Plant Physiol. (1988) 86, 922-9260032-0889/88/86/0922/05/$0 1.00/0

Biosynthesis of the Snowdrop (Galanthus nivalis) Lectin inRipening Ovaries1

Received for publication September 11, 1987 and in revised form November 23, 1987

ELS J. M. VAN DAMME* AND WILLY J. PEUMANSLaboratorium voor Plantenbiochemie, Facultein der Landbouwwetenschappen, Willem de Croylaan 42, B-3030 Leuven, Belgium

ABSTRACT

The biosynthesis and processing of the Galanthus nivalis agglutinin werestudied in vivo in ripening snowdrop ovaries. Using labeling and pulsechase labeling experiments it could be demonstrated that the snowdroplectin is synthesized as a precursor of relative molecular weight (Mr) 15,000which is posttranslationally converted into the authentic lectin polypeptideOfMr 13,000 with a half-life of about 6 hours. Gel filtration of an extractof [3HJleucine labeled ovaries on Sepharose 4B showed that a significantportion of the newly synthesized lectin is associated with the particulatefraction. When the organeliar fraction was fractionated on isopycnic su-crose gradients this lectin banded in the same density region as the en-doplasmic reticulum (ER) marker enzyme NADH cytochrome c reductase.Both radioactivity in lectin and in enzyme activity shifted towards a higherdensity in the presence of 2 miliimolar Mg-acetate indicating that thelabeled lectin was associated with the rough ER. Labeled lectin could bechased from the ER with a half-life of 4 hours and then accumulated inthe soluble fraction. Whereas the ER-associated lectin contains exclusivelypolypeptides of M, 15,000 the soluble fraction contains both precursormolecules and mature lectin polypeptides. The snowdrop lectin in the ERis fully capable of binding immobilized mannose. It is associated intotetramers with an appropriate molecular weight of 60,000. These resultsindicate that newly synthesized snowdrop lectin is transiently associatedwith the ER before transport and processing.

Until now most of the information about the biosynthesis andprocessing of plant lectins has been obtained with seed lectins.In vivo and in vitro studies of lectin synthesis in soybean (Glycinemax) (14), field bean (Vicia faba) (4), pea (Pisum sativum) (5,6), french bean (Phaseolus vulgaris) (1), castor bean (Ricinuscommunis) (10), rice (Oryza sativa), and wheat (Triticum aes-tivum) (9) have shown that these lectins are all synthesized ashigher mol wt precursors which are subsequently processed atthe co- and/or posttranslational levels.

Lectins occur also in different types of vegetative tissues ofmany dicotyledonous as well as monocotyledonous species wherethey can represent a significant part of the total protein content.Recently we isolated a mannose-specific lectin from bulbs ofsnowdrop (Galanthus nivalis) (12). Further studies have shownthat the lectin is not confined to the underground parts of theplant but also occurs in leaves and stems as well as in certain

' Supported in part by grants of the National Fund for Scientific Re-search (Belgium) of which W. P. is a Senior Research Associate. E. V.D. acknowledges the receipt of a Fellowship of the Belgian Instituut totAanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en

Landbouw.

parts of the flower, especially the ovary (E Van Damme, un-published data). The GNA' is a tetrameric protein built up offour identical subunits of M, 13,000, which does not containcarbohydrate (12).

In this paper we report the biosynthesis of the snowdrop lectinin ripening ovaries. We show evidence that this lectin is synthe-sized in the ER as a precursor of M, 15,000 which is posttrans-lationally modified.

MATERIALS AND METHODS

Material. Flowering plants of Galanthus nivalis were collectedlocally. Unless used immediately they were kept in water toprevent wilting. Ovaries were excised immediately before theonset of the experiments.

Radioactive Labeling of the Ovaries. Ovaries were cut longi-tudinally and each half incubated with a small droplet (10 ,l)containing 10 ,Ci [3H]leucine for appropriate times. Five ovarieswere used for each experimental condition. In chase experimentsovaries were labeled with [3H]leucine for 2 h, washed free oflabeled precursor, and incubated for different times in the pres-ence of 20 Al of a saturated solution of unlabeled leucine. Duringthe experiments ovaries were kept on a parafilm sheet in closedPetri dishes at 20°C. To prevent dessication moistened filter pa-per was placed below the parafilm sheets on which the ovarieswere kept.

Tissue Homogenization. After labeling for the appropriate timesthe ovaries were rinsed with distilled water and blotted dry. Twodifferent media were used for homogenization and isolation ofthe organelles. Both contained 100 mm Tris-HCl (pH 7.8) and12% sucrose; medium A contained in addition 1 mM EDTAwhereas medium B contained in addition 2 mM Mg-acetate. Theovaries were homogenized in a cold mortar with medium A orB and the homogenate was centrifuged at 3000g for 1 min toremove nuclei and cell wall debris. Small aliquots of the extractswere withdrawn for SDS-PAGE, protein estimation, and deter-mination of [3H]leucine uptake and incorporation in TCA-in-soluble material. The remainder of the extract was used for fur-ther analyses.

Isolation of Organelles. The organelles were separated fromthe soluble proteins and small molecules on Sepharose 4B col-umns (1.7 x 12 cm) (Pharmacia, Uppsala, Sweden) as describedby Van der Wilden et al. (13). Briefly, homogenates made inhomogenization medium A or B were applied to Sepharose 4Bcolumns equilibrated with medium A or B, respectively. Thecolumns were eluted with the homogenization medium and frac-tions of 1 ml each collected. Fractions containing the light-scat-tering material (organelles) in the first peak and those containingthe soluble proteins (which eluted in the second peak) werepooled and used for further analyses.

2 Abbreviation: GNA, Galanthus nivalis agglutinin.922

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BIOSYNTHESIS OF THE SNOWDROP LECTIN

Fractionation of ER on Sucrose Gradients. Linear 16 to 48%(w/w) sucrose gradients were formed in medium A or B with aBeckman density gradient former on top of a 1 ml cushion of70% sucrose. Gradients were loaded with 2 ml of organelle frac-tion (from the Sepharose 4B column) and centrifuged for 2.5 hat 38,000 rpm in a Beckman SW 40 rotor. Fractions of 0.5 mlwere collected with an ISCO (Palo Alto, CA) gradient fraction-ator and used for determination of total [3H]leucine incorpora-tion (into TCA-insoluble material), [3H]leucine incorporation inthe lectin, NADH Cyt c reductase and Cyt c oxidase activity.Sucrose concentrations were determined with a refractometer.

Isolation of the Lectin by Affinity Chromatography. Fractionsfrom Sepharose 4B or sucrose gradients were brought at 0.5%Triton X-100 and 0.5 M (NH4)2SO4, and applied to small (0.2 mlbed volume) columns of immobilized mannose (Selectin 10 fromPierce Chemical Co., Rockford, IL) equilibrated with 0.5 M(NH4)2SO4. Unbound proteins were washed off with 5 ml of 0.5M (NH4)2SO4. Then the lectin was desorbed with 4 ml of un-buffered 2,3 diaminopropane, frozen at - 80°C and lyophilized.These lyophilized fractions were dissolved in 1 ml of distilledwater and the lectin precipitated with TCA (10% final concen-tration). Finally the precipitate was dissolved in sample bufferfor subsequent SDS-PAGE and fluorography (and for deter-mination of [3H]leucine incorporation in the lectin).SDS-PAGE and Fluorography. SDS-PAGE was done on 12.5

to 25% acrylamide gradient gels using a discontinuous system asdescribed by Laemmli (7). After fixing and destaining gels wereimmersed in amplify (Amersham, U.K.) for 30 min, dried underpartial vacuum, and exposed to preflashed x-ray films (FUJI RX,Japan).

Analytical Methods. NADH Cyt c reductase and Cyt c oxidasewere assayed as described by Bowles and Kauss (2) and Sottocasaet al. (11), respectively. Agglutination assays using trypsin-treatedrabbit erythrocytes were carried out as described previously (12).Protein contents of extracts were determined by the method ofBradford (3) using BSA as a standard.

RESULTSChoice of Material. Ripening ovaries of snowdrop represent a

favorable system for a study of the lectin synthesis for severalreasons. First, the plant material is easy to prepare and to handle.We preferred ovaries over bulbs since extracts from bulbs arevery viscous and rapidly turn brown. In addition, the lectin con-centration within the ovaries is very high and the lectin canreadily be purified. The reliability of the affinity purificationprocedure as described in the "Materials and Methods" sectionis quite high. As shown in Figure 1 the lectin fraction obtainedafter affinity chromatography is pure (lane 3). In addition, thesame figure shows that the lectin polypeptides (which are themajor labeled bands on the fluorogram of the crude extract [lane1]) are completely removed by affinity chromatography (lane 2).GNA is Synthesized in Vivo as a Precursor. Snowdrop ovaries

were labeled with [3H]leucine for different times and the (total)lectin was isolated by affinity chromatography on immobilizedmannose. Subsequent analyses by SDS-PAGE and fluorographyrevealed that the first product to be synthesized is a polypeptidewith an apparent Mr or 15,000 (Fig. 2). After longer labelingtimes radioactivity also appears in the lectin polypeptide with aMr of 13,000. This sequence of appearance of radioactivity indifferent polypeptides suggests that GNA is synthesized as aprecursor of Mr 15,000 which is subsequently converted into the13,000 Mr lectin polypeptide.

Kinetics of the Conversion of the Lectin Precursor into theAuthentic Lectin. To follow the fate of the precursor and deter-mine the kinetics of its processing pulse-chase experiments werecarried out. Snowdrop ovaries were labeled for 2 h with [3H]leucineand further incubated in excess of unlabeled leucine for different

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1 2 3FIG. 1. SDS-PAGE and fluorography of labeled snowdrop ovaries.

Lane 1 shows a crude extract of snowdrop ovaries labeled with [3H]leucinefor 8 h. The lectin isolated from this extract is shown in lane 3. Lane 2shows a protein pattern of the proteins which are not bound to the affinitycolumn.

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FIG. 2. SDS-PAGE and fluorography of affinity-purified lectin re-covered from snowdrop ovaries incubated with [3H]leucine for the timesindicated. M, reference protein was the mature snowdrop lectin. Itsposition is indicated by the arrowhead.

times. Under these conditions the processing of the precursorinto other polypeptides can be followed. Isolation of the totallectin fraction after different labeling times has shown that thelabeling of the precursor decreased as a function of chase timewhereas radioactivity in the lectin polypeptide of M, 13,000 in-creased (Fig. 3). Densitometric analysis of the x-ray plate usinga gel scanner allowed a quantitative interpretation of the resultsof the chase experiment. The half-life of the precursor was cal-culated to be about 6 h (Fig. 4).Sugar Binding Activity of the Lectin Precursor. In all experi-

ments described above lectin was isolated by affinity chroma-tography on immobilized mannose. Since the precursor is re-tained on the affinity column it is evident that the GNA precursorexhibits the same carbohydrate-binding activity as the lectin it-self. It appears therefore that the sequence, which is cleaved offwhen the precursor is processed into the lectin, is not essentialfor the carbohydrate-binding properties of the lectin precursor.

Organelle-Associated Lectin. Snowdrop ovaries were pulse la-beled with [3H]leucine for 2 h, homogenized in medium A, andfractionated on a Sepharose 4B column in the same medium to

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VAN DAMME AND PEUMANS Plant Physiol. Vol. 86, 1988

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FIG. 3. SDS-PAGE and fluorography of affinity-purified lectin re-

covered from snowdrop ovaries labeled with [3H]leucine for 2 h and thenfurther incubated with unlabeled leucine for the times indicated. Theposition of the Mr marker proteins is indicated by arrowheads. They are

in order of increasing Mr: myoglobin I (8,200), lysozyme (14,000), myo-

globin I + II (14,600), myoglobin (intact, 17,200), soybean trypsin in-hibitor (21,000), carbonic anhydrase (30,000), ovalbumin (45,000), BSA(68,000), and phosphorylase b (94,000).

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FIG. 5. Fractionation of pulse-labeled snowdrop ovaries on Sephar-ose 4B. Snowdrop ovaries were labeled with [3H]leucine (20 ,uCi per

ovary) for 2 h, homogenized in medium A, and fractionated on Sepharose4B in the same medium. Fractions (1 ml) were collected and the agglu-tination activity of each fraction determined. Then (NH4)2SO4 and TritonX-100 were added to the fractions, aliquots were withdrawn for deter-mination of radioactivity in TCA-insoluble material, and the lectin iso-

lated by affinity chromatography. The radioactivity in the lectin was alsodetermined. Agglutination activity is expressed as the titer (highest di-lution at which agglutination still occurs) of the fractions. Radioactivityin both total protein and lectin are expressed as cpm per fraction.

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FIG. 4. Kinetics of the processing of precursor into authentic lectin.To quantitate the fluorographic results shown in Figure 2 the x-ray platewas cut into strips and scanned with a Gilford spectrophotometer (equippedwith a gel scanning device at 600 nm) for subsequent determination ofpeak areas. Results are expressed as percentage of total radioactivity (inprecursor + lectin) which was found in the precursor and as the logvalues of these values. The half-life of the precursor was determinedusing the log values.

separate the soluble proteins from the particulate components.Fractions were collected and small aliquots were withdrawn fordetermination of [3Hlleucine incorporated in TCA-insoluble ma-terial. To the remainder Triton X-100 and (NH4)2SO4 were addedand the lectin isolated. The elution position of the agglutinationactivity shows that the bulk of lectin elutes with the solubleproteins. This does not exclude that the lectin might occur inorganelles since the conditions of homogenization may disruptprotein bodies so that their contents become part of the solubleprotein fraction. Labeled lectin was found in the soluble fraction

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FIG. 6. Fluorography of newly synthesized snowdrop lectin fraction-ated by SDS-PAGE. Snowdrop ovaries were labeled for 6 h with[3H]leucine. Extracts were made and the homogenates fractionated on

Sepharose 4B. The lectin was isolated by affinity chromatography, con-

centrated by lyophilization, and dissolved in sample buffer for subsequentSDS-PAGE. Lane 1, lectin recovered from the organelle fraction; Lane2. lectin recovered from the soluble fraction. The Mr of the polypeptidesas indicated was determined using mol wt marker proteins (as in Fig. 3).

as well as in the organellar fraction which indicates that somelectin is associated with the organelles (Fig. 5).Comparison of Organelle and Soluble Lectin. Ovaries of snow-

drop were pulse labeled for different times and homogenized inmedium A. For each time condition the organellar fraction andthe soluble fraction were recovered (by gel filtration on Se-pharose 4B) and the lectin isolated. The lectin polypeptides wereanalyzed by SDS-PAGE and detected by fluorography. As shownin Figure 6 the lectin polypeptide isolated from the organellarfraction corresponds to the precursor lectin whereas the precur-sor as well as the authentic lectin are found in the soluble fraction.These data suggest that the precursor is not processed in the ERas it is still present in the soluble fraction.

Lectin in ER. To determine whether newly synthesized GNA

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BIOSYNTHESIS OF THE SNOWDROP LECTIN

is associated with the ER or with some other organelle, ovarieswere labeled for 1 h using [3H]leucine. Labeled ovaries weredivided into two portions and homogenized in medium A or B.The homogenates were fractionated on Sepharose 4B and theorganellar fraction recovered. The organelles were then sepa-rated on isopycnic sucrose gradients in the same media. Thegradients were fractionated and aliquots of each fraction wereused for determination of (a) total radioactivity in TCA-insol-uble protein, (b) sucrose concentration, (c) NADH Cyt c re-ductase activity (a marker enzyme for the ER), (d) Cyt c oxidaseactivity (a marker enzyme for the mitochondria), and(e) [3H]leucine incorporation in the lectin. For the isolation ofthe lectin sucrose gradient fractions were diluted by addition of2 volumes of 0.7 M (NH4)2SO4, brought to 0.5% Triton X-100,and passed through Selectin 10 columns.

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The organelles were separated on 16 to 48% (w/w) sucrose gradients inthe same media, medium A (A) and medium B (B). Sucrose gradientswere centrifuged at 38,000 rpm for 2.5 h, fractions were collected, andaliquots of each fraction used for determination of (a) total incorporated[3H]leucine into 10% TCA-insoluble material, (b) radioactivity in affin-ity-purified lectin, and (c) NADH-Cyt c reductase activity. NADH-Cytc reductase activity is expressed as /v A55,, h per fraction. Radioactivityis expressed as cpm per fraction. The density of the ER and the organelle-

In a medium with Mg with ribosomes remain attached to theER whereas in a medium with EDTA the ribosomes are removedfrom the ER. As a result, the ER shifts to a lower density in thepresence of EDTA. The distribution of the total radioactivity inthe gradient fractions, the radioactivity incorporated in the lectin,and the position of NADH Cyt c reductase activity are shownfor sucrose gradients built up in both media in Figure 7. TheNADH Cyt c reductase activity banded at an average density of1.09 g/cm3 in the presence of EDTA whereas the lectin precursorwas found at an average density of 1.10 g/cm3. In a medium withMg the ER marker enzyme formed a broad band in the gradientwith a density range of 1.09 to 1.13 g/cm3. The lectin was alsomore dispersed when Mg was in the medium. Besides one largepeak with a density of 1.11 to 1.12 g/cm3 there was also a smallerpeak at a density of 1.15 g/cm3.The position of the mitochondrial marker enzyme Cyt c oxi-

dase was the same in both gradients (1.16 g/cm3). Other organ-elles are also known to remain at the same density in both ho-mogenization buffers (8). Taken together these data indicate thatthe newly synthesized GNA is associated with the rough ER.Since the peak of incorporated radioactivity in the lectin bandsat a slightly higher density (in the absence of Mg) than the peakof NADH Cyt c reductase activity this could mean that only asubset of the ER cisternae is engaged in lectin synthesis.

ER-Associated Lectin is a Tetramer of Four Precursor Chains.Although the results shown in Figure 6 demonstrate that thelectin in the rough ER corresponds to the precursor form of thisprotein, they do not allow us to decide whether this lectin occursas a monomer, a dimer, or a tetramer. Therefore, lectin wasisolated from the organellar fraction of 2 h labeled ovaries andchromatographed on a Superose 12 gel filtration column. Asshown in Figure 8 the lectin from the ER elutes well before theauthentic (or mature) lectin. Its mol wt was estimated, usingmarker proteins, to be 60,000. It can be concluded therefore thatthe lectin associated with the rough ER consists of four precursorchains. In a control experiment with labeled lectin from thesoluble fraction of 24 h chased ovaries label co-eluted with theauthentic lectin.

Kinetics of Lectin Transport Out of ER. To follow the fate ofER-associated lectin, snowdrop ovaries were labeled with[3Hlleucine for different times. After 2 h some ovaries wererinsed with water and further incubated with unlabeled leucinefor different chase times. Then they were homogenized in me-dium A and the homogenates fractionated by gel filtration on aSepharose 4B column in the same medium. The fractions con-taining the organelles and the soluble proteins were collected,the lectin isolated by affinity chromatography, and the incor-poration of [3H]leucine in each lectin sample determined. Fromthe thus obtained data the distribution of total label in the lectinover organellar and soluble fraction was calculated. Figure 9Ashows that newly synthesized lectin is first associated with theorganelles but is then quickly transferred to the soluble fraction.When radioactivity is chased (Fig. 9B) GNA disappears fromthe ER with a half-life of about 4 h and then accumulates in thesoluble fraction. The radioactivity in the soluble proteins con-tinued to increase for the entire period of the chase (24 h) butleveled off towards the end. These results indicate that newlysynthesized GNA is transiently associated with the ER and thenaccumulates in the soluble fraction.

DISCUSSIONA detailed study of the in vivo synthesis and processing of the

Galanthus nivalis agglutinin has shown that this lectin is synthe-sized as a precursor of Mr 15,000 which is posttranslationallyconverted into the lectin polypeptide of Mr 13,000. A fraction-ation of the different organelles on sucrose gradients indicatedthat the newly synthesized lectin is associated with the rough ER

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associated lectin band is indicated by the arrows.

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FIG. 8. Gel filtration of ER-associated and soluble lectin on a Su-perose 12 column. Snowdrop ovaries were labeled with [3H]leucine, ho-mogenized, and fractionated on Sepharose 4B into organellar and solublefraction. Lectin was isolated from both fractions and applied to a Su-perose 12 column mounted on a Pharmacia Fast Protein Liquid Chro-matography system (Pharmacia, Uppsala, Sweden). The column was

eluted with PBS containing 0.2 M mannose at a flow rate of 40 ml h- '.The elution position of the lectin was determined by measuring the A-,8,of the eluate with a Delsi Enica 21 integrator (Delsi, Suresnes, France).Fractions of 100 Al were collected and used for determination of theradioactivity. Mr marker proteins were: aldolase (Ald., Mr 160,000),bovine serum albumin (BSA, M, 68,000), chymotrypsinogen (Chym.,Mr 25,000), and Cyt c (Cyt. c, Mr 12,500). Their elution position was

determined in a separate run. In (A) 100 ,ul purified GNA was addedto trace the elution position of the authentic lectin. A, Labeled lectinrecovered from the organellar fraction; B, labeled lectin recovered fromthe soluble fraction.

since the elution positions of both the ER marker enzyme andthe lectin shifted towards a higher density in the presence of Mg.

Precursor molecules of higher mol wt have been reported formany lectins. However, in the case of GNA the difference in Mrbetween precursor and lectin is rather small. In some cases car-

bohydrate moieties of glycoproteins synthesized in ER are sub-sequently modified resulting in polypeptides with a slightly dif-ferent Mr. In contrast with these lectins the snowdrop lectin isnot glycosylated and since further experiments have shown thatthe GNA precursor does not bind to concanavalin A-Sepharose(results not shown) it is very unlikely that the precursor is gly-cosylated. Moreover, a difference in Mr of about 2000 can hardlyaccount for a possible glycosylation. Therefore, the differencesin mobility between precursor and lectin probably reside in thepolypeptide chains. This posttranslational modification could bedue to the removal of a small peptide.

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Plant Physiol. Vol. 86, 1988

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FIG. 9. Pulse and pulse-chase labeling of snowdrop ovaries. A, Fiveovaries of snowdrop were incubated with [3H]leucine (20 ,u Ci per ovary)for the times indicated. Extracts were prepared and fractionated on

Sepharose 4B. Organelles and soluble fractions were collected, the lectinisolated by affinity chromatography, and the radioactivity determined.Results are expressed as percentage of total radioactivity in lectin in theorganellar and soluble fraction. B, Five ovaries of snowdrop were labeledfor 1 h with [3H]leucine, then further incubated with unlabeled leucinefor the times indicated. The ovaries were extracted and fractionated,and the radioactivity in lectin in organelle and soluble fraction was de-termined. Results are expressed as in (A).

Since the soluble fraction contains the cytosolic proteins aswell as the contents of vacuoles and vacuole-derived organelles(which were ruptured upon homogenization) we assume that thelabeled lectin which leaves the ER is transported to some or-

ganelles where it is processed, as is the case for many lectins andstorage proteins.

LITERATURE CITED

1. BOLLINI R. MJ CHRISPEELS 1979 The rough endoplasmic reticulum is the sitcof reserve-protein synthesis in developing Phaseolus vulgaris cotyledons.Planta 146: 487-501

2. BOWLEs DJ, H KAUSS 1976 Characterization, enzymatic and lectin propertiesof isolated membranes from Phaseolus aureus. Biochim Biophys Acta 443:360-374

3. BRADFORD M 1976 A rapid and sensitive method for quantitation of microgramquantities of protein utilizing the principle of protein-dye binding. AnalBiochem 72: 248-254

4. HEMPERLEY JJ, KE MOSTOV, BA CUNNINGHAM 1982 In vitro translation andprocessing of a precursor form of favin, a lectin from Viciafaba. J Biol Chem257: 7903-7909

5. HIGGINs TJV, PM CHANDLER, G. ZURAWSKI, SC BUTTON, D SPENCER 1983The biosynthesis and primary structure of pea seed lectin. J Biol Chem 258:9548-9549

6. HIGGINs TJV, MJ CHRISPEELS, PM CHANDLER. D SPENCER 1983 Intracellularsites of synthesis and processing of lectin in developing pea cotyledons. JBiol Chem 258: 9550-9552

7. LAEMMLI UK 1970 Cleavage of structural proteins during the assembly of thehead of bacteriophage T4. Nature 227: 680-685

8. LORD JM. T KAGAWA, TS MOORE, H BEEVERS 1973 Endoplasmic reticulumas the site of lecithin formation in castor bean endosperm. J Cell Biol 57:659-667

9. PEUMANS WJ, HM STINtSSEN 1983 Gramineae lectins: occurrence, molecularbiology and physiological function. In IJ Goldstein, ME Etzler. eds, Chem-ical Taxonomy, Molecular Biology and Function of Plant Lectins. Alan R.Liss, Inc., New York, pp 99-1 16

10. ROBERTS LM, JM LORD 1981 The synthesis of Ricinus communis agglutiniin.Eur J Biochem 119: 31-41

11. SOTTOCASA GL, B KUYLENSTIERNA, L ERNSTER, A BERGSTRAND 1967 Anelectron-transport system associated with the outer membrane of liver mi-tochondria. A. Biochemical and morphological study. J Cell Biol 32: 415-438

12. VAN DAMME EJM, AK ALLEN, WJ PEUMANS 1987 Isolation and character-ization of a lectin with exclusive specificity towards mannose from snowdrop(Galanthus nivalis) bulbs. FEBS Lett 215: 140-144

13. VAN DER WILDEN W, NR GILKES. MJ CHRISPEELS 1980 The endoplasmicreticulum of mung bean cotyledons. Role in the accumulation of hydrolasesin protein bodies during seedling growth. Plant Physiol 66: 390-394

14. VODKIN LO 1981 Isolation and characterization of messenger RNAs for seedlectin and Kunitz trypsin inhibitor in soybeans. Plant Physiol 68: 766-771

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