 30 p1 of 10 mM Tris-HCI, pH 7.6, 150 mM Lic1, 6 M urea, 0.4 M mer- captoethanol prior to injection onto a 300-A pore C3 silica column (Ultrapore RPSC, Beckman). A, elution profile. B, polypeptide composition of the HPLC- purified ferritin. Lane 1, 2 pg of protein contained in the peak shown in A; lane 2 ,4 p g of marker.

1 2 3 4

"

7 21 35 49 Tlme (mn)

FIG. 4. Determination of the molecular mass of pea seed ferritin using nondenaturing gradient gel electrophoresis. Lanes I and 4 , 10 pg of horse spleen ferritin; lane 2, 10 pg of pea seed ferritin; lane 3, 10 pg of horse spleen ferritin mixed with 10 pg of pea seed ferritins. Gel was a linear 5-10% polyacrylamide gradient in Tris borate buffer. It was run at 50 volts for 63 h, after which time no movement of the ferritin could be detected.

extract has been determined (legend of Fig. 7) . Iron is present in extracts of the four different organs analyzed. The higher concentration of iron has been measured in flowers where ferritins are the most abundant (Fig. 7B, lune 4 ) . However, it is clear that the total iron of an organ is not necessarily correlated with ferritin iron. For example, 150 pmol of iron/ pg protein were found in leaves in which no ferritins were detected. On the other hand, ferritins were detected in roots which contain less total iron than flowers (119 pmol of iron/ pg protein).

A

94 67.-

4 3 .

30-

20.

v 94

67 s

4

FIG. 5. Two-dimensional gel electrophoresis of HPLC-pu- rified ferritins from pea ( A ) and maize ( B ) seeds. Arrows indicate the 28- and 26.5-kDa subunits and the four pea seed isoforms of the 28-kDa subunit. pH gradient in the first dimension was from 4.6 to 7.2.

We noticed that in fruits a faint second polypeptide is present below the predominant one. In flowers (Fig. 7B, lune 4 , two faint polypeptides are located below the major one. High molecular weight components are also immunologically detected in Fig. 7B, lanes 2,4, and 5. Their origin is unknown, but they could be cross-linked subunits as observed in ferritin of low iron content from horse and sheep spleen.

In conclusion, pea ferritins are not uniformly distributed in all the different organs of a plant, and the relative abundance of ferritins seems to be organ-regulated.

DISCUSSION

So far, plant ferritins have been purified from sorghum roots (8) and soyabean (7), bean (6), pea, and lentil seeds (5). They were partially characterized biochemically from the four legume seeds. I t was established from these studies that apoferritin may be organized in a 24-mer of a unique subunit,

Characterization of Ferritins from Plants 10293

TABLE I1 Comparison of the amino acid composition of the pea and maize

seed ferritins ND. not determined.

A B

I 2 3 4 5 1 2 3 4 5 c-

% Residues in acids pea seed ferritin

% Residues in maize seed ferritin

Ala Arg Asx CYS Glx GlY His Ile Leu LYS Met Phe Pro Ser Thr

8.8 3.6

10.3 0

16.1 6.8 5 3.9 9.7 6.5 1.3 5.3 3 4.2 2.4

Trp ND TY r 3.8 Val 8.9

1

9.5 5.1 1 0 7 7.2 3.3 4.3 0.7 7.3 1.8 5.6 0 2.8 1.8

ND 4.1 8.3

14 "."

1

FIG. 6. Characterization of antibodies raised against the pea seed ferritins purified by reverse phase HPLC. A, Coo- massie staining of a 12.5% polyacrylamide-SDS gel loaded with: 4 pg of marker (lane I ) , 5 pg of pea seed ferritins (lane 21, soyabean seed ferritins (lane 3) , maize seed ferritins (lane 4 ) , and horse spleen ferritins (lane 5) . E , Western blot analysis of a duplicate of the gel shown in A using pea seed ferritin antibodies diluted a t 1/500 and anti-rabbit IgG sheep immunoglobulins coupled with peroxidase di- luted a t 1/800.

the mass of which is different in the four plants analyzed (5- 7), although the existence of different subunits has been mentioned in the case of bean leaf ferritin (9).

In this paper, it is shown that whatever the procedure used, two polypeptides of 28 and 26.5 kDa are found in ferritins from two dicotyledons (pea and soyabean) and one monoco- tyledon (maize). It is important to notice that the phytofer- ritins purified elute as a homogeneous peak on gel filtration columns and that pea seed ferritin gives a well defined band on a native polyacrylamide gel a t a molecular mass of 540 kDa. These observations are reminiscent of those of Sczek- man and Joshi (7) with heavy soyabean ferritin which has a molecular mass of 572 kDa but which is built from a unique 28-kDa polypeptide. This raises the question of the origin of the 26.5-kDa polypeptide in our preparation. Sczekman and Joshi (7) have shown that the polypeptides smaller than 28 kDa (26, 23.5, and 22 kDa) are related to ferritin obtained in the supernatant of a MgC12 precipitation from soyabean seeds soaked for 72 h in water a t room temperature. They have

FIG. 7. Distribution of ferritins in various organs of pea plants. A , Coomassie staining of a 12.5% polyacrylamide-SDS gel loaded with: 4 pg of marker (lane I ), 15 pg of total proteins extracted from roots (lane 2) , leaves (lane 3) , flowers (lane 4 ) , and fruits (lane 5) . B, Western blot analysis of a duplicate of the gel shown in A using pea seed ferritin antibodies diluted a t 1/500 and anti-rabbit IgG sheep immunoglobulins coupled with peroxidase diluted a t 1/800. 200 pl of each extract was mineralized and the iron content determined ac- cording to Ref. 19. The results obtained are as follow: root extract, 119 pmol of ironlpg protein; leave extract, 150 pmol of iron/pg protein; flower extract, 305 pmol of iron/pg protein; fruit extract, 165 pmol of iron/pg protein.

clearly shown that the 22-kDa polypeptide results from a cleavage of the 28-kDa polypeptide and is used to build up heterogeneous polymers equivalent to animal hemosiderin. They have not analyzed the 26 and 23.5-kDa polypeptides. Under our conditions (soaking the seeds for 48 h at 4 "C with constant air bubbling prior to extraction), we failed to detect the 22kDa-polypeptide even by Western blot analysis (a very sensitive method), but we always observed the 26.5-kDa poly- peptide. There are two possibilities to explain the presence of this polypeptide within homogeneous phytoferritin prepara- tions. The first postulates that isoforms of phytoferritins could exist in relation to the step of development or the organ of a plant which is considered. This hypothesis would involve different genes for phytoferritin subunit synthesis as is the case for the H and L subunits in animal ferritins (18). The second postulates that a specific cleavage of some of the 28- kDa polypeptides could occur, leading to hidden breaks within the native oligomer. The product of these hidden breaks could be the 26.5-kDa polypeptide. If this was the case, the 28/26.5 kDa ferritin we have characterized is a functional intermedi- ate in the pathway leading from the 28-kDa ferritin to the 22- kDa ferritin described by Sczekman and Joshi (7). Work is in progress to investigate these two possibilities.

From immunological studies and determination of the amino acid composition, we can conclude that phytoferritins of monocotyledons and dicotyledons share structural similar- ities. However, a t least one difference has been observed because isoforms of the higher mass polypeptide exist in pea seed ferritins and not in maize.

The new procedure developed to purify the pea seed ferritin to homogeneity is fast (no dialysis step) and allows prepara- tion of 50-80 mg of pure phytoferritin/kg of dry seeds. The 10-fold higher yield by this procedure, compared to that obtained with Crichton's method (5) can be explained by the fact that mercaptoethanol, a strong reducer, is not present in the extraction buffers used. Therefore, iron ferritin is not reduced and not released from ferritin. As a consequence, less ferritin molecules are lost in the supernatant during the high speed centrifugation step of the purification. I t is important

10294 Characterization of Ferritins from Plants

to obtain large amounts of pure phytoferritin. This enables biophysical studies and investigations into the nature of the mechanisms of iron release and uptake by ferritins from plants.

Finally, the difference in the relative amount of ferritin observed in different organs of pea plants raises questions about the nature of the molecular mechanisms controlling these amounts. Ferritins were not detected in pea leaves. However, immunoprecipitation of in vitro translation prod- ucts from poly(A+) RNA of bean leaves with bean seed ferritin antibodies indicates that translatable ferritin mRNAs are present in this organ (11). We have performed the same kind of experiments with i n vitro translation products of RNAs isolated from pea leaves, flowers, and fruits. Preliminary results indicate that translatable ferritin mRNAs are present in similar amounts in the three organs we have analyzed (not shown). Work is in progress to further investigate this prob- lem, but it seems that the variations observed in the amount of ferritin in different organs of normal plants (ie. not iron- loaded) could be post-transcriptionally regulated.

Acknowledgments-We are indebted to Drs. 0. Massenet and R. Stejiades for their help with HPLC, amino acid analysis, and two- dimensional gel electrophoresis. We wish to thank Pr. Marguerie for the use of the amino acid analyzer located in his laboratory.

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