Planta 9 by Springer-Verlag 1980
Planta 150, 82 88 (1980)
What is Pea Legumin - Is it Glycosylated? William J. Hurkman and Leonard Beevers Department of Botany and Microbiology,Universityof Oklahoma, Norman, OK 73019, USA
Abstract. Since there is some question as to whether
or not legumin is glycosylated, this storage protein was isolated by various procedures from developing cotyledons of P i s u m s a t i v u m L. supplied with [14C]labeled glucosamine and analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Legumin isolated by the classical method of Danielsson [(1949) Biochem. J. 44, 387-400] a procedure in which globulins extracted with a buffered salt solution are precipitated with ammonium sulfate (70% saturation) and legumin separated from vicilin by isoelectric precipitation, was labeled. The glucosamine incorporated into legumin was associated with low-molecular-weight polypeptides. In contrast, legumin isolated by the method of Casey [(1979) Biochem. J. 177, 509-520], a procedure where legumin is prepared by zonal isoelectric precipitation from globulins precipitated with 40-70% ammonium sulfate, was not labeled. However, the globulin fraction precipitated with 40% ammonium sulfate was labeled and the radioactive glucosamine was associated with low-molecular-weight polypeptides. Legumin isolated from protein bodies [Thomson et al. (1978) Aust. J. Plant Physiol. 5, 263279] was not extensively labeled. However, the saltinsoluble fraction of protein body extracts was labeled and the radioactivity was associated with low-molecular-weight polypeptides. These results indicate that protein bodies contain a glycoprotein of low-molecular-weight that co-purifies with legumin isolated by the method of Danielsson but that is discarded when isolation methods developed more recently are used. Key words: Legumin Storage protein.
Pisum
-
Protein (storage) -
Introduction
Historically, the name legumin was first given by Braconnot (1827) to the major protein fraction extracted
0032-0935/80/0150/0082/$01.40
with water from legume seeds. Seed proteins were later found to be salt-soluble globulins (Weyl 1876, 1877). Osborne and Campbell (1898) used ammonium-sulfate precipitation and repeated dilution and dialysis to separate globulins of pea seeds into two major protein fractions, legumin and vicilin, the former insoluble in dilute salt solutions and not coagulated when heated to 100 ~ C, the latter soluble in dilute salt solutions and coagulated when heated to 95~ Danielsson (1949) found that pea legumin could be more effectively separated from vicilin by isoelectric precipitation. Basha and Beevers (1976) showed that acid hydrolysates of legumin isolated by the procedure of Danielsson (1949) from developing pea seeds contained glucosamine, an amino sugar, and mannose, a neutral sugar, and Browder and Beevers (1978) demonstrated the occurrence of glucosaminyl-asparagine in glycopeptides isolated from legumin prepared by this method. These results indicated that pea legumin, as prepared by the Danielsson procedure, was glycosylated. Recently, zonal isoelectric precipitation has been utilized for separation of legumin from vicillin (Casey 1979; Scholz et al. 1974; Wright and Boulter 1974). Davey and Dudman (1979) found that pea legumin isolated from protein bodies by isoelectric precipitation followed by zonal isoelectric precipitation was glycosylated and the amounts of glucosamine and neutral sugars they found in their preparations were in good agreement with those previously reported by Basha and Beevers (1976). However, Casey (1979) reported that legumin prepared from mature pea seeds by zonal isoelectric precipitation and purified by diethylaminoethyl (DEAE)-cellulose chromatography and sucrose gradient centrifugation was not glycosylated. Similar results were reported by Gatehouse et al. (1980) for legumin prepared from pea seeds by hydroxylapatite chromatography. It thus, appears that legumin prepared by the pro-
W.J. Hurkman and L. Beevers: Glycosylation of Pea Legumin cedure of Danielsson
(1949) is g l y c o s y l a t e d w h e r e a s
l e g u m i n p r e p a r e d b y m o r e r e c e n t m e t h o d s is n o t . In order to determine the causes for this discrepancy, we have prepared legumin from the cotyledons of p e a s e e d s b y t h e m e t h o d s o f D a n i e l s s o n (1949), C a s e y (1979), a n d T h o m s o n e t al. (1978), a n d a n a l y z e d t h e preparations for glycosylated peptides. This was done using cotyledons supplied with [14C]glucosamine ( [ 1 4 C ] G l c N H 2 ) w h i c h is k n o w n t o b e i n c o r p o r a t e d i n t o l e g u m i n ( B a s h a a n d B e e v e r s 1976). T h e l a b e l e d l e g u m i n f r a c t i o n s , as w e l l as u n l a b e l e d l e g u m i n f r a c t i o n s p r e p a r e d f r o m m a t u r e seeds, w e r e a n a l y z e d b y s o d i u m d o d e c y l s u l f a t e ( S D S ) - p o l y a c r y l a m i d e gel electrophoresis. Our results showed that pea cotyledons contain a glycosylated component labeled with [ 1 4 C ] G l c N H 2 t h a t is a s s o c i a t e d w i t h p r o t e i n b o d i e s and co-purifies with legumin isolated by the proced u r e o f D a n i e l s s o n (1949) b u t w h i c h is d i s c a r d e d i n more recent procedures.
Materials and Methods Plant Material. Cotyledons were collected from developing peas (Pisum sativum L. cv. Burpeeana; Burpee Seed Co., Warminster, Pa. USA) cultured as described in Basha and Beevers (1976), or from mature seeds soaked in water overnight. Testas and embryos were removed. Preparation of [14CJGlucosamine-Labeled Proteins. Thirty cotyledons from seeds 24 or 28 d post-anthesis were collected and each cotyledon was sliced, vertically to the axis, into five pieces. Ten gl of [14C]GlcNHH2 (1.057.10 l~ Bq/mmo}.=286 mCi/mmol and 37.104 Bq/400 gl= 10 gCi/400 gl; Amersham, Arlington Heights, II1., USA) diluted 1:20 with water were placed on each slice. The slices were incubated in the light for 4 h, collected, and homogenized in the appropriate buffer. Cotyledons extracted by the methods of Danielsson (1949) and Casey (1979) (details below) were homogenized with a Polytron homogenizer (Brinkman Instruments, Westbury, N.Y., USA). Cotyledons 28 d post-anthesis were used for the method of Thomson et al. (1978) (details below); they were homogenized with the mechanical chopping device described by Beevers and Mense (1977). An additional 20 g of cotyledons were added to labeled cotyledons to be extracted by the methods of Casey and Thomson et al. in order to facilitate recovery of proteins separated by column chromatography. Protein Isolation by the Method of Danielsson (1949). Cotyledons were homogenized in Buffer A (1.0 M NaC1, 20 mM KzHPO 4 KH2PO4, pH 7.0; 7 ml buffer/g cotyledons). When proteins were prepared from mature seeds, 30 g of cotyledons were homogenized in a precooled Waring blender (four 15-s bursts). The brei was stirred for 30 rain on ice, filtered through fov.r layers of cheesecloth, and centrifuged (19,000 .g, 15 min; unless otherwise stated, all centrifugations were done at 0~ with an SS-34 rotor in a Sorvall RC 2B centrifuge; DuPont Co., Newtown, Conn. USA). The pellet was extracted in the same way twice more with Buffer A (20 ml). Solid (NH4)2SO4 was slowly added with stirring to the pooled supernatants to 70% saturation; the solution was stirred for 15 min on ice and centrifuged (3,000.g, 20 min). The supernatant was discarded. The 70% (NH4)zSO4 precipitate was suspended in Buffer B (0.2 M NaC1, 5 mM Kzt-IPO 4 KHzPO4, pH 7.0, 20 mI) and dialyzed against distilled water for 48 h at 4 ~ C. The dialysate
83 was centrifuged (19,000 .g, 15 min) and the supernatant discarded. The pellet was suspended in Buffer C (0.2 M NaC1, 5 mM K2HPO4--KH2PO4, pH 4.5; 20 ml), stirred overnight at 4 ~ C, and centrifuged (3,000.g, 10 rain). The pellet was washed twice with Buffer C (20 ml) and then suspended in Buffer B (20 ml). The suspended pellet, containing legumin, and the pooled supernatants, containing vicilin, were dialyzed against water for 48 h at 4 ~ C. Legumin and vicilin were recovered by centrifugation (19,000. g, 15 min) and stored at - I 0 ~ C. Protein Isolation by the Method of Casey (1979). Cotyledons were homogenized as above, but in Buffer D (0.5 M NaC1, 0.1 mM dithiolthreitol (DTT), 0.02% NAN3, 0.05 M tris(hydroxymethyl) aminomethane (Tris)-HCl, pH 7.0). The brei was stirred on ice for 15 min, filtered, and centrifuged (19,000-g, i0 min). The pellet was discarded and (NH4)2SO e was added to the supernatant to 40% saturation. After stirring for 15 min at room temperature, the solution was centrifuged (19,000-g, 10 min), the pellet discarded, and (NH4)2SO4 added to the supernatant to 75% saturation. After 15 min, the solution was centrifuged (19,000 .g, 10 min) and the supernatant discarded. The 40-75% (NH4)zSO4 pellet was dispersed in 5 ml of Buffer E (0.2 M NaC1, 0.1 M DTT, 0.02% NAN3, 0.05 M Tris-HC1, pH 8.0) and desalted by chromatography on a column (2.5 cm diameter, 100 cm long) of Sephadex G-25 (Sigma Chemical Co., St. Louis, Mo., USA) equilibrated in Buffer E. Globulins were eluted at room temperature with Buffer E (60 ml/ h) and concentrated by dialysis against polyethylene glycol (20,000 molecular weight). Legumin and vicilin were separated by zonal isoelectric precipitation : globulins were applied to a Sephadex G-50 column (3 cm diameter, 60 cm long) equilibrated with Buffer F (0.2 M NaC1, 0.1 M DTT, 0.02% NaN> 0.05 M citric acid-NaOH, pH 4.8) and eluted at room temperature with Buffer E (60 ml/h) and ultraviolet (UV)-absorbance monitored (280 nm, ISCO Model UA-2 UV Analyzer; Instrumentation Specialties Co., Lincoln, Neb., USA). Fractions corresponding to the first peak (vicilin) and those corresponding to the tailing half of the second peak (legumin) were pooled, respectively, and dialyzed against water for 48 h at 4 ~ C. Legumin and vicilin were recovered by centrifugation (19,000.g, 10 min) and stored at - 1 0 ~ C. The same procedure was used to obtain legumin and vicilin from protein bodies, except that precipitation with 40% (NH4)2 SO4 was omitted. Protein bodies were isolated from mature seeds by discontinuous gradient centrifugation according to the method of Larkins and Hurkman (1978). Protein Isolation by the Method of Thomson et al. (1978). Protein bodies, isolated in water and separated by differential centrifugation at 17,500-g (Thomson et al. 1978), were extracted with 1.0 M NaC1, 0.02% NaNa, 0.1 M K2HPO4--KH2PO4 (pH 7.2) for 1 h at 0 ~ C. The extract was centrifuged (19,000.g, 10 min) and the pellet retained. The supernatant was dialyzed at 4~ overnight to 0.2 M NaC1 in McIlvaine buffer, pH 4.8 (0.2 M Na2PO4, 0.1 M citric acid; see Scholz et al. 1974). The extract was centrifuged (19,000.g, 10rain) and the supernatant (vicilin) was dialyzed against water for 48 h at 4 ~ C. The pellet (legumin) was dissolved in 20 ml McIivaine buffer, pH 8.0, and applied to a Sephadex G-75 column (2.5 cm diameter, 80 cm long) equilibrated with McIlvaine buffer, pH 4.0. Protein was eluted (60 ml/h) at room temperature with McIlvaine buffer, pH 8.0. The retarded fractions were pooled and dialyzed against water for 48 h at 4 ~ C. Legumin and vicilin were recovered by centrifugation (19,000.g, 10 min) and stored at 10~ C. Acid Hydrolysis of La~eled Proteins. Forty gl of labeled proteins (approx. 9,000 cpm) were incubated in 1 ml of 4 N HC1 for 3 h in a boiling water bath. Following incubation, chloride was precipitated by addition of solid silver nitrate to the hydrolysate. The samples were dried with a stream of air and then rinsed with
84 water and redried; the water rinse was repeated twice more. The hydrolysates were dissolved in 100 gl of 80% (v/v) ethanol and analyzed by thin layer chromatography on cellulose (Chromagram 13255; Eastman-Kodak, Rochester, N.Y., USA) in bntanolpyridine-water (6:4:3, v/v). Chromatograms were divided into 1-cm strips and transferred into vials. Scintillation fluid was added and radioactivity determined (LS-100 scintillation counter; Beckman Instruments, Irvine, Cal., USA).
Gel Electrophoresis. Protein samples were analyzed on slab gels with a sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis system similar to that described by Laemmli (1970). Gels were 1.5 mm thick and consisted of a 9-cm running gel of i2.5% acrylamide (acrylamide/bisacrylamide=75:l, v/v) in 3.75mM Tris-HCl (pH 8.9), 0.058 mM N,N,N',N'-tetramethylethylenediamine (TEMED), and 0.075% SDS. The running gel was overlaid with a 2.5 cm stacking gel of 5% acrylamide. Freshly prepared ammonium persulfate was added to a final concentration of 0.035% immediately before each gel layer was poured. Proteins to be applied to the gel were dissolved by boiling in sample buffer (0.024 M Tris-HC1, pH 8.3, 1% SDS, 1% 2-mercaptoethanol, 0.002% bromophenol blue, 10% glycerol). Electrophoresis was done at room temperature at 15 mA constant current until the tracking dye had traversed the stacking gel, and then at 25 mA constant current until the dye had reached the bottom of the running gel. The gels were stained overnight in a solution of 0.1% Coomassie blue, 50% methanol, 10% acetic acid, and destained in 15% methanol7% acetic acid. The protein samples contained approximately 30 gg protein. Protein content was determined by the procedure of Lowry et al. (1951) with bovine serum albumin as standard. Phosphorylase B (molecular weight 94,000), bovine serum albumin (68,000), pyruvate kinase (57,000), horseradish peroxidase (40,000), carbonic anhydrase (30,000), trypsin inhibitor (21,000), and cytochrome c (11,700) were used as marker proteins for SDS-polyacrylamide gel electrophoresis. Mobilities of markers were calculated relative to cytochrome c and the apparent molecular weights of seed proteins were determined by reference to the mobilities of the markers (Weber and Osborn 1969). The values obtained, being approximations, were used to facilitate the identification of proteins and are not intended to represent precise molecular weights. Acrylamide, bisacrylamide and TEMED were obtained from EastmanKodak, Rochester, N.Y., USA, and ammonium persulfate, bromophenol blue, Coomassie blue, 2-mercaptoethanol, SDS, and the proteins used as standards from Sigma Chemical Co., St. Louis, Mo., USA. The distribution of radioactivity among polypeptides separated by gel electrophoresis was determined by the method of Davies and Delmer (1979). A 1-cm strip was removed from the center of the gel and stained with Coomassie blue. The rest of the gel was sliced and the slices were incubated in 3% (v/v) Protosol (New England Nuclear, Boston, Mass., USA) in ACS (aqueous counting scintillant; Amersham, Arlington Heights, Ill., USA) for 48 h at 50 ~ C.
Results
Distribution of Radioactivity Among Protein Fractions Isolated from Cotyledons Supplied with [14C]Glucosamine. When developing pea cotyledons were labeled with [14C]GlcNH2, radioactivity became associated with legumin and vicilin fractions isolated by the methods of Danielsson (1949), Casey (1979), and Thomson et al. (1978) (Table 1). In the globulins prepared according to Danielsson, the legumin fraction
W.J. Hurkman and L. Beevers : Glycosylation of Pea Legumin Table 1. Distribution of radioactivity among protein fractions prepared by various procedures from developing pea cotyledons supplied with [14C]glucosamine. The protein fractions prepared by isoelectric precipitation (Methods 1,3~) are termed legumin and vicilin after Danielsson (1949). The fractions prepared by zonal isoelectric precipitation (Method 2) are termed legumin and vicilin after Scholz et al. (1974). The 40% (NH4)2SO 4 fractions were precipitated from the salt soluble fraction obtained by Casey's (1979) method. The salt insoluble fraction was the residual material remaining after salt extraction of protein bodies prepared by the method of Thomson et al. (1978) Method
Protein fraction Vicilin
Legumin
1. Danielsson (1949)
12,700a 251,500
2. Casey (1979)
19,000
3. Danielsson on 19,000 40 70% (NH4)2SO4 fraction 4. Danielsson on 40% (NH~)2SO4 fraction 5. Thomson et al. (1979) a
4,800
40% Salt (NH4)zSO4 insoluble
676,760
10.900
5,700
306,200
52,900
17,600
272,900
Values are expressed as cpm/fraction
was much more extensively labeled than the vicilin fraction (Table 1, Method 1), a result consistent with previous observations by Basha and Beevers (1976). In contrast, radioactivity was found to be much less extensively incorporated in globulin fractions isolated by the procedure of Casey, and the vicilin fraction contained the greatest amount of label (Table 1, Method 2). Radioactivity was associated with globulins isolated from protein bodies by the procedure of Thomson et al. and vicilin was more extensively labeled than legumin (Table 1, Method 5). The ratio of label between vicilin and legumin was similar in the proteins isolated by the methods of Casey and Thomson et al. Acid hydrolysis of legumin samples prepared by the various procedures indicated that the hydrolysates contained radioactivity that co-chromatographed with authentic [14C]GlcNH2. Thus, the label associated with legumin fractions was due to glycosyl components and cannot be attributed to conversion of the applied glucosamine to amino-acid residues. Since legumin prepared according to the classical procedure of Danielsson was extensively labeled, analyses were performed to account for the low association of radioactivity with legumin fractions prepared by the more recent methods of Casey and of Thomson et al. A major difference between the proce-
W.J. Hurkman and L. Beevers: Glycosylation of Pea Legumin
A I II
I
|11
10 20 30 SLICE NUMBER
IB I
I
,I ,11 7
85
It,, III1,11 I~1
10 20 3R SLICE NUMRER
10 20 30 SLICE NUMBER
Fig. 1A-C. Distribution of radioactivity and protein components following SDS-polyacrylamide gel electrophoresis of [14C]glucosaminelabeled protein preparations from pea cotyledons 24 d post-anthesis. A Legumin prepared by the procedure of Danielsson (1949). B Protein precipitated with 40% (NH4)2SO4 that is normally discarded in the method of Casey (1979). C Protein from the insoluble residue fraction of protein bodies isolated and extracted by the method of Thomson et al. (1978). O, origin; 20 and 40, 20,000 and 40,000 molecular weight components, respectively
dure of Danielsson and that of Casey is that the latter method utilizes a 40 75% (NH~)2SO4 precipitate as a source of globulins whereas the former method utilizes a 0-70% (NH4)2SO4 precipitate. It was found that the globulin fraction prepared by the Danielsson method was difficult to disperse in the suspending buffers whereas that prepared by Casey's method (40-75% (NH4)2SO4) was more readily solubilized. When the 0-40% (NH~)2SO4 fraction normally discarded in the Casey method was recovered and its radioactivity determined, this fraction was found to be extensively labeled (Table 1, Method 2). When differential (NH~)2SO4 fractionation was performed on proteins isolated by Danielsson's method, it was found that within the 40-70% fraction vicilin was most extensively labeled (Table 1, Method 3) whereas in the 0-40% fraction the greatest proportion of radioactivity was in legumin (Table 1, Method 4). The greater recovery of radioactivity in globulins recovered by procedures where the 0 4 0 % (NH4)2SO4 fraction was included, in contrast to the lower level recovered from globulins isolated from protein bodies (Table 1, Method 5), indicated either that the 0-40% precipitable protein was not associated with protein bodies or that it was not solubilized by salt extraction of these organelles. The analyses showed that the saltinsoluble fraction of protein bodies was extensively labeled (Table 1, Method 5).
SDS-Polyacrylamide Gel Analysis of Proteins Labeled with [14C]Glucosamine. The information in Table 1 indicates that the major discrepancy concerning the glycosylation of legumin can be accounted for by the fact that, when the newer methods of Casey and of Thomson et al. are used, a glycosylated component is discarded. This concept was confirmed when labeled legumin fractions were analyzed by SDS-poly-
acrylamide gel electrophoresis (Fig. 1 A C). Legumin isolated by the procedure of Danielsson contained six major polypeptides with two prominent components that had molecular weights of approx. 20,000 and 40,000 (Fig. 1 A). Radioactivity was associated principally with smaller components of approx. 12,000 14,000 molecular weight. This association of radioactivity with low-molecular-weight polypeptides of legumin is consistent with the earlier observations of Basha and Beevers (1976). The limited extent of labeling of the legumin fraction prepared by the procedure of Casey precluded definitive conclusions concerning the association of radioactivity with specific polypeptides. When the 0-40% (NH4)2SO4 fraction, nromally discarded in Casey's procedure, was analyzed by SDS-polyacrylamide gel electrophoresis, it was found that this fraction contained several polypeptides and radioactivity was associated with those of low molecular weight (Fig. 1 B). Electrophoretic analysis of the insoluble residue fraction from protein bodies showed the presence of several polypeptides; the low-molecular-weight components contained the majority of the radioactivity (Fig. 1 C). The rapid migration of radioactivity in the legumin fraction (Fig. 1A), the 0-40% (NH4)2SO~ fraction (Fig. 1B), and the insoluble fraction of protein bodies (Fig. 1 C) is similar to that of a low-molecular-weight component produced when particulate fractions of mungbean hypocotyls are incubated with labeled glucosamine (Roberts and Pollard 1975). Roberts and Pollard (1975) suggested that the radioactivity was associated with a lipid fraction, thus raising the possibility that the labeled component isolated during legumin fractionation may be a glycolipid rather than a glycoprotein. However, repeated attempts to recover radioactivity from the protein preparation by such lipid solvents as chloroform-methanol (2:1) and chloro-
86
W.J. Hurkman and L. Beevers: Glycosylationof Pea Legumin
Fig. 2. SDS-polyacrylamidegel analysis of globulins prepared from pea cotyledons by the methods of Casey(1979) (C) and Danielsson (1949) (D) and of globulins isolated by the method of Casey (1979) (P) from protein bodies recovered by discontinuous gradient centrifugation. S, standards; L, legumin; V, vicilin; arrows, origin; MW, molecular weight x 10-3 Fig. 3. SDS-polyacrylamidegel analysis of pea legumin prepared from globulins precipitated with 40 70% ammonium sulfate by the method of Casey (1979) (C), 0-70% ammonium sulfate by the method of Danielsson (1949) (D1), and 40 70% ammonium sulfate by the method of Danielsson (D2). S, standards; MW, molecular weight x 10-3 Fig. 4. SDS-polyacrylamidegel analysis of globulins from protein bodies of pea cotyledons prepared and extracted by the method of Thomson et al. (1978). V, vicilin, and L, Iegumin, prepared by isoelectric precipitation; R, insoluble residue fraction of extracted protein bodies; S, standards; MW, molecular weight x 10-3
form-methanol-water (1:1:0.3) were unsuccessful. This observation coupled with the occurrence of a glucosamine-asparagine linkage in polypeptides isolated from legumin prepared by the procedure of Danielsson (Browder and Beevers 1978) indicates that the radioactivity from glucosamine is associated with glycoprotein. SDS-Polyacrylamide Gel Analyses of Proteins Isolated from Mature Seeds. Since developing seeds were used in our study whereas mature seeds were used in the studies of Casey (1979), Thomson et al. (1978), and Davey and Dudman (1979), it was necessary to determine the polypeptide composition of fractions prepared from mature seeds by the procedures of Table 1. Analyses by SDS-polyacrylamide gel electrophoresis showed that proteins prepared by each method had different polypeptide patterns (Figs. 2, 4). In general, legumin had two principal components with molecular weights of 37,000 and 18,000 (Fig. 2, lanes CL, DL, PL; Fig. 4, lane L), polypeptides that probably corresponded to the 40,000 and 20,000 components generally reported for pea legumin (Casey 1979 ; Croy et al. 1979; Davey and Dudman 1979). Legumin also contained four other polypeptides; two of these, 25,000 and 93,000 molecular weight, were specific for legumin whereas those of 54,000 and 74,000 molecular weight were similar to those encountered in the vicilin fraction. The 54,000 and 74,000 molecular weight polypeptides were present in legumin prepared by isoelectric precipitation (Fig. 2, lane DL) or by zonal
isoelectric precipitation from seeds (Fig. 2, lane CL) or protein bodies (Fig. 2, lane PL; Fig. 4, lane L). In addition, these polypeptides occurred in legumin initially obtained by zonal isoelectric precipitation and further purified by chromatography on DEAEcellulose by the method of Casey (1979) (data not shown). Analyses by SDS-polyacrylamide gel electrophoresis indicated that the 37,000 and 18,000 molecular weight components were less prominent in legumin isolated by the method of Danielsson than in legumin prepared by the method of Casey (Fig. 3, lanes C and D1). When legumin was prepared by a modified method of Danielsson, where the fraction precipitated with 40% (NH4)2SO ~ was discarded, the polypeptide pattern was similar to that of legumin isolated by the method of Casey (Fig. 3, lanes C and D2). The salt-insoluble material obtained from protein bodies extracted by the method of Thomson et al. contained some polypeptides that corresponded in molecular weight to those present in vicilin (Fig. 4, compare lanes R and V) and included several lowmolecular-weight components which were radioactively labeled in the in-vivo incorporation system (see Fig. 1 C).
Discussion
Based on studies of solubility and coagulation properties of pea globulins, Osborne and Campbell (1898)
w.J. Hurkman and L. Beevers: Glycosylationof Pea Legumin defined legumin as a protein insoluble in dilute salt solutions and not coagulated when heated to 100 ~ C, and vicilin as a protein soluble in salt solutions and coagulated when heated to 95 ~ C. Danielsson (1949), using the method of Osborne and Campbell (1898) to prepare pea globulins (i.e., extraction with salt, precipitation with 70% (NH~)2SO4, and dialysis) demonstrated by ultracentrifugation that globulins sedimented as two components with sedimentation coefficients of 12.6 S and 8.1 S. He found that the components could be separated by isoelectric precipitation at pH 4.5 and by coagulation experiments demonstrated that the 12.6 S component corresponded to legumin and the 8.1 S component to vicilin. Danielsson also examined the seed globulins prepared by this procedure from 34 species of the Leguminosae and found that nearly all contained two components of approx. 11 S and 7 S, which he termed legumin and vicilin, respectively. Legumin can therefore be defined as a protein fraction of legume seeds that is water-insoluble, saline-soluble, and precipitable from saline solution at pH 4.5. Legumin-like and vicilin-like proteins have been isolated from a number of dicotyledonous seeds (review: Derbyshire et al. 1976). Legumin is generally separated from vicilin by isoelectric precipitation at pHs near 4.5, essentially as described by Danielsson (1949). This procedure must be repeated several times for complete separation of legumin from vicilin (Danielsson 1949; Millerd 1975). Zonal isoelectric precipitation is a more rapid method that produces protein preparations that are relatively pure (Casey 1979; Scholz et al. 1974; Wright and Boulter 1974). However, as indicated in Fig. 2, legumin prepared by zonal isoelectric precipitation contains polypeptides with molecular weights common to those of vicilin. In our experience, zonal isoelectric precipitation can be utilized for pea globulins only when a protein fraction precipitated with 40% (NH4)2SO4 is discarded, a requirement in agreement with the method of Casey (1979). The protein isolated by the method of Casey is then only a fraction of the legumin prepared by the method of Danielsson. Since it contains only a fraction of the legumin defined and characterized by Osborne and Campbell and by Danielsson, the term legumin should not be applied to the protein prepared by zonal isoelectric precipitation. Perhaps this problem in terminology can be resolved by use of a speciesspecific term, such as pisin, to define the protein purified from legumin, as has been done for legumin-like proteins of soy bean (glycinin) and peanut (arachin), for example. A similar terminology has evolved for the prolamines of maize (zein) and barley (hordein). Legumin prepared from peas by the procedure of Danielsson (1949) is glycosylated, as previously
87 reported by Basha and Beevers (1976). However, the protein isolated by the procedure of Casey (1979) is not glycosylated and this is consistent with the lack of glycosylation reported for pea (Casey 1979; Gatehouse et al. 1980) and bean ( V i c i a f a b a L., Bailey and Boulter 1970) legumin (see also Croy et al. 1979). Our study shows that the contrasting results of Basha and Beevers (1976) and Casey (1979) on the glycosylation status of legumin were a consequence of the presence or absence of the proteins precipitated with 40% (NH4)2NO 4. The glycosylated component of legumin is precipitable with 40% (NH,~)2SO4, is present in protein bodies as a salt-insoluble component when they are isolated in aqueous extraction medium, and appears to be associated with polypeptides of approx. 12,00014,000 molecular weight. Since the component can be isolated with a buffered salt solution from intact cotyledons, it appears to be salt-soluble prior to exposure to water. The low solubility of this component following exposure to water could account for the difficulty in dissolving globulin fractions prepared by the Danielsson procedure. The solubility properties of this component could account for the observations of Davey and Dudman (1979) that polypeptides of legumin and vicilin prepared from protein bodies are glycosylated. This researchwas supported by National ScienceFoundation grant PCM7728273.
References
Bailey, C.J., Boulter, D. (1970)The structure of legumin, a storage protein of broad bean (Vicia faba) seeds. Eur. J. Biochem. 17, 460 466 Basha, S.M.M., Beevers,L. (1975) The developmentof proteolytic activity and protein degradation during germination of Pisum sativurn L. Planta 124, 77-87 Basha, S.M.M., Beevers, L. (1976) Glycoprotein metabolism in the cotyledonsof Pisurn sativum during developmentand germination. Plant Physiol. 57, 93-97 Beevers,L., Mense, R.M. (1977) Glycoproteinbiosynthesisin cotyledons of Pisurn sativum L.: Involvementof lipid-linked intermediates. Plant Physiol. 60, 703~08 Braconnot, H. (1827) Memoire sur un principe particular aux graines de la famille des legumineuses, et analyse des pois et des haricots. Ann. Chim. Phys. 34, 68-85 Browder, S.K., Beevers,L. (1978) Characterizationof the glycopeptide bond in legumin from Pisum sativum E. FEBS Lett. 89, 145-148 Casey, R. (1979) Immunoaffinitychromatography as a means of purifying legumin from Pisurn (pea) seeds. Biochem. J. 177, 509~20 Croy, R.R.D., Derbyshire, E., Krishma, T.G., Boulter, D. (1979) Legumin of Pisum sativurn and Vicia faba. New Phytologist 83, 29-35 Danielsson, L.E. (1949) Seed globulins of the gramineae and Ieguminosae. Biochem.J. 44, 387400
88 Davey, R.A., Dudman, W.F. (1979) The carbohydrate of storage glycoproteins from seeds of Pisum sativum: Characterization and distribution of component polypeptides. Aust. J. Plant Physiol. 6, 435~447 Davies, H.M., Delmer, D.P. (1979) Seed reserve-protein glycosylation in an in vitro preparation from developing cotyledons of Phaseolus vulgaris. Planta 146, 513 520 Derbyshire, E., Wright, D.J., Boulter, D. (1976) Legumin and vicilin, storage proteins of legume seeds. Phytochemistry 15, 3-24 Gatehouse, J.A., Croy, R.R.D., Boulter, D. (1980) Isoelectric-focusing properties and carbohydrate content of pea (Pisum sat# rum) legumin. Biochem. J. 185, 497-503 Larkins, B.A., Hurkman W.J. (1978) Synthesis and deposition of zein in protein bodies of maize endosperm. Plant Physiol. 62, 256-263 Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275 Millerd, A. (1975) Biochemistry of legume seed proteins. Ann. Rev. Plant Physiol. 26, 53 72 Osborne, T.B., Campbell, G.F. (1898) Proteids of the pea. J. Am. Chem. Soc. 20, 348-362
W.J. Hurkman and L. Beevers : Glycosyiation of Pea Legumin Roberts, R.M., Pollard, W.E. (i975) The incorporation of D-glucosamine into glycolipids and glycoproteins of membrane preparations from Phaseolus aureus hypocotyls. Plant Physiol. 55, 431M36 Scholz, G., Richter, J., Manteuffel, R. (1974) Studies on seed globulins from legumes. I. Separation and purification of legumin and vicilin from Viciafaba L. by zone precipitation. Biochem. Physiol. Pflanzen 166, 163-172 Thomson, J.A., Schroeder, H.E., Dudman, W.F. (1978) Cotyledonary storage proteins in Pisum sativum I. Molecular heterogeneity. Aust. J. Plant Physiol. g, 263~79 Weber, K., Osborn, M. (1969) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406M412 WeyI, T. (1876) Beitrfige zur Kenntniss thierischer und pflanzlicher Eiweissk6rper. Pflfigers Arch. 12, 635~638 WeyI, T. (1877) Beitrfige zur Kenntniss thierischer und pflanzlicher Eiweissk6rper. Z. Physiol. Chem. 1, 72-100
Received 5 May; accepted 7 July 1980
Planta 150, 82 88 (1980)
What is Pea Legumin - Is it Glycosylated? William J. Hurkman and Leonard Beevers Department of Botany and Microbiology,Universityof Oklahoma, Norman, OK 73019, USA
Abstract. Since there is some question as to whether
or not legumin is glycosylated, this storage protein was isolated by various procedures from developing cotyledons of P i s u m s a t i v u m L. supplied with [14C]labeled glucosamine and analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Legumin isolated by the classical method of Danielsson [(1949) Biochem. J. 44, 387-400] a procedure in which globulins extracted with a buffered salt solution are precipitated with ammonium sulfate (70% saturation) and legumin separated from vicilin by isoelectric precipitation, was labeled. The glucosamine incorporated into legumin was associated with low-molecular-weight polypeptides. In contrast, legumin isolated by the method of Casey [(1979) Biochem. J. 177, 509-520], a procedure where legumin is prepared by zonal isoelectric precipitation from globulins precipitated with 40-70% ammonium sulfate, was not labeled. However, the globulin fraction precipitated with 40% ammonium sulfate was labeled and the radioactive glucosamine was associated with low-molecular-weight polypeptides. Legumin isolated from protein bodies [Thomson et al. (1978) Aust. J. Plant Physiol. 5, 263279] was not extensively labeled. However, the saltinsoluble fraction of protein body extracts was labeled and the radioactivity was associated with low-molecular-weight polypeptides. These results indicate that protein bodies contain a glycoprotein of low-molecular-weight that co-purifies with legumin isolated by the method of Danielsson but that is discarded when isolation methods developed more recently are used. Key words: Legumin Storage protein.
Pisum
-
Protein (storage) -
Introduction
Historically, the name legumin was first given by Braconnot (1827) to the major protein fraction extracted
0032-0935/80/0150/0082/$01.40
with water from legume seeds. Seed proteins were later found to be salt-soluble globulins (Weyl 1876, 1877). Osborne and Campbell (1898) used ammonium-sulfate precipitation and repeated dilution and dialysis to separate globulins of pea seeds into two major protein fractions, legumin and vicilin, the former insoluble in dilute salt solutions and not coagulated when heated to 100 ~ C, the latter soluble in dilute salt solutions and coagulated when heated to 95~ Danielsson (1949) found that pea legumin could be more effectively separated from vicilin by isoelectric precipitation. Basha and Beevers (1976) showed that acid hydrolysates of legumin isolated by the procedure of Danielsson (1949) from developing pea seeds contained glucosamine, an amino sugar, and mannose, a neutral sugar, and Browder and Beevers (1978) demonstrated the occurrence of glucosaminyl-asparagine in glycopeptides isolated from legumin prepared by this method. These results indicated that pea legumin, as prepared by the Danielsson procedure, was glycosylated. Recently, zonal isoelectric precipitation has been utilized for separation of legumin from vicillin (Casey 1979; Scholz et al. 1974; Wright and Boulter 1974). Davey and Dudman (1979) found that pea legumin isolated from protein bodies by isoelectric precipitation followed by zonal isoelectric precipitation was glycosylated and the amounts of glucosamine and neutral sugars they found in their preparations were in good agreement with those previously reported by Basha and Beevers (1976). However, Casey (1979) reported that legumin prepared from mature pea seeds by zonal isoelectric precipitation and purified by diethylaminoethyl (DEAE)-cellulose chromatography and sucrose gradient centrifugation was not glycosylated. Similar results were reported by Gatehouse et al. (1980) for legumin prepared from pea seeds by hydroxylapatite chromatography. It thus, appears that legumin prepared by the pro-
W.J. Hurkman and L. Beevers: Glycosylation of Pea Legumin cedure of Danielsson
(1949) is g l y c o s y l a t e d w h e r e a s
l e g u m i n p r e p a r e d b y m o r e r e c e n t m e t h o d s is n o t . In order to determine the causes for this discrepancy, we have prepared legumin from the cotyledons of p e a s e e d s b y t h e m e t h o d s o f D a n i e l s s o n (1949), C a s e y (1979), a n d T h o m s o n e t al. (1978), a n d a n a l y z e d t h e preparations for glycosylated peptides. This was done using cotyledons supplied with [14C]glucosamine ( [ 1 4 C ] G l c N H 2 ) w h i c h is k n o w n t o b e i n c o r p o r a t e d i n t o l e g u m i n ( B a s h a a n d B e e v e r s 1976). T h e l a b e l e d l e g u m i n f r a c t i o n s , as w e l l as u n l a b e l e d l e g u m i n f r a c t i o n s p r e p a r e d f r o m m a t u r e seeds, w e r e a n a l y z e d b y s o d i u m d o d e c y l s u l f a t e ( S D S ) - p o l y a c r y l a m i d e gel electrophoresis. Our results showed that pea cotyledons contain a glycosylated component labeled with [ 1 4 C ] G l c N H 2 t h a t is a s s o c i a t e d w i t h p r o t e i n b o d i e s and co-purifies with legumin isolated by the proced u r e o f D a n i e l s s o n (1949) b u t w h i c h is d i s c a r d e d i n more recent procedures.
Materials and Methods Plant Material. Cotyledons were collected from developing peas (Pisum sativum L. cv. Burpeeana; Burpee Seed Co., Warminster, Pa. USA) cultured as described in Basha and Beevers (1976), or from mature seeds soaked in water overnight. Testas and embryos were removed. Preparation of [14CJGlucosamine-Labeled Proteins. Thirty cotyledons from seeds 24 or 28 d post-anthesis were collected and each cotyledon was sliced, vertically to the axis, into five pieces. Ten gl of [14C]GlcNHH2 (1.057.10 l~ Bq/mmo}.=286 mCi/mmol and 37.104 Bq/400 gl= 10 gCi/400 gl; Amersham, Arlington Heights, II1., USA) diluted 1:20 with water were placed on each slice. The slices were incubated in the light for 4 h, collected, and homogenized in the appropriate buffer. Cotyledons extracted by the methods of Danielsson (1949) and Casey (1979) (details below) were homogenized with a Polytron homogenizer (Brinkman Instruments, Westbury, N.Y., USA). Cotyledons 28 d post-anthesis were used for the method of Thomson et al. (1978) (details below); they were homogenized with the mechanical chopping device described by Beevers and Mense (1977). An additional 20 g of cotyledons were added to labeled cotyledons to be extracted by the methods of Casey and Thomson et al. in order to facilitate recovery of proteins separated by column chromatography. Protein Isolation by the Method of Danielsson (1949). Cotyledons were homogenized in Buffer A (1.0 M NaC1, 20 mM KzHPO 4 KH2PO4, pH 7.0; 7 ml buffer/g cotyledons). When proteins were prepared from mature seeds, 30 g of cotyledons were homogenized in a precooled Waring blender (four 15-s bursts). The brei was stirred for 30 rain on ice, filtered through fov.r layers of cheesecloth, and centrifuged (19,000 .g, 15 min; unless otherwise stated, all centrifugations were done at 0~ with an SS-34 rotor in a Sorvall RC 2B centrifuge; DuPont Co., Newtown, Conn. USA). The pellet was extracted in the same way twice more with Buffer A (20 ml). Solid (NH4)2SO4 was slowly added with stirring to the pooled supernatants to 70% saturation; the solution was stirred for 15 min on ice and centrifuged (3,000.g, 20 min). The supernatant was discarded. The 70% (NH4)zSO4 precipitate was suspended in Buffer B (0.2 M NaC1, 5 mM Kzt-IPO 4 KHzPO4, pH 7.0, 20 mI) and dialyzed against distilled water for 48 h at 4 ~ C. The dialysate
83 was centrifuged (19,000 .g, 15 min) and the supernatant discarded. The pellet was suspended in Buffer C (0.2 M NaC1, 5 mM K2HPO4--KH2PO4, pH 4.5; 20 ml), stirred overnight at 4 ~ C, and centrifuged (3,000.g, 10 rain). The pellet was washed twice with Buffer C (20 ml) and then suspended in Buffer B (20 ml). The suspended pellet, containing legumin, and the pooled supernatants, containing vicilin, were dialyzed against water for 48 h at 4 ~ C. Legumin and vicilin were recovered by centrifugation (19,000. g, 15 min) and stored at - I 0 ~ C. Protein Isolation by the Method of Casey (1979). Cotyledons were homogenized as above, but in Buffer D (0.5 M NaC1, 0.1 mM dithiolthreitol (DTT), 0.02% NAN3, 0.05 M tris(hydroxymethyl) aminomethane (Tris)-HCl, pH 7.0). The brei was stirred on ice for 15 min, filtered, and centrifuged (19,000-g, i0 min). The pellet was discarded and (NH4)2SO e was added to the supernatant to 40% saturation. After stirring for 15 min at room temperature, the solution was centrifuged (19,000-g, 10 min), the pellet discarded, and (NH4)2SO4 added to the supernatant to 75% saturation. After 15 min, the solution was centrifuged (19,000 .g, 10 min) and the supernatant discarded. The 40-75% (NH4)zSO4 pellet was dispersed in 5 ml of Buffer E (0.2 M NaC1, 0.1 M DTT, 0.02% NAN3, 0.05 M Tris-HC1, pH 8.0) and desalted by chromatography on a column (2.5 cm diameter, 100 cm long) of Sephadex G-25 (Sigma Chemical Co., St. Louis, Mo., USA) equilibrated in Buffer E. Globulins were eluted at room temperature with Buffer E (60 ml/ h) and concentrated by dialysis against polyethylene glycol (20,000 molecular weight). Legumin and vicilin were separated by zonal isoelectric precipitation : globulins were applied to a Sephadex G-50 column (3 cm diameter, 60 cm long) equilibrated with Buffer F (0.2 M NaC1, 0.1 M DTT, 0.02% NaN> 0.05 M citric acid-NaOH, pH 4.8) and eluted at room temperature with Buffer E (60 ml/h) and ultraviolet (UV)-absorbance monitored (280 nm, ISCO Model UA-2 UV Analyzer; Instrumentation Specialties Co., Lincoln, Neb., USA). Fractions corresponding to the first peak (vicilin) and those corresponding to the tailing half of the second peak (legumin) were pooled, respectively, and dialyzed against water for 48 h at 4 ~ C. Legumin and vicilin were recovered by centrifugation (19,000.g, 10 min) and stored at - 1 0 ~ C. The same procedure was used to obtain legumin and vicilin from protein bodies, except that precipitation with 40% (NH4)2 SO4 was omitted. Protein bodies were isolated from mature seeds by discontinuous gradient centrifugation according to the method of Larkins and Hurkman (1978). Protein Isolation by the Method of Thomson et al. (1978). Protein bodies, isolated in water and separated by differential centrifugation at 17,500-g (Thomson et al. 1978), were extracted with 1.0 M NaC1, 0.02% NaNa, 0.1 M K2HPO4--KH2PO4 (pH 7.2) for 1 h at 0 ~ C. The extract was centrifuged (19,000.g, 10 min) and the pellet retained. The supernatant was dialyzed at 4~ overnight to 0.2 M NaC1 in McIlvaine buffer, pH 4.8 (0.2 M Na2PO4, 0.1 M citric acid; see Scholz et al. 1974). The extract was centrifuged (19,000.g, 10rain) and the supernatant (vicilin) was dialyzed against water for 48 h at 4 ~ C. The pellet (legumin) was dissolved in 20 ml McIivaine buffer, pH 8.0, and applied to a Sephadex G-75 column (2.5 cm diameter, 80 cm long) equilibrated with McIlvaine buffer, pH 4.0. Protein was eluted (60 ml/h) at room temperature with McIlvaine buffer, pH 8.0. The retarded fractions were pooled and dialyzed against water for 48 h at 4 ~ C. Legumin and vicilin were recovered by centrifugation (19,000.g, 10 min) and stored at 10~ C. Acid Hydrolysis of La~eled Proteins. Forty gl of labeled proteins (approx. 9,000 cpm) were incubated in 1 ml of 4 N HC1 for 3 h in a boiling water bath. Following incubation, chloride was precipitated by addition of solid silver nitrate to the hydrolysate. The samples were dried with a stream of air and then rinsed with
84 water and redried; the water rinse was repeated twice more. The hydrolysates were dissolved in 100 gl of 80% (v/v) ethanol and analyzed by thin layer chromatography on cellulose (Chromagram 13255; Eastman-Kodak, Rochester, N.Y., USA) in bntanolpyridine-water (6:4:3, v/v). Chromatograms were divided into 1-cm strips and transferred into vials. Scintillation fluid was added and radioactivity determined (LS-100 scintillation counter; Beckman Instruments, Irvine, Cal., USA).
Gel Electrophoresis. Protein samples were analyzed on slab gels with a sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis system similar to that described by Laemmli (1970). Gels were 1.5 mm thick and consisted of a 9-cm running gel of i2.5% acrylamide (acrylamide/bisacrylamide=75:l, v/v) in 3.75mM Tris-HCl (pH 8.9), 0.058 mM N,N,N',N'-tetramethylethylenediamine (TEMED), and 0.075% SDS. The running gel was overlaid with a 2.5 cm stacking gel of 5% acrylamide. Freshly prepared ammonium persulfate was added to a final concentration of 0.035% immediately before each gel layer was poured. Proteins to be applied to the gel were dissolved by boiling in sample buffer (0.024 M Tris-HC1, pH 8.3, 1% SDS, 1% 2-mercaptoethanol, 0.002% bromophenol blue, 10% glycerol). Electrophoresis was done at room temperature at 15 mA constant current until the tracking dye had traversed the stacking gel, and then at 25 mA constant current until the dye had reached the bottom of the running gel. The gels were stained overnight in a solution of 0.1% Coomassie blue, 50% methanol, 10% acetic acid, and destained in 15% methanol7% acetic acid. The protein samples contained approximately 30 gg protein. Protein content was determined by the procedure of Lowry et al. (1951) with bovine serum albumin as standard. Phosphorylase B (molecular weight 94,000), bovine serum albumin (68,000), pyruvate kinase (57,000), horseradish peroxidase (40,000), carbonic anhydrase (30,000), trypsin inhibitor (21,000), and cytochrome c (11,700) were used as marker proteins for SDS-polyacrylamide gel electrophoresis. Mobilities of markers were calculated relative to cytochrome c and the apparent molecular weights of seed proteins were determined by reference to the mobilities of the markers (Weber and Osborn 1969). The values obtained, being approximations, were used to facilitate the identification of proteins and are not intended to represent precise molecular weights. Acrylamide, bisacrylamide and TEMED were obtained from EastmanKodak, Rochester, N.Y., USA, and ammonium persulfate, bromophenol blue, Coomassie blue, 2-mercaptoethanol, SDS, and the proteins used as standards from Sigma Chemical Co., St. Louis, Mo., USA. The distribution of radioactivity among polypeptides separated by gel electrophoresis was determined by the method of Davies and Delmer (1979). A 1-cm strip was removed from the center of the gel and stained with Coomassie blue. The rest of the gel was sliced and the slices were incubated in 3% (v/v) Protosol (New England Nuclear, Boston, Mass., USA) in ACS (aqueous counting scintillant; Amersham, Arlington Heights, Ill., USA) for 48 h at 50 ~ C.
Results
Distribution of Radioactivity Among Protein Fractions Isolated from Cotyledons Supplied with [14C]Glucosamine. When developing pea cotyledons were labeled with [14C]GlcNH2, radioactivity became associated with legumin and vicilin fractions isolated by the methods of Danielsson (1949), Casey (1979), and Thomson et al. (1978) (Table 1). In the globulins prepared according to Danielsson, the legumin fraction
W.J. Hurkman and L. Beevers : Glycosylation of Pea Legumin Table 1. Distribution of radioactivity among protein fractions prepared by various procedures from developing pea cotyledons supplied with [14C]glucosamine. The protein fractions prepared by isoelectric precipitation (Methods 1,3~) are termed legumin and vicilin after Danielsson (1949). The fractions prepared by zonal isoelectric precipitation (Method 2) are termed legumin and vicilin after Scholz et al. (1974). The 40% (NH4)2SO 4 fractions were precipitated from the salt soluble fraction obtained by Casey's (1979) method. The salt insoluble fraction was the residual material remaining after salt extraction of protein bodies prepared by the method of Thomson et al. (1978) Method
Protein fraction Vicilin
Legumin
1. Danielsson (1949)
12,700a 251,500
2. Casey (1979)
19,000
3. Danielsson on 19,000 40 70% (NH4)2SO4 fraction 4. Danielsson on 40% (NH~)2SO4 fraction 5. Thomson et al. (1979) a
4,800
40% Salt (NH4)zSO4 insoluble
676,760
10.900
5,700
306,200
52,900
17,600
272,900
Values are expressed as cpm/fraction
was much more extensively labeled than the vicilin fraction (Table 1, Method 1), a result consistent with previous observations by Basha and Beevers (1976). In contrast, radioactivity was found to be much less extensively incorporated in globulin fractions isolated by the procedure of Casey, and the vicilin fraction contained the greatest amount of label (Table 1, Method 2). Radioactivity was associated with globulins isolated from protein bodies by the procedure of Thomson et al. and vicilin was more extensively labeled than legumin (Table 1, Method 5). The ratio of label between vicilin and legumin was similar in the proteins isolated by the methods of Casey and Thomson et al. Acid hydrolysis of legumin samples prepared by the various procedures indicated that the hydrolysates contained radioactivity that co-chromatographed with authentic [14C]GlcNH2. Thus, the label associated with legumin fractions was due to glycosyl components and cannot be attributed to conversion of the applied glucosamine to amino-acid residues. Since legumin prepared according to the classical procedure of Danielsson was extensively labeled, analyses were performed to account for the low association of radioactivity with legumin fractions prepared by the more recent methods of Casey and of Thomson et al. A major difference between the proce-
W.J. Hurkman and L. Beevers: Glycosylation of Pea Legumin
A I II
I
|11
10 20 30 SLICE NUMBER
IB I
I
,I ,11 7
85
It,, III1,11 I~1
10 20 3R SLICE NUMRER
10 20 30 SLICE NUMBER
Fig. 1A-C. Distribution of radioactivity and protein components following SDS-polyacrylamide gel electrophoresis of [14C]glucosaminelabeled protein preparations from pea cotyledons 24 d post-anthesis. A Legumin prepared by the procedure of Danielsson (1949). B Protein precipitated with 40% (NH4)2SO4 that is normally discarded in the method of Casey (1979). C Protein from the insoluble residue fraction of protein bodies isolated and extracted by the method of Thomson et al. (1978). O, origin; 20 and 40, 20,000 and 40,000 molecular weight components, respectively
dure of Danielsson and that of Casey is that the latter method utilizes a 40 75% (NH~)2SO4 precipitate as a source of globulins whereas the former method utilizes a 0-70% (NH4)2SO4 precipitate. It was found that the globulin fraction prepared by the Danielsson method was difficult to disperse in the suspending buffers whereas that prepared by Casey's method (40-75% (NH4)2SO4) was more readily solubilized. When the 0-40% (NH~)2SO4 fraction normally discarded in the Casey method was recovered and its radioactivity determined, this fraction was found to be extensively labeled (Table 1, Method 2). When differential (NH~)2SO4 fractionation was performed on proteins isolated by Danielsson's method, it was found that within the 40-70% fraction vicilin was most extensively labeled (Table 1, Method 3) whereas in the 0-40% fraction the greatest proportion of radioactivity was in legumin (Table 1, Method 4). The greater recovery of radioactivity in globulins recovered by procedures where the 0 4 0 % (NH4)2SO4 fraction was included, in contrast to the lower level recovered from globulins isolated from protein bodies (Table 1, Method 5), indicated either that the 0-40% precipitable protein was not associated with protein bodies or that it was not solubilized by salt extraction of these organelles. The analyses showed that the saltinsoluble fraction of protein bodies was extensively labeled (Table 1, Method 5).
SDS-Polyacrylamide Gel Analysis of Proteins Labeled with [14C]Glucosamine. The information in Table 1 indicates that the major discrepancy concerning the glycosylation of legumin can be accounted for by the fact that, when the newer methods of Casey and of Thomson et al. are used, a glycosylated component is discarded. This concept was confirmed when labeled legumin fractions were analyzed by SDS-poly-
acrylamide gel electrophoresis (Fig. 1 A C). Legumin isolated by the procedure of Danielsson contained six major polypeptides with two prominent components that had molecular weights of approx. 20,000 and 40,000 (Fig. 1 A). Radioactivity was associated principally with smaller components of approx. 12,000 14,000 molecular weight. This association of radioactivity with low-molecular-weight polypeptides of legumin is consistent with the earlier observations of Basha and Beevers (1976). The limited extent of labeling of the legumin fraction prepared by the procedure of Casey precluded definitive conclusions concerning the association of radioactivity with specific polypeptides. When the 0-40% (NH4)2SO4 fraction, nromally discarded in Casey's procedure, was analyzed by SDS-polyacrylamide gel electrophoresis, it was found that this fraction contained several polypeptides and radioactivity was associated with those of low molecular weight (Fig. 1 B). Electrophoretic analysis of the insoluble residue fraction from protein bodies showed the presence of several polypeptides; the low-molecular-weight components contained the majority of the radioactivity (Fig. 1 C). The rapid migration of radioactivity in the legumin fraction (Fig. 1A), the 0-40% (NH4)2SO~ fraction (Fig. 1B), and the insoluble fraction of protein bodies (Fig. 1 C) is similar to that of a low-molecular-weight component produced when particulate fractions of mungbean hypocotyls are incubated with labeled glucosamine (Roberts and Pollard 1975). Roberts and Pollard (1975) suggested that the radioactivity was associated with a lipid fraction, thus raising the possibility that the labeled component isolated during legumin fractionation may be a glycolipid rather than a glycoprotein. However, repeated attempts to recover radioactivity from the protein preparation by such lipid solvents as chloroform-methanol (2:1) and chloro-
86
W.J. Hurkman and L. Beevers: Glycosylationof Pea Legumin
Fig. 2. SDS-polyacrylamidegel analysis of globulins prepared from pea cotyledons by the methods of Casey(1979) (C) and Danielsson (1949) (D) and of globulins isolated by the method of Casey (1979) (P) from protein bodies recovered by discontinuous gradient centrifugation. S, standards; L, legumin; V, vicilin; arrows, origin; MW, molecular weight x 10-3 Fig. 3. SDS-polyacrylamidegel analysis of pea legumin prepared from globulins precipitated with 40 70% ammonium sulfate by the method of Casey (1979) (C), 0-70% ammonium sulfate by the method of Danielsson (1949) (D1), and 40 70% ammonium sulfate by the method of Danielsson (D2). S, standards; MW, molecular weight x 10-3 Fig. 4. SDS-polyacrylamidegel analysis of globulins from protein bodies of pea cotyledons prepared and extracted by the method of Thomson et al. (1978). V, vicilin, and L, Iegumin, prepared by isoelectric precipitation; R, insoluble residue fraction of extracted protein bodies; S, standards; MW, molecular weight x 10-3
form-methanol-water (1:1:0.3) were unsuccessful. This observation coupled with the occurrence of a glucosamine-asparagine linkage in polypeptides isolated from legumin prepared by the procedure of Danielsson (Browder and Beevers 1978) indicates that the radioactivity from glucosamine is associated with glycoprotein. SDS-Polyacrylamide Gel Analyses of Proteins Isolated from Mature Seeds. Since developing seeds were used in our study whereas mature seeds were used in the studies of Casey (1979), Thomson et al. (1978), and Davey and Dudman (1979), it was necessary to determine the polypeptide composition of fractions prepared from mature seeds by the procedures of Table 1. Analyses by SDS-polyacrylamide gel electrophoresis showed that proteins prepared by each method had different polypeptide patterns (Figs. 2, 4). In general, legumin had two principal components with molecular weights of 37,000 and 18,000 (Fig. 2, lanes CL, DL, PL; Fig. 4, lane L), polypeptides that probably corresponded to the 40,000 and 20,000 components generally reported for pea legumin (Casey 1979 ; Croy et al. 1979; Davey and Dudman 1979). Legumin also contained four other polypeptides; two of these, 25,000 and 93,000 molecular weight, were specific for legumin whereas those of 54,000 and 74,000 molecular weight were similar to those encountered in the vicilin fraction. The 54,000 and 74,000 molecular weight polypeptides were present in legumin prepared by isoelectric precipitation (Fig. 2, lane DL) or by zonal
isoelectric precipitation from seeds (Fig. 2, lane CL) or protein bodies (Fig. 2, lane PL; Fig. 4, lane L). In addition, these polypeptides occurred in legumin initially obtained by zonal isoelectric precipitation and further purified by chromatography on DEAEcellulose by the method of Casey (1979) (data not shown). Analyses by SDS-polyacrylamide gel electrophoresis indicated that the 37,000 and 18,000 molecular weight components were less prominent in legumin isolated by the method of Danielsson than in legumin prepared by the method of Casey (Fig. 3, lanes C and D1). When legumin was prepared by a modified method of Danielsson, where the fraction precipitated with 40% (NH4)2SO ~ was discarded, the polypeptide pattern was similar to that of legumin isolated by the method of Casey (Fig. 3, lanes C and D2). The salt-insoluble material obtained from protein bodies extracted by the method of Thomson et al. contained some polypeptides that corresponded in molecular weight to those present in vicilin (Fig. 4, compare lanes R and V) and included several lowmolecular-weight components which were radioactively labeled in the in-vivo incorporation system (see Fig. 1 C).
Discussion
Based on studies of solubility and coagulation properties of pea globulins, Osborne and Campbell (1898)
w.J. Hurkman and L. Beevers: Glycosylationof Pea Legumin defined legumin as a protein insoluble in dilute salt solutions and not coagulated when heated to 100 ~ C, and vicilin as a protein soluble in salt solutions and coagulated when heated to 95 ~ C. Danielsson (1949), using the method of Osborne and Campbell (1898) to prepare pea globulins (i.e., extraction with salt, precipitation with 70% (NH~)2SO4, and dialysis) demonstrated by ultracentrifugation that globulins sedimented as two components with sedimentation coefficients of 12.6 S and 8.1 S. He found that the components could be separated by isoelectric precipitation at pH 4.5 and by coagulation experiments demonstrated that the 12.6 S component corresponded to legumin and the 8.1 S component to vicilin. Danielsson also examined the seed globulins prepared by this procedure from 34 species of the Leguminosae and found that nearly all contained two components of approx. 11 S and 7 S, which he termed legumin and vicilin, respectively. Legumin can therefore be defined as a protein fraction of legume seeds that is water-insoluble, saline-soluble, and precipitable from saline solution at pH 4.5. Legumin-like and vicilin-like proteins have been isolated from a number of dicotyledonous seeds (review: Derbyshire et al. 1976). Legumin is generally separated from vicilin by isoelectric precipitation at pHs near 4.5, essentially as described by Danielsson (1949). This procedure must be repeated several times for complete separation of legumin from vicilin (Danielsson 1949; Millerd 1975). Zonal isoelectric precipitation is a more rapid method that produces protein preparations that are relatively pure (Casey 1979; Scholz et al. 1974; Wright and Boulter 1974). However, as indicated in Fig. 2, legumin prepared by zonal isoelectric precipitation contains polypeptides with molecular weights common to those of vicilin. In our experience, zonal isoelectric precipitation can be utilized for pea globulins only when a protein fraction precipitated with 40% (NH4)2SO4 is discarded, a requirement in agreement with the method of Casey (1979). The protein isolated by the method of Casey is then only a fraction of the legumin prepared by the method of Danielsson. Since it contains only a fraction of the legumin defined and characterized by Osborne and Campbell and by Danielsson, the term legumin should not be applied to the protein prepared by zonal isoelectric precipitation. Perhaps this problem in terminology can be resolved by use of a speciesspecific term, such as pisin, to define the protein purified from legumin, as has been done for legumin-like proteins of soy bean (glycinin) and peanut (arachin), for example. A similar terminology has evolved for the prolamines of maize (zein) and barley (hordein). Legumin prepared from peas by the procedure of Danielsson (1949) is glycosylated, as previously
87 reported by Basha and Beevers (1976). However, the protein isolated by the procedure of Casey (1979) is not glycosylated and this is consistent with the lack of glycosylation reported for pea (Casey 1979; Gatehouse et al. 1980) and bean ( V i c i a f a b a L., Bailey and Boulter 1970) legumin (see also Croy et al. 1979). Our study shows that the contrasting results of Basha and Beevers (1976) and Casey (1979) on the glycosylation status of legumin were a consequence of the presence or absence of the proteins precipitated with 40% (NH4)2NO 4. The glycosylated component of legumin is precipitable with 40% (NH,~)2SO4, is present in protein bodies as a salt-insoluble component when they are isolated in aqueous extraction medium, and appears to be associated with polypeptides of approx. 12,00014,000 molecular weight. Since the component can be isolated with a buffered salt solution from intact cotyledons, it appears to be salt-soluble prior to exposure to water. The low solubility of this component following exposure to water could account for the difficulty in dissolving globulin fractions prepared by the Danielsson procedure. The solubility properties of this component could account for the observations of Davey and Dudman (1979) that polypeptides of legumin and vicilin prepared from protein bodies are glycosylated. This researchwas supported by National ScienceFoundation grant PCM7728273.
References
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Received 5 May; accepted 7 July 1980
