Planta (Berl.) 124, 77--87 (1975) 9 by Springer-Verlag 1975
The Development of Proteolytic Activity and Protein Degradation during the Germination of Pisum sativum L. S. M. M. Basha* and Leonard Beevers Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma 73069, USA Received 17 December 1974; accepted 16 February 1975 Summary. The change in protein content and composition of the cotyledons of Pisum sativum L. cv. Burpeeana during germination was studied. Protein depletion from the coty-
ledons was slow during the first 4 days of germination but became rapid on the 5th day and by the 16th day the majority of the protein had disappeared. During the first 4 days the depletion of the globuliI)s exceeded that of the albumins; legumin appeared to be degraded slightly more rapidly than vicilin during the early phase of germination. Sodium-dodecylsulfate (SDS) electrophoresis of SOS- and dithiothreitol-dissociated globulins indicated that before rapid protein depletion there were marked changes in the component composition of the major globulins legumin and vieilin. The onset of rapid protein depletion was associated with an increase in the level of an aeid-sulfhydryl protease in the cotyledons. These findings indicate that the reserve globulins undergo modifications prior to their eventual hydrolysis. Introduction During the germination of pea seeds the nitrogen content of the cotyledons decreases while t h a t of the growing axis increases (Larson and Beevers, 1965; Beevers and Guernsey, 1966). The accumulation of soluble amino acids in cotyledons and axis, which accompanies the depletion of nitrogen in the former (Beevers and Guernsey, 1966), indicates t h a t the alterations in nitrogen content are caused by a hydrolysis of the reserve proteins in the cotyledon followed b y a transport of the products to the developing axis (Beevers and Splittstoesser, 1968). The mechanism by which the cotyledonary proteins are degraded is little understood. Danielson (1951) reported t h a t the globulins were broken down more rapidly than the albumins in germinating peas. The degradation of globulins in soybeans (Catsimpoolas et al., 1968) and peanuts (Daussant et al., 1969) is accompanied b y a change in the electrophoretic mobility of these proteins. Thus an early feature of reserve utilization appears to be the removal of amido groups b y the action of deamidase (Catsimpoolas et al., 1968; Daussant etal., 1969). However, there have been no reports of deamidases in the cotyledons. I n contrast there have been m a n y demonstrations of the presence of proteolytic enzymes in the cotyledons of germinating seeds (Ryan, 1973 ; Dechary, 1970), but in m a n y instances, the role of these enzymes in the mobilization of reserve protein remains to be established. I t was originally reported t h a t proteolytic activity did not change in the cotyledons of peas during germination (Young and Varner, 1959; Henshall and * Present Address: Department of Food Science, Georgia Agricultural Experiment Station, Experiment, Georgia, 30212, USA.
78
S.M.M. Basha and L. Beevers
Goodwin, 1964). H o w e v e r , m o r e r e c e n t l y it has been d e m o n s t r a t e d t h a t proteolyric a c t i v i t y , m e a s u r e d as t h e c a p a c i t y to h y d r o l y s e casein (Beevers a n d S p l i t t stoesser, 1968; Beevers, 1968; u a n d Varner, 1973) or a z a g l o b u l i n ( H o b d a y et al., 1973), increases d u r i n g g e r m i n a t i o n . I n ~ddition, m a t u r e p e a cotyledons c o n t a i n e d a high level of p e p t i d a s e a c t i v i t y , c a p a b l e of h y d r o l y s i n g leucine-pn i t r o a n i l i d e a n d b e n z o y l a r g i n y l - p - n i t r o a n i l i d c , a n d this a c t i v i t y declined d u r i n g g e r m i n a t i o n (Beevers, 1968). P u s t z a i a n d D u n c a n (1971) a n d Y o m o a n d S r i n i v a s a n (1973) h a v e r e p o r t e d similar changes in p r o t e o l y t i c a c t i v i t y d u r i n g t h e g e r m i n a t i o n of Phaseolus vulgaris; however, p e p t i d a s e a c t i v i t y r e m a i n e d r e l a t i v e l y c o n s t a n t t h r o u g h o u t t h e p e r i o d of s t u d y (Pustzai a n d Duncan, 1971). A p a r t from t h e i n v e s t i g a t i o n s of H o b d a y et al. (1973), who utilized a modified p l a n t p r o t e i n (azaglobulin) as s u b s t r a t e all studies of p r o t e o l y t i c a c t i v i t y d u r i n g seed g e r m i n a t i o n were m a d e w i t h casein as s u b s t r a t e , a n d it can be a r g u e d t h a t t h e p r o t e o l y t i c a c t i v i t y t h u s m e a s u r e d bears little r e l a t i o n s h i p to t h e e n z y m e s i n v o l v e d in t h e h y d r o l y s i s of n a t i v e reserve proteins. W e h a v e therefore u s e d p e a p r o t e i n s labeled w i t h t r i t i a t e d a m i n o acids as s u b s t r a t e s for t h e a s s a y of p r o t e o l y t i c enzymes. W e h a v e followed t h e f l u c t u a t i o n s in p r o t e o t y l i e a c t i v i t y d u r i n g germin a t i o n a n d r e l a t e d t h e m to changes in t h e p r o t e i n s of t h e cotyledon.
Material and Methods Plant. Material. Pea seeds (Pisum sativum L. cv. Burpeeana, W. Atlee Burpee Comp., Clinton, Iowa, USA) were germinated at 25 ~ in the dark in sterile vermiculite and the seedlings irrigated with deionized water. The addition of water to the dry seeds is taken as zero (0) times germination. Cotyledons were obtained from the seedling at different stages of germination. Protein Extraction and Yractionation. The testa and embryo were removed from the seed and protein was ffactionated from the cotyledons by a procedure modified from Danielson (1949). The cotyledons were homogenized in cold 1 M NaC1, 20 mM sodium-phosphate buffer, pit 7.0, with a Virtis tissue homogenizer at high speed for 2 rain and at low speed for 8 rain. The homogenate was stirred for 30 min and then centrifuged at 20000 • for 15 min. The resulting pellet was extracted twice with the same buffer, and centrifuged. The resulting three supernatants were pooled and made to 70% saturation by adding solid (NH4)2SOa. After standing for 1 h the mixture was centrifuged at 30000• for 20 rain. The supernatant was discarded and the pellet suspended in 0.2 M NaC1, 5 mM phosphate buffer, p i t 7.0, and dialysed against distilled water for 2 days. The dialysates were centrifuged at 20000 • for 15 min. The supernatant fraction was designated as albumin and the pellet as globulin. The globulin fraction was further suspended in 0.2 M NaCI, 5 mM phosphate buffer, pH 4.5, and stirred overnight at 4 ~ This globulin suspension was centrifuged at 3000 • g for 10 rain. The resulting pellet was washed twice with the same buffer and centrifuged. The three supernatants were pooled and dialysed against distilled water for 2 days. The material precipitated during the dialysis was designated as vicilin. The remaining pellet from the globulin fraction was suspended in 0.2 M NaC1, 5 mM phosphate buffer, pH 7.0 and dialysed against distilled water for 2 days. The dialysate was centrifuged at 20000 • g for 15 rain. The supernatant was discarded and the pellet collected as legumin. The legumin and vicilin pellets were suspended in 0.2 M NaC1, 20 mM phosphate buffer, pH 7.0, and stored at -- 10% Protein Determination. The protein content of the different fractions (Total protein, albumin, globulin, legumin and vicilin) of cotyledon extracts was determined by the method of Lowry et al. (1951) with bovine serum albumin as a standard. Polyavrylamide Gel Electrophoresis. Gel eleetrophoresis in 7.5% polyacrylamide was performed according to the method of Davis (1964). The protein samples suspended in 0.21~
Proteolysis in Cotyledons of Germinating Peas
79
NaCI, 20 mM phosphate buffer, pit 7.0, were made to 5 % sucrose concentration and 100-200 ~zg of protein applied per gel (6 • 90 ram). Electrophoresis was run at a constant current of 4 mA/tube for 2 h, or until the tracking dye reached ca. 1 cm above the bottom of the gel. Mter electrophoresis the gels were stained with 1% amido black stain in 7.5% acetic acid for 1 h. The gels were destained electrophoretically in 7.5% acetic acid, and scanned in a Gilford instrument linear transport system at 620 rim. SDS Gel Eleetrophoresis. Legumin and vicilin were dissociated essentially by the method of Palmiter et al. (1971). The protein was precipitated with trichloroacetic acid and a freshly made solution containing 1.2% Tris (pH unadjusted), 1.5% dithiothreitol, 1.0% sodium dodeeyt sulfate and 20% glycerol was added to the precipitate to give a final protein concentration of 2 mg/ml. The protein was dissociated by mixing the sample with a glass rod for 1-5 min while heating in a boiling water bath. SDS gel electrophoresis was carried out in 5% polyacrylamide gels by the procedure of Weber and Osbom (1969) by applying 200 fzg of dissociated protein per gel. The electrophoresis was carried out for 4 h and then the gels were removed from the Plexiglass tubes and fixed in 20% sulfosalieylic acid for 18 h. The gels were stained with 0.25 % Coomassie blue in water for 3 h; destained by repeated washings with 7.5% acetic acid, and scanned at 620 nm. Preparation o/Enzyme Extracts. The cotyledons from germinating pea seeds were homogenized for 2 rain at high speed in a Virtis tissue homogenizer with 0.02 M sodium-phosphate buffer, pH 7.2, with or without 5 mlY[2-mercaptoethanol. The tissue-to-buffer ratio was 1:4 (w/v). The homogenates were squeezed through a layer of cheesecloth and Miracloth (Calbiochem, La Jolla, Calif., USA) and centrifuged at 20000 • for 20 min. The supernatant from this eentrifugation was used in the protease assays. Standard Protease Assay. Proteolytic activity was measured using [3H]albuminm, [SH]legumin or [aH]vicilin, prepared from peas, as substrate. Crude enzyme extract (1 ml) was incubated with 0.2 ml [3H]legumin (4.4 • 10-3 cpm/ml) or 0.2 ml [3H]vicilin (2.7 • 10-~ cpm/ml) or 0.5 ml [all]albumin (1.1 • 10-5 cpm/ml) and 1.0 ml of 0.2 M sodium-citrate buffer, pH 5.0, in a shaker bath at 40~ for 3 h. The reaction was terminated by addition of 0.8 ml (in legumin and vicilin assays) or 0.5 ml (in albumin assays) of 1% casein, pH 7.0, followed by 1.0 ml of 20% triehloroacetio acid to give a final volume of 4.0 ml. A reaction mixture in which casein and 20 % trichloroacetie acid was added immediately after the addition of the enzyme extract served as the zero-time control. After the addition of trichloroacetic acid the samples were allowed to precipitate overnight at 4~ and then the insoluble material was removed by centrifugation. A 1-ml aliquot was taken from the triehloroacetic acid soluble supernatant and after addition of 12 ml of scintillation fluid [8.0 g PPO (2,5-diphenyloxazole) in 2 1 toluene, 1 1 Triton X-100] the radioactivity was determined in a Beckman LS-100 scintillation counter. Appropriate modifications of the standard assay were made in order to establish optimal pH and sulfhydryl requirements for proteolytie activity. In studies of pH effects 0.2 M sodinm-phosphate buffer was used from pH 6.5 to 9.0, and 0.2 M sodium citrate buffer between pH 3.0 and 6.0. The sulfhydryl content of the reaction mixture was modified by the addition of 2-mercaptoethanol. Preparation ot 3H-Labeled Substrates. 5 Ezl of [all]amino acids (1 mCi/ml tritiated aminoacid mixture TRK 440, Amersham Searle Corp., Arlington Heights, Ill., USA) were injected with a Hamilton syringe into each cotyledon of 20 peas in the pods, 21 days after flowering. The pods were left attached to the plant and the cotyledons were collected at maturity, 36 days after flowering. The protein was extracted from the cotyledons and ffactionated into albumins, vieilin, and legumin. Results
Course o/Protein Disappearance P r o t e i n disappearance from the cotyledons was slow d u r i n g the first 4 days of g e r m i n a t i o n , b u t became r a p i d after the 5th d a y (Fig. 1). D u r i n g the first 4 days the depletion of the globulins exceeded t h a t of the a l b u m i n s ; after 5-6 days of g e r m i n a t i o n b o t h the a l b u m i n a n d globulin b r e a k d o w n increased rapidly,
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and by the 16th day the majority of the protein had disappeared. Legumin appeared to be degraded slightly more rapidly than vicilin during the initial phases of germination. These findings are similar to those of Danielson (1951). Gel Electrophoresis o/Albumin
Numerous protein bands were evident after electrophoresis and amido-black staining of the albumin fraction. The composition of this fraction began to change after 2 days of germination, with a dramatic alteration in the protein components occurring after day 4 (Fig. 2). Some of the proteins disappeared during germination but there was also a production of new components as indicated by the development of new absorbing peaks in the stained gels. The albumin fraction contains enzymatic proteins (Danielson, 1956) and in view of the altered metabolic activity of the cotyledon during germination an alteration in the component proteins is to be expected. Polyacrylamide gel electrophoresis of the legumin fraction, followed by staining with amido-black, indicated that this fraction contained only one detectable component. There was a change in the electrophoretic mobility of the fraction prepared from pea cotyledon at successive stages of germination (Table 1), the l~m increasing from 0.17 at 0 days of germination to 0.22 after 8 days of germination. This change in electrophoretic mobility is similar, although of less magnitude, to that reported for soybean proteins by Catsimpoolas et al. (1968) and proteins from peanut cotyledons by Daussant et al. (1969).
Proteolysis in Cotyledons of Germinating Peas
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and 8 of germination whereas in vieilin they were observed between days 10 and 12. Catsimpoolas et al. (1968) and Daussant et al. (1969) attribute the change in electrophoretic mobility to a deamidation of the reserve protein during germination. Our data indicate that the change in charge of the reserve proteins occurs after the commencement of protein depletion. Deamidation of legumin and deearboxylation of vicilin appear therefore to play no role in the initiation of protein degradation during pea seed germination.
SDS Gel Electrophoresis o/Legumin and Vicilin SDS electrophoresis of the globulin fractions, prepared from mature seeds and dissociated in the presence of SDS and dithiothreitol, indicated that legumin was composed of three major and two minor components while vicilin contained five major components. During germination the component composition of these reserve proteins underwent marked changes. Component I I I of legumin began to decline at day 2 of seed germination and components I, I I and IV had disappeared by day 6 while a fraction with the mobility of component V was still present 14 days after germination, and more slowly migrating components appeared on days 12 and 14 of germination (Fig. 3). Changes in the components of legumin were evident before alterations were observed in the composition of vicilin (Fig. 4). This is consistent with the slower initial depletion of vicilin (Fig. 1).
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Fig. 4. Optical density scans of Coomassie-blue-stainedSDS polyacrylamide gels after electrophoresis of SDS- and dithiothreitol-dissociated vicilin prepared from pea cotyledons 0, 2, 4, 6, 8, 10, 12 and 14 days after seed germination Major changes in the composition of vicilin were observed after day 4; component I disappeared first, followed by II and III. Components IV and V appeared to be degraded slowly and made up an increasing proportion of the vicilin in the late stages of seed germination. Some new, slower-migrating components not present in vieilin preparations from dry seeds were seen after day 10 of germination.
Proteolytic Activity In extracts prepared from cotyledons 13 days after germination it was possible to demonstrate the presence of enzymes capable of releasing trichloroacetic-acid soluble radioactivity from sit-labeled albumins, vicilin and legumin (Fig. 5). Maximal hydrolytic activity with Jail]albumin as substrate, but only limited hydrolysis of [aH]vicilin and [aH]legumin, was observed at pH 5.0 in extracts prepared in the absence of 2-mercaptoethanol. In contrast, enzyme extracts prepared in the presence of 2-mercaptoethanol were able to hydrolyse legumin and vicilin with optimal activity at pH 5.0 and also showed enhanced activity with albumin as substrate. Trichloroacetic-aeid-soluble radioactivity was released from [SH]vicilin at pH 3.0; however, this may be due to the dissociation of the proteins at low pH rather than being caused by enzymatic hydrolyses.
Requirement ]or Sul]hydryl The enhanced hydrolytic activity observed in extracts prepared in the presence of 2-mercaptoethanol may be explained in two ways: (1) 2-mercaptoethanol may 6*
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serve as a p r o t e c t a n t to p r e v e n t d e t e r i o r a t i o n or i n a c t i v a t i o n of t h e p r o t e i n during isolation, (2) s u l f h y d r y l groups m a y be a necessary, i n t e g r a l c o m p o n e n t of t h e p r o t e o l y t i c reaction. A series of e x p e r i m e n t s was p e r f o r m e d to d e t e r m i n e t h e role of s u l f h y d r y l (Fig. 6 A - D ) . T h e e n z y m e p r e p a r a t i o n (C) showed o n l y l i m i t e d p r o t e o l y t i e a c t i v i t y b u t was s t i m u l a t e d b y inclusion of 5 m M 2 - m e r c a p t o -
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Fig. 7A and B. Changes during germination of the capacity of extracts from pea cotyledons to hydrolyse [3H]legumin (A) and [3H]vicilin (B) at pH 5.0 (9 and pH 7.5 (o). :Noteexpansion of scale in early stages of germination
ethanol in the reaction mixture. Thus although the enzyme(s) had been protected by the presence of 2-mercaptoethanol during extraction the proteolytie activity was reduced in reaction mixtures devoid of sulfhydryl. (Some mercaptoethanol may have been carried over in the ammonium-sulfate precipitate but this was insufficient to sustain maximum hydrolytic activity.) Extracts prepared in the absence of 2-mercaptoethanol showed limited hydrolysis in the absence of sulfhydryl (B) but were much more active in its presence (B) and (D). The results indicate that sulfhydryl functions as a component of the hydrolytic reaction, rather than as a proteetant during enzyme isolation. The greater activity of extracts prepared in the absence of mercaptoethanol towards [3H]albumin in comparison to legumin and vicilin may be related to the fact that albumins have a geater content of sulfur-containing amino acids than globulins (Boulter, 1973).
Proteolytic Activity during Seed Germination Enzyme extracts prepared from the cotyledons of germinating seeds of different ages showed varying capacities to hydrolyse all-labeled proteins at pH 5.0. Very low enzyme activity was present in the extracts from the cotyledons until day 3 of germination (Fig. 7). Between days 3 and 5 of germination the enzyme activity increased slowly, but between days 5 and 15 there was a rapid enhancement in the hydrolytic capacity of the extracts. A similar time course was also obtained for albumin hydrolysis (data not shown). Beevers (1968) and Beevers and Splittstoesser (1968) had observed caseolytic activity in extracts from pea cotyledons at p i t 5.5 and pH 7.0, with greater activity at the lower pit. Although
86
S.M. NI. Basha and L. Beevers
the studies using extracts from cotyledons 13 days after germination (Fig. 5) had failed to show any evidence of a peak of hydrolytic activity at pH 7.0 when pea proteins were used as substrate it is nevertheless possible that enzymes with optimal activity near pH 7 are present in extracts from pea cotyledons at other stages of germination. Thus, it was found that enzyme preparations from cotyledons after day 9 of germination did show proteolytic activity at pH 7.5. However, the consistently lower activity, and the overall similarity of the time course of the development of hydrolytic activity during germination indicate that enzymatic activity measured at pH 7.5 may merely represent the residual activity of the same enzyme that has optimal activity at pH 5.0. It is thus possible that the proteolytic activity in the cotyledonary extracts of peas is mediated by one acid sulfhydryl protease which is capable of hydrolysing the albumins as well as the globulins, legumin and vicilin. Conclusions Although the most rapid depletion of protein from the cotyledons did not occur until after day 5 of seed germination there were marked changes in the component proteins prior to this time. There were major changes in the distribution of components produced by SDS and dithiothreitol dissociation of legumin and vicilin. The components of legumin are believed to be constituent polypeptide subunits of a polymeric protein (Wright and Boulter, 1974). The enrichment of the legumin in component V and its persistence during germination indicate that this subunit is resistant to proteolysis whereas the other components are readily degraded. However component III is degraded early, prior to the build-up of the acid sulfhydryl protease. If component III is extensively degraded in the early stages of germination, i.e. between days 0 and 4, one would expect a marked depletion of the legumin content of the cotyledon in the early phases of germination (Fig. 1). This was however not observed. It is possible that the hydrolysed components produce fractions which, following SDS eleetrophoresis, migrate at the same rate as component V. Such a dissociation of subunits, capable of interaction, preceeding the metabolic utilization of the complex reserve protein molecules, was proposed by Catsimpoolas et al. (1968). Decomposition of vieilin, which commenced later than that of legumin, also appeared to involve either a preferential utilization of components I, II and III, or a resistance of subunRs IV and V, or a progressive conversion of I, II and III into IV and V. It is clear that the reserve proteins undergo considerable modification prior to the period of rapid depletion. The period of rapid decline in protein content of the cotyledon after day 5 of germination coincides with the increased accumulation of an acid sulfhydryl protease capable of hydrolysing pea proteins. The coincidence of these events suggests that this enzyme plays an important role in the protein depletion in the cotyledon. Similarly, proteolysis in Phaseolus vulgaris (u and Srinivasan, 1973) and in corn endosperm (Harvey and Oaks, 1974) is achieved by acid sulfhydryl proteases. However, since the alteration in component composition of the reserve proteins of the pea cotyledon occurs before the maior accumulation of the acid sulfhydryl protease(s) the involvement of this
Proteolysis in Cotyledons of Germinating Peas
87
e n z y m e in t h e initial p r o t e i n modifications seems unlikely, a n d t h e m e c h a n i s m s of these e a r l y events in t h e m o d i f i c a t i o n of t h e r e s e r v e p r o t e i n s of p e a c o t y l e d o n s r e m a i n t o be resolved. Supported by grant GB 27318A from the National Science Foundation. References Beevers, L. : Protein degradation and proteolytie activity in the cotyledons of germinating pea seeds Pisum sativum. Phytoehem. 7, 1837-1844 (1968) Beevers, L., Guernsey, F. S. : Changes in some nitrogenous components during the germination of pea seeds. Plant Physiol. 41, 1455-1458 (1966) Beevers, L., Splittstoesser, W. E. : Protein and nucleic acid metabolism in germinating peas. J. exp. Bet. 19, 698-711 (1968) Boulter, D. : Flowering plant proteins. In: Phytochemistry, vol. II, p. 30-60, Miller, L. P., ed. New York: Van Nostrand-Reinhold 1973 Catsimpoolas, N., Campbell, T. G., Mayer, E . W . : Immunochemical study of changes in reserve proteins of germinating soybean seeds. Plant Physiol. 48, 799-805 (1968) Danielson, C. E. : Seed globulins of the Gramineae and Leguminosae. Biochem. J. 44, 387-400 (1969) Danielson, C. E. : The breakdown of the high molecular weight reserve protein of peas during germination. Acta chem. scand. 5, 541-544 (1951) Danielson, C. E. : Plant proteins. Ann. Rev. Plant Physiol. 7, 215-236 (1956) Daussant, J., Neucere, N. J., Conkerton, E. J. : Immunochemical studies on Arachis hypogaea proteins with particular reference to the reserve proteins. II. Protein modification during germination. Plant Physiol. 44, 480-489 (1969) Davis, B . J . : Disc electrophoresis. II. Method and application to human serum proteins. Ann. N. Y. Aead. Sci. 121, 404-427 (1964) Dechary, J. M. : Seed proteases and protease inhibitors. Econ. Bet. 24, 113-122 (1970) Harvey, B. M. R., Oaks, A. : The hydrolysis of endosperm protein in Zea mays. Plant Physiol. 53, 453--457 (1974) Henshall, J. D., Goodwin, T. W. : Amino acid activating enzymes in germinating pea seedlings. Phytochem. 8, 677-691 (1964) ttobday, S. M., Thurman, D. A., Barber, D. J. : Proteolytic and trypsin inhibitory activities in extracts of germinating Pisum sativum seeds. Phytoehem. 12, 1041-1046 (1973) Larson, L. A., Beevers, H. : Amino acid metabolism in young pea seedlings. Plant Physiol. 40, 424-432 (1965) Lowry, O.H., Rosebrough, N. J., Farr, A.L., Randall, R. J.: Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265-275 (1951) Palmiter, R. D., Oka, T., Schimke, 1~. T. : Modulation of ovalbumin synthesis by estradiol-17 B and aetinomycin D as studied in explants of chick oviduct in culture. J. biol. Chem. 246, 724-737 (1971) Pusztai, A., Duncan, I.: Changes in proteolytie enzyme activities and transformation of nitrogenous compounds in the germinating seeds of kidney bean, Phaseolus vulgaris. Planta (Berl.) 96, 317-325 (1971) Ryan, C. J. : Proteolytie enzymes and their inhibitors in plants. Ann. Rev. Plant Physiol. 24, 173-196 (1973) Weber, K., Osborn, M. : The reliability of molecular weight determinations by sodium dodecy] sulfate polyacrylamide gel eleetrophoresis. J. biol. Chem. 244, 4406-4412 (1969) Wright, D. J., Boulter, D.: Purification and subunit structure of legumin of Vicia /aba, broad bean. Bioehem. J. 141, 413-418 (1974) Yomo, H., Srinivasan, K.: Protein breakdown and formation of protease in attached and detached cotyledons of Phaseolus vulgarls L. Plant Physiol. 52, 671-673 (1973) Yomo, It., Varner, J. E. : Control of the formation of amylase and protease in the cotyledons of germinating peas. Plant Physiol. 51, 708-713 (1973) Young, J. L., Varner, J. E. : Enzyme synthesis in the cotyledons of germinating seeds. Arch. Biochem. Biophys. 84, 71-78 (1959)
The Development of Proteolytic Activity and Protein Degradation during the Germination of Pisum sativum L. S. M. M. Basha* and Leonard Beevers Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma 73069, USA Received 17 December 1974; accepted 16 February 1975 Summary. The change in protein content and composition of the cotyledons of Pisum sativum L. cv. Burpeeana during germination was studied. Protein depletion from the coty-
ledons was slow during the first 4 days of germination but became rapid on the 5th day and by the 16th day the majority of the protein had disappeared. During the first 4 days the depletion of the globuliI)s exceeded that of the albumins; legumin appeared to be degraded slightly more rapidly than vicilin during the early phase of germination. Sodium-dodecylsulfate (SDS) electrophoresis of SOS- and dithiothreitol-dissociated globulins indicated that before rapid protein depletion there were marked changes in the component composition of the major globulins legumin and vieilin. The onset of rapid protein depletion was associated with an increase in the level of an aeid-sulfhydryl protease in the cotyledons. These findings indicate that the reserve globulins undergo modifications prior to their eventual hydrolysis. Introduction During the germination of pea seeds the nitrogen content of the cotyledons decreases while t h a t of the growing axis increases (Larson and Beevers, 1965; Beevers and Guernsey, 1966). The accumulation of soluble amino acids in cotyledons and axis, which accompanies the depletion of nitrogen in the former (Beevers and Guernsey, 1966), indicates t h a t the alterations in nitrogen content are caused by a hydrolysis of the reserve proteins in the cotyledon followed b y a transport of the products to the developing axis (Beevers and Splittstoesser, 1968). The mechanism by which the cotyledonary proteins are degraded is little understood. Danielson (1951) reported t h a t the globulins were broken down more rapidly than the albumins in germinating peas. The degradation of globulins in soybeans (Catsimpoolas et al., 1968) and peanuts (Daussant et al., 1969) is accompanied b y a change in the electrophoretic mobility of these proteins. Thus an early feature of reserve utilization appears to be the removal of amido groups b y the action of deamidase (Catsimpoolas et al., 1968; Daussant etal., 1969). However, there have been no reports of deamidases in the cotyledons. I n contrast there have been m a n y demonstrations of the presence of proteolytic enzymes in the cotyledons of germinating seeds (Ryan, 1973 ; Dechary, 1970), but in m a n y instances, the role of these enzymes in the mobilization of reserve protein remains to be established. I t was originally reported t h a t proteolytic activity did not change in the cotyledons of peas during germination (Young and Varner, 1959; Henshall and * Present Address: Department of Food Science, Georgia Agricultural Experiment Station, Experiment, Georgia, 30212, USA.
78
S.M.M. Basha and L. Beevers
Goodwin, 1964). H o w e v e r , m o r e r e c e n t l y it has been d e m o n s t r a t e d t h a t proteolyric a c t i v i t y , m e a s u r e d as t h e c a p a c i t y to h y d r o l y s e casein (Beevers a n d S p l i t t stoesser, 1968; Beevers, 1968; u a n d Varner, 1973) or a z a g l o b u l i n ( H o b d a y et al., 1973), increases d u r i n g g e r m i n a t i o n . I n ~ddition, m a t u r e p e a cotyledons c o n t a i n e d a high level of p e p t i d a s e a c t i v i t y , c a p a b l e of h y d r o l y s i n g leucine-pn i t r o a n i l i d e a n d b e n z o y l a r g i n y l - p - n i t r o a n i l i d c , a n d this a c t i v i t y declined d u r i n g g e r m i n a t i o n (Beevers, 1968). P u s t z a i a n d D u n c a n (1971) a n d Y o m o a n d S r i n i v a s a n (1973) h a v e r e p o r t e d similar changes in p r o t e o l y t i c a c t i v i t y d u r i n g t h e g e r m i n a t i o n of Phaseolus vulgaris; however, p e p t i d a s e a c t i v i t y r e m a i n e d r e l a t i v e l y c o n s t a n t t h r o u g h o u t t h e p e r i o d of s t u d y (Pustzai a n d Duncan, 1971). A p a r t from t h e i n v e s t i g a t i o n s of H o b d a y et al. (1973), who utilized a modified p l a n t p r o t e i n (azaglobulin) as s u b s t r a t e all studies of p r o t e o l y t i c a c t i v i t y d u r i n g seed g e r m i n a t i o n were m a d e w i t h casein as s u b s t r a t e , a n d it can be a r g u e d t h a t t h e p r o t e o l y t i c a c t i v i t y t h u s m e a s u r e d bears little r e l a t i o n s h i p to t h e e n z y m e s i n v o l v e d in t h e h y d r o l y s i s of n a t i v e reserve proteins. W e h a v e therefore u s e d p e a p r o t e i n s labeled w i t h t r i t i a t e d a m i n o acids as s u b s t r a t e s for t h e a s s a y of p r o t e o l y t i c enzymes. W e h a v e followed t h e f l u c t u a t i o n s in p r o t e o t y l i e a c t i v i t y d u r i n g germin a t i o n a n d r e l a t e d t h e m to changes in t h e p r o t e i n s of t h e cotyledon.
Material and Methods Plant. Material. Pea seeds (Pisum sativum L. cv. Burpeeana, W. Atlee Burpee Comp., Clinton, Iowa, USA) were germinated at 25 ~ in the dark in sterile vermiculite and the seedlings irrigated with deionized water. The addition of water to the dry seeds is taken as zero (0) times germination. Cotyledons were obtained from the seedling at different stages of germination. Protein Extraction and Yractionation. The testa and embryo were removed from the seed and protein was ffactionated from the cotyledons by a procedure modified from Danielson (1949). The cotyledons were homogenized in cold 1 M NaC1, 20 mM sodium-phosphate buffer, pit 7.0, with a Virtis tissue homogenizer at high speed for 2 rain and at low speed for 8 rain. The homogenate was stirred for 30 min and then centrifuged at 20000 • for 15 min. The resulting pellet was extracted twice with the same buffer, and centrifuged. The resulting three supernatants were pooled and made to 70% saturation by adding solid (NH4)2SOa. After standing for 1 h the mixture was centrifuged at 30000• for 20 rain. The supernatant was discarded and the pellet suspended in 0.2 M NaC1, 5 mM phosphate buffer, p i t 7.0, and dialysed against distilled water for 2 days. The dialysates were centrifuged at 20000 • for 15 min. The supernatant fraction was designated as albumin and the pellet as globulin. The globulin fraction was further suspended in 0.2 M NaCI, 5 mM phosphate buffer, pH 4.5, and stirred overnight at 4 ~ This globulin suspension was centrifuged at 3000 • g for 10 rain. The resulting pellet was washed twice with the same buffer and centrifuged. The three supernatants were pooled and dialysed against distilled water for 2 days. The material precipitated during the dialysis was designated as vicilin. The remaining pellet from the globulin fraction was suspended in 0.2 M NaC1, 5 mM phosphate buffer, pH 7.0 and dialysed against distilled water for 2 days. The dialysate was centrifuged at 20000 • g for 15 rain. The supernatant was discarded and the pellet collected as legumin. The legumin and vicilin pellets were suspended in 0.2 M NaC1, 20 mM phosphate buffer, pH 7.0, and stored at -- 10% Protein Determination. The protein content of the different fractions (Total protein, albumin, globulin, legumin and vicilin) of cotyledon extracts was determined by the method of Lowry et al. (1951) with bovine serum albumin as a standard. Polyavrylamide Gel Electrophoresis. Gel eleetrophoresis in 7.5% polyacrylamide was performed according to the method of Davis (1964). The protein samples suspended in 0.21~
Proteolysis in Cotyledons of Germinating Peas
79
NaCI, 20 mM phosphate buffer, pit 7.0, were made to 5 % sucrose concentration and 100-200 ~zg of protein applied per gel (6 • 90 ram). Electrophoresis was run at a constant current of 4 mA/tube for 2 h, or until the tracking dye reached ca. 1 cm above the bottom of the gel. Mter electrophoresis the gels were stained with 1% amido black stain in 7.5% acetic acid for 1 h. The gels were destained electrophoretically in 7.5% acetic acid, and scanned in a Gilford instrument linear transport system at 620 rim. SDS Gel Eleetrophoresis. Legumin and vicilin were dissociated essentially by the method of Palmiter et al. (1971). The protein was precipitated with trichloroacetic acid and a freshly made solution containing 1.2% Tris (pH unadjusted), 1.5% dithiothreitol, 1.0% sodium dodeeyt sulfate and 20% glycerol was added to the precipitate to give a final protein concentration of 2 mg/ml. The protein was dissociated by mixing the sample with a glass rod for 1-5 min while heating in a boiling water bath. SDS gel electrophoresis was carried out in 5% polyacrylamide gels by the procedure of Weber and Osbom (1969) by applying 200 fzg of dissociated protein per gel. The electrophoresis was carried out for 4 h and then the gels were removed from the Plexiglass tubes and fixed in 20% sulfosalieylic acid for 18 h. The gels were stained with 0.25 % Coomassie blue in water for 3 h; destained by repeated washings with 7.5% acetic acid, and scanned at 620 nm. Preparation o/Enzyme Extracts. The cotyledons from germinating pea seeds were homogenized for 2 rain at high speed in a Virtis tissue homogenizer with 0.02 M sodium-phosphate buffer, pH 7.2, with or without 5 mlY[2-mercaptoethanol. The tissue-to-buffer ratio was 1:4 (w/v). The homogenates were squeezed through a layer of cheesecloth and Miracloth (Calbiochem, La Jolla, Calif., USA) and centrifuged at 20000 • for 20 min. The supernatant from this eentrifugation was used in the protease assays. Standard Protease Assay. Proteolytic activity was measured using [3H]albuminm, [SH]legumin or [aH]vicilin, prepared from peas, as substrate. Crude enzyme extract (1 ml) was incubated with 0.2 ml [3H]legumin (4.4 • 10-3 cpm/ml) or 0.2 ml [3H]vicilin (2.7 • 10-~ cpm/ml) or 0.5 ml [all]albumin (1.1 • 10-5 cpm/ml) and 1.0 ml of 0.2 M sodium-citrate buffer, pH 5.0, in a shaker bath at 40~ for 3 h. The reaction was terminated by addition of 0.8 ml (in legumin and vicilin assays) or 0.5 ml (in albumin assays) of 1% casein, pH 7.0, followed by 1.0 ml of 20% triehloroacetio acid to give a final volume of 4.0 ml. A reaction mixture in which casein and 20 % trichloroacetie acid was added immediately after the addition of the enzyme extract served as the zero-time control. After the addition of trichloroacetic acid the samples were allowed to precipitate overnight at 4~ and then the insoluble material was removed by centrifugation. A 1-ml aliquot was taken from the triehloroacetic acid soluble supernatant and after addition of 12 ml of scintillation fluid [8.0 g PPO (2,5-diphenyloxazole) in 2 1 toluene, 1 1 Triton X-100] the radioactivity was determined in a Beckman LS-100 scintillation counter. Appropriate modifications of the standard assay were made in order to establish optimal pH and sulfhydryl requirements for proteolytie activity. In studies of pH effects 0.2 M sodinm-phosphate buffer was used from pH 6.5 to 9.0, and 0.2 M sodium citrate buffer between pH 3.0 and 6.0. The sulfhydryl content of the reaction mixture was modified by the addition of 2-mercaptoethanol. Preparation ot 3H-Labeled Substrates. 5 Ezl of [all]amino acids (1 mCi/ml tritiated aminoacid mixture TRK 440, Amersham Searle Corp., Arlington Heights, Ill., USA) were injected with a Hamilton syringe into each cotyledon of 20 peas in the pods, 21 days after flowering. The pods were left attached to the plant and the cotyledons were collected at maturity, 36 days after flowering. The protein was extracted from the cotyledons and ffactionated into albumins, vieilin, and legumin. Results
Course o/Protein Disappearance P r o t e i n disappearance from the cotyledons was slow d u r i n g the first 4 days of g e r m i n a t i o n , b u t became r a p i d after the 5th d a y (Fig. 1). D u r i n g the first 4 days the depletion of the globulins exceeded t h a t of the a l b u m i n s ; after 5-6 days of g e r m i n a t i o n b o t h the a l b u m i n a n d globulin b r e a k d o w n increased rapidly,
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and by the 16th day the majority of the protein had disappeared. Legumin appeared to be degraded slightly more rapidly than vicilin during the initial phases of germination. These findings are similar to those of Danielson (1951). Gel Electrophoresis o/Albumin
Numerous protein bands were evident after electrophoresis and amido-black staining of the albumin fraction. The composition of this fraction began to change after 2 days of germination, with a dramatic alteration in the protein components occurring after day 4 (Fig. 2). Some of the proteins disappeared during germination but there was also a production of new components as indicated by the development of new absorbing peaks in the stained gels. The albumin fraction contains enzymatic proteins (Danielson, 1956) and in view of the altered metabolic activity of the cotyledon during germination an alteration in the component proteins is to be expected. Polyacrylamide gel electrophoresis of the legumin fraction, followed by staining with amido-black, indicated that this fraction contained only one detectable component. There was a change in the electrophoretic mobility of the fraction prepared from pea cotyledon at successive stages of germination (Table 1), the l~m increasing from 0.17 at 0 days of germination to 0.22 after 8 days of germination. This change in electrophoretic mobility is similar, although of less magnitude, to that reported for soybean proteins by Catsimpoolas et al. (1968) and proteins from peanut cotyledons by Daussant et al. (1969).
Proteolysis in Cotyledons of Germinating Peas
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and 8 of germination whereas in vieilin they were observed between days 10 and 12. Catsimpoolas et al. (1968) and Daussant et al. (1969) attribute the change in electrophoretic mobility to a deamidation of the reserve protein during germination. Our data indicate that the change in charge of the reserve proteins occurs after the commencement of protein depletion. Deamidation of legumin and deearboxylation of vicilin appear therefore to play no role in the initiation of protein degradation during pea seed germination.
SDS Gel Electrophoresis o/Legumin and Vicilin SDS electrophoresis of the globulin fractions, prepared from mature seeds and dissociated in the presence of SDS and dithiothreitol, indicated that legumin was composed of three major and two minor components while vicilin contained five major components. During germination the component composition of these reserve proteins underwent marked changes. Component I I I of legumin began to decline at day 2 of seed germination and components I, I I and IV had disappeared by day 6 while a fraction with the mobility of component V was still present 14 days after germination, and more slowly migrating components appeared on days 12 and 14 of germination (Fig. 3). Changes in the components of legumin were evident before alterations were observed in the composition of vicilin (Fig. 4). This is consistent with the slower initial depletion of vicilin (Fig. 1).
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Proteolytic Activity In extracts prepared from cotyledons 13 days after germination it was possible to demonstrate the presence of enzymes capable of releasing trichloroacetic-acid soluble radioactivity from sit-labeled albumins, vicilin and legumin (Fig. 5). Maximal hydrolytic activity with Jail]albumin as substrate, but only limited hydrolysis of [aH]vicilin and [aH]legumin, was observed at pH 5.0 in extracts prepared in the absence of 2-mercaptoethanol. In contrast, enzyme extracts prepared in the presence of 2-mercaptoethanol were able to hydrolyse legumin and vicilin with optimal activity at pH 5.0 and also showed enhanced activity with albumin as substrate. Trichloroacetic-aeid-soluble radioactivity was released from [SH]vicilin at pH 3.0; however, this may be due to the dissociation of the proteins at low pH rather than being caused by enzymatic hydrolyses.
Requirement ]or Sul]hydryl The enhanced hydrolytic activity observed in extracts prepared in the presence of 2-mercaptoethanol may be explained in two ways: (1) 2-mercaptoethanol may 6*
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serve as a p r o t e c t a n t to p r e v e n t d e t e r i o r a t i o n or i n a c t i v a t i o n of t h e p r o t e i n during isolation, (2) s u l f h y d r y l groups m a y be a necessary, i n t e g r a l c o m p o n e n t of t h e p r o t e o l y t i c reaction. A series of e x p e r i m e n t s was p e r f o r m e d to d e t e r m i n e t h e role of s u l f h y d r y l (Fig. 6 A - D ) . T h e e n z y m e p r e p a r a t i o n (C) showed o n l y l i m i t e d p r o t e o l y t i e a c t i v i t y b u t was s t i m u l a t e d b y inclusion of 5 m M 2 - m e r c a p t o -
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ethanol in the reaction mixture. Thus although the enzyme(s) had been protected by the presence of 2-mercaptoethanol during extraction the proteolytie activity was reduced in reaction mixtures devoid of sulfhydryl. (Some mercaptoethanol may have been carried over in the ammonium-sulfate precipitate but this was insufficient to sustain maximum hydrolytic activity.) Extracts prepared in the absence of 2-mercaptoethanol showed limited hydrolysis in the absence of sulfhydryl (B) but were much more active in its presence (B) and (D). The results indicate that sulfhydryl functions as a component of the hydrolytic reaction, rather than as a proteetant during enzyme isolation. The greater activity of extracts prepared in the absence of mercaptoethanol towards [3H]albumin in comparison to legumin and vicilin may be related to the fact that albumins have a geater content of sulfur-containing amino acids than globulins (Boulter, 1973).
Proteolytic Activity during Seed Germination Enzyme extracts prepared from the cotyledons of germinating seeds of different ages showed varying capacities to hydrolyse all-labeled proteins at pH 5.0. Very low enzyme activity was present in the extracts from the cotyledons until day 3 of germination (Fig. 7). Between days 3 and 5 of germination the enzyme activity increased slowly, but between days 5 and 15 there was a rapid enhancement in the hydrolytic capacity of the extracts. A similar time course was also obtained for albumin hydrolysis (data not shown). Beevers (1968) and Beevers and Splittstoesser (1968) had observed caseolytic activity in extracts from pea cotyledons at p i t 5.5 and pH 7.0, with greater activity at the lower pit. Although
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S.M. NI. Basha and L. Beevers
the studies using extracts from cotyledons 13 days after germination (Fig. 5) had failed to show any evidence of a peak of hydrolytic activity at pH 7.0 when pea proteins were used as substrate it is nevertheless possible that enzymes with optimal activity near pH 7 are present in extracts from pea cotyledons at other stages of germination. Thus, it was found that enzyme preparations from cotyledons after day 9 of germination did show proteolytic activity at pH 7.5. However, the consistently lower activity, and the overall similarity of the time course of the development of hydrolytic activity during germination indicate that enzymatic activity measured at pH 7.5 may merely represent the residual activity of the same enzyme that has optimal activity at pH 5.0. It is thus possible that the proteolytic activity in the cotyledonary extracts of peas is mediated by one acid sulfhydryl protease which is capable of hydrolysing the albumins as well as the globulins, legumin and vicilin. Conclusions Although the most rapid depletion of protein from the cotyledons did not occur until after day 5 of seed germination there were marked changes in the component proteins prior to this time. There were major changes in the distribution of components produced by SDS and dithiothreitol dissociation of legumin and vicilin. The components of legumin are believed to be constituent polypeptide subunits of a polymeric protein (Wright and Boulter, 1974). The enrichment of the legumin in component V and its persistence during germination indicate that this subunit is resistant to proteolysis whereas the other components are readily degraded. However component III is degraded early, prior to the build-up of the acid sulfhydryl protease. If component III is extensively degraded in the early stages of germination, i.e. between days 0 and 4, one would expect a marked depletion of the legumin content of the cotyledon in the early phases of germination (Fig. 1). This was however not observed. It is possible that the hydrolysed components produce fractions which, following SDS eleetrophoresis, migrate at the same rate as component V. Such a dissociation of subunits, capable of interaction, preceeding the metabolic utilization of the complex reserve protein molecules, was proposed by Catsimpoolas et al. (1968). Decomposition of vieilin, which commenced later than that of legumin, also appeared to involve either a preferential utilization of components I, II and III, or a resistance of subunRs IV and V, or a progressive conversion of I, II and III into IV and V. It is clear that the reserve proteins undergo considerable modification prior to the period of rapid depletion. The period of rapid decline in protein content of the cotyledon after day 5 of germination coincides with the increased accumulation of an acid sulfhydryl protease capable of hydrolysing pea proteins. The coincidence of these events suggests that this enzyme plays an important role in the protein depletion in the cotyledon. Similarly, proteolysis in Phaseolus vulgaris (u and Srinivasan, 1973) and in corn endosperm (Harvey and Oaks, 1974) is achieved by acid sulfhydryl proteases. However, since the alteration in component composition of the reserve proteins of the pea cotyledon occurs before the maior accumulation of the acid sulfhydryl protease(s) the involvement of this
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e n z y m e in t h e initial p r o t e i n modifications seems unlikely, a n d t h e m e c h a n i s m s of these e a r l y events in t h e m o d i f i c a t i o n of t h e r e s e r v e p r o t e i n s of p e a c o t y l e d o n s r e m a i n t o be resolved. Supported by grant GB 27318A from the National Science Foundation. References Beevers, L. : Protein degradation and proteolytie activity in the cotyledons of germinating pea seeds Pisum sativum. Phytoehem. 7, 1837-1844 (1968) Beevers, L., Guernsey, F. S. : Changes in some nitrogenous components during the germination of pea seeds. Plant Physiol. 41, 1455-1458 (1966) Beevers, L., Splittstoesser, W. E. : Protein and nucleic acid metabolism in germinating peas. J. exp. Bet. 19, 698-711 (1968) Boulter, D. : Flowering plant proteins. In: Phytochemistry, vol. II, p. 30-60, Miller, L. P., ed. New York: Van Nostrand-Reinhold 1973 Catsimpoolas, N., Campbell, T. G., Mayer, E . W . : Immunochemical study of changes in reserve proteins of germinating soybean seeds. Plant Physiol. 48, 799-805 (1968) Danielson, C. E. : Seed globulins of the Gramineae and Leguminosae. Biochem. J. 44, 387-400 (1969) Danielson, C. E. : The breakdown of the high molecular weight reserve protein of peas during germination. Acta chem. scand. 5, 541-544 (1951) Danielson, C. E. : Plant proteins. Ann. Rev. Plant Physiol. 7, 215-236 (1956) Daussant, J., Neucere, N. J., Conkerton, E. J. : Immunochemical studies on Arachis hypogaea proteins with particular reference to the reserve proteins. II. Protein modification during germination. Plant Physiol. 44, 480-489 (1969) Davis, B . J . : Disc electrophoresis. II. Method and application to human serum proteins. Ann. N. Y. Aead. Sci. 121, 404-427 (1964) Dechary, J. M. : Seed proteases and protease inhibitors. Econ. Bet. 24, 113-122 (1970) Harvey, B. M. R., Oaks, A. : The hydrolysis of endosperm protein in Zea mays. Plant Physiol. 53, 453--457 (1974) Henshall, J. D., Goodwin, T. W. : Amino acid activating enzymes in germinating pea seedlings. Phytochem. 8, 677-691 (1964) ttobday, S. M., Thurman, D. A., Barber, D. J. : Proteolytic and trypsin inhibitory activities in extracts of germinating Pisum sativum seeds. Phytoehem. 12, 1041-1046 (1973) Larson, L. A., Beevers, H. : Amino acid metabolism in young pea seedlings. Plant Physiol. 40, 424-432 (1965) Lowry, O.H., Rosebrough, N. J., Farr, A.L., Randall, R. J.: Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265-275 (1951) Palmiter, R. D., Oka, T., Schimke, 1~. T. : Modulation of ovalbumin synthesis by estradiol-17 B and aetinomycin D as studied in explants of chick oviduct in culture. J. biol. Chem. 246, 724-737 (1971) Pusztai, A., Duncan, I.: Changes in proteolytie enzyme activities and transformation of nitrogenous compounds in the germinating seeds of kidney bean, Phaseolus vulgaris. Planta (Berl.) 96, 317-325 (1971) Ryan, C. J. : Proteolytie enzymes and their inhibitors in plants. Ann. Rev. Plant Physiol. 24, 173-196 (1973) Weber, K., Osborn, M. : The reliability of molecular weight determinations by sodium dodecy] sulfate polyacrylamide gel eleetrophoresis. J. biol. Chem. 244, 4406-4412 (1969) Wright, D. J., Boulter, D.: Purification and subunit structure of legumin of Vicia /aba, broad bean. Bioehem. J. 141, 413-418 (1974) Yomo, H., Srinivasan, K.: Protein breakdown and formation of protease in attached and detached cotyledons of Phaseolus vulgarls L. Plant Physiol. 52, 671-673 (1973) Yomo, It., Varner, J. E. : Control of the formation of amylase and protease in the cotyledons of germinating peas. Plant Physiol. 51, 708-713 (1973) Young, J. L., Varner, J. E. : Enzyme synthesis in the cotyledons of germinating seeds. Arch. Biochem. Biophys. 84, 71-78 (1959)
