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The action of the proteolytic enzymes of dry vetch seeds on their own reserve proteins
Plant Science Letters, 4 (1975) 309--313
309
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
THE ACTION OF THE PROTEOLYTIC ENZYMES OF DRY VETCH SEEDS ON THEIR OWN RESERVE PROTEINS
T.N. KOROLYOVA, A.D. SHUTOV and I.A. VAINTRAUB Laboratory of Protein Chemistry, V.L Lenin State University of Kishinev (U.S.S.R.)
(Received December 14th, 1974)
SUMMARY Legumin (L) and vicflin (V), the reserve proteins of dry vetch seeds (DS) are not affected by proteolytic enzymes of DS, but L and V isolated from germinated seeds (GS) are hydrolysed by these enzymes. Supporting evidence of modification of reserve proteins during germination demonstrated earlier comes from these results. This modification can apparently be considered as the stage preceding the hydrolysis of reserve proteins.
INTRODUCTION During the germination of vetch seed their reserve proteins, L and V, are modified. This is manifest by changes in electrophoretic mobility and chromatographic behaviour [1]. Similar changes of g~ound-nut [2, 3] and kidney bean [4] reserve proteins were also observed. This modification is a result of at least two processes [5]. One of them is the deamidation of L and V [ 1]. The nature of the second is not yet ascertained. In the present work we have investigated the action of proteolytic enzymes of vetch DS [6] on L and V isolated from DS and GS. Comparison of the results is necessary to elucidate the significance of the modification of reserve proteins during germination. MATERIALS AND METHODS
To obtain the enzyme preparation the meal of vetch (Vicia sativa L.) seed cotyledons (harvest of 1972 or 1973) was extracted by 0.5 M NaC1 containing 1 ml/1 of 2-mercaptoethanol. The addition of mercaptoethanol considerably enhances the proteolytic activity in the range of pH 3.0 to 5.0 (these results were obtained using casein as substrate). To remove low-molecular-weight substances the extract was run through a Sephadex G-50 column and then Abbreviations: DS, dry seeds; GS, germinated seeds; L, legumin; TCA, trichloroacetic acid; V, vieilin.
310 diluted until a mesa: extract ratio of 1:20 (w/v) was reached. The final solution was 0.01 M in mercaptoethanol. L and V preparations were isolated from DS as described earlier [7]. Preparations obtained by the method of Danielson [8] were also investigated. The vetch seeds were germinated in the dark at 25 ° on wet filter paper for 8.5 days. L and V from GS were separated by zonal isoelectric precipitation on a Sephadex G-50 column [9]. L was further purified by gradient chromatography on DEAE~eUulose [7] and V by stepwise chromatography on CM-cellulose [10]. The methods of isolation lead to chromatographically and electrophoreticsaly homogeneous preparations [7, 8, 10]. According to ultracentrifugation and gel filtration (on Sephadex G-100) data none of the preparations contained admixtures of proteins of lower molecular weight. L and V of DS isolated by Danielson's method were contaminated by each other, but the content of admixtures did not exceed 10%. The sum of L and V of DS and GS practically devoid of non-reserve proteins was obtained by ssating-out with (NH4)2SO4 (50 to 90% saturation) and subsequent gel filtration on a Sephadex G-100 column. To determine the proteolytic activity 0.05 ml of 1% solution of substrate, 0.05 ml of phosphate-citrate buffer [11] of appropriate pH and 0.05 ml of enzyme preparation were incubated at 40 ° for 2 h. As demonstrated by preliminary experiments with casein as substrate under these conditions the increase of free NH2-groups in TCA filtrate is nearly linear with time. After the incubation 0.05 ml of 15% solution of TCA was added and the mixture was centrifuged. 0.05 ml of 7 mM N-ethylmaleimide was added to 0.1 ml of the supernatant to block the sulfhydryl groups of mercaptoethanol; the amount of free NH2-groups was determined with 2, 4, 6-trinitrobenzenesulfonic acid according to Langner et al. [12]. In control experiments the substrate was added after the incubation and addition of TCA. In calculating the concentration of NH2-groups the molar absorbance of trinitrophenyl derivatives of the amines was taken A340=13 500 (calculated according to the data of Langner et sa. [12] ). The activity determinations were carried out in the pH range 3.0--7.7. The values of the buffer's pH were corrected for the changes caused by the addition of substrate and enzyme preparation. With each of the substrates the pH-activity curve was determined 3--4 times each time a new enzyme preparation was used. The values of activity in parallel determinations varied markedly but the shape of pH-activity curves was reproducible. RESULTS At least 4 proteolytic enzymes were found in vetch DS extracts with casein as substrate [6]. Their pH optimums were at pH 3.8, 4.8, 5.7 and 7.2. All these enzymes were inactive with L and V of DS as substrates in the investigated range of pH (Fig.l). Several preparations of L and V of DS isolated by different methods and the sum of L and V from DS gave similar results. Both
311
.
_q
Caaei.n,pa 5.g . L, p~47.0
300
60
.~
E
"~,V,GS
"I
%% 200 E:ZL
/?,~"
/"
/ . zr 100 fl~/,/--
:£ z 3o I
2
3
5
7
pH
__ /------"
-- V,
j~
4
CaseLn,pN7.2
~
time (h)
8
Fig.1. Hydrolysis at different pH o f L and V from DS and GS by DS proteases. The m a x i m u m departure from 0 for V o f DS corresponds to the experimental value of extinction 0.012. Fig.2. Comparison o f hydrolysis velocities o f reserve proteins from GS and o f casein by DS proteases.
e n z y m e preparations from freshly harvested seeds and from those stored during a year were inactive. L and V from GS, however, are hydrolysed by s o m e o f the DS proteinases. With V as substrate t w o broad m a x i m a at pH 4 . 5 - - 5 . 0 and 5.5--6.2 were observed. With L there is o n e major m a x i m u m around pH 7.0 and a small but reproducible activity at pH 4 . 0 - - 4 . 5 ( F i g . l ) . At neutral pH the velocity o f hydrolysis o f L from GS is more than double the rate for casein. The initial rate o f hydrolysis o f V from GS at pH 5.9 also markedly exceeds that o f casein, but it declines rapidly while the rate o f casein hydrolysis is almost constant for 5 h (Fig.2). When proteases o f DS act u p o n the sum of L and V isolated from seeds germinated for 2.5 days, the pH-activiW curve characteristic o f V was obtain-
90 1
j / ~ 4 . 5 da95
a
5
7
pH
Fig.3. The hydrolysis o f the s u m of L and V isolated from seeds germinated during different periods by DS proteases.
312 ed, while after 4.5 days' germination a m-~imum in the neutral pH range characteristic of L is revealed as well (Fig.3). DISCUSSION The data on the action of DS proteases on seed reserve proteins are contradictory [13--19]. As a rule these data were obtained by studying globulins or their fractions which may contain n o t only the reserve proteins [20]. However, we have observed, some non-reserve proteins of DS are hydrolysed by DS proteases. Our data obtained with purified reserve proteins as substrates demonstrate that DS proteases do not act on the L and V from DS, b u t hydrolyse the same proteins which were isolated from GS. These results support previous reports on the modification of reserve proteins during germinatoin [1--5] and show that their availability to hydrolysis by DS proteases may be used as a criterion of modification of reserve proteins. According to this criterion the modification of V during germination begins earlier than the 2.5th day, and that of L between the 2.5th day. Evidently, factors modifying the reserve proteins are synthesized or activated during germination. Only after modification do the reserve proteins become available to the DS proteases. There are no appreciable changes of the molecular weight of reserve proteins during modification. This was demonstrated earlier [1] and confirmed in the present work when~hamcterizing L and V isolated from GS. Thus, preliminary dissociation as an initial step~in the hydrolysis of reserve proteins as postulated by several authors [21--24], is not always necessary. It cannot be asserted that the described way of hydrolysis is the only possible one. New proteolytic enzymes which can hydrolyse unmodified reserve proteins may be synthesized or activated during germination. Moreover, it can be assumed that the newly formed proteases act as modifying factors through limited proteolysis. ACKNOWLEDGEMENT
The authors express their sincere gratitude to Dr. E.M. Dobmskina for her help in the translation of this paper.
REFERENCES 1 A.D. Shutov and I.A. Vaintrsub, Fiziol. Rut., 20 (1973) 504. 2 J.M. Dechary, K.F. Talluto, W.J. Evans, W.B. Carney and A.M. Alt~hul, Nature, 190 (1961) 1125. 3 J. Dammant, N.J. Neueere and E.J. Conkerton, Plant Physiol., 44 (1969) 480. 4 J. Kloz, A. Turkova and E. Klozova, Biol. Flantarum Acad. Svi. Bohemoelov., 8 (1966) 164. 5 A.D. Shutov and Nguen Chong Tkhin, Fiziol. R u t , (in press).
313 6 T.N. Korolyova, M.V. Alekseeva, A.D. Shutov and LA. Vaintraub, Fiziol. Rast., 20 (1973) 769, 7 A.D. Shutov and I.A. Vaintraub, Biokhimiya, 31 (1966) 726. 8 C.E. Danielson, Acta Chem. Scan&, 4 (1950) 762. 9 A.D. Shutov and I.A. Vaintraub, Ukr. Biokhim. Zh., 37 (1965) 177. 10 V.S. Shvarz, A.D. Shutov and I.A. Vaintraub, Biol. Nauki, 11 N12 (1968) 99. 11 P.J. Elving, J.M. Markowitz and I. Rosenthal, Anal. Chem., 28 (1956) 1179. 12 J. Langner, S. Ansorge, P. Bohley, H. Kirschke and H. Hanson, Acta Biol. Med. Ger., 26 (1971) 935. 13 M. Mergentine and E.H. Wigand, Fruit Products J., 26 (1946) 72. 14 G. Tazawa and T. Hirokawa, J. Biochem., 43 (1956) 785. 15 S. Takagi, Nippon Nogei Kagaku Kaishi, 34 (1960) 357, quoted after CA, 58 (1963) 10456e. 16 S. Shinano and K. Fukushima, Agr. Biol. Chem., 33 (1969) 1236. 17 G.F.G. Morris, D.A. Thurman and D. Boulter, Phytochemistry, 9 (1970) 1707. 18 A.G.S. Angelo, R.L. Ory and H.G. Hanson, Phytochemistry, 9 (1970) 1933. 19 G.L. Guardiola and G.F. Sutcliffe, Ann. Botany (London), 35 (1971) 791. 20 I.A. Vaintraub and A.D. Shutov, Tr. Khim. Prir. Soedin., 7 (1968) 23. 21 V.L. Kretovich and T.I. Smirnova, Biokhim. Zerna i Khlebopecheniya, 6 (1960) 66. 22 V. Ghetie, Rev. Roum. Biochim., 3 (1963) 353. 23 N. Catsimpoolas, T.G. Campbell and E.M. Mayer, Plant Physiol., 43 (1968) 799. 24 M.A. Belozerskii, Dokl. Akad. Nauk SSSR, 199 (1971) 468.
309
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
THE ACTION OF THE PROTEOLYTIC ENZYMES OF DRY VETCH SEEDS ON THEIR OWN RESERVE PROTEINS
T.N. KOROLYOVA, A.D. SHUTOV and I.A. VAINTRAUB Laboratory of Protein Chemistry, V.L Lenin State University of Kishinev (U.S.S.R.)
(Received December 14th, 1974)
SUMMARY Legumin (L) and vicflin (V), the reserve proteins of dry vetch seeds (DS) are not affected by proteolytic enzymes of DS, but L and V isolated from germinated seeds (GS) are hydrolysed by these enzymes. Supporting evidence of modification of reserve proteins during germination demonstrated earlier comes from these results. This modification can apparently be considered as the stage preceding the hydrolysis of reserve proteins.
INTRODUCTION During the germination of vetch seed their reserve proteins, L and V, are modified. This is manifest by changes in electrophoretic mobility and chromatographic behaviour [1]. Similar changes of g~ound-nut [2, 3] and kidney bean [4] reserve proteins were also observed. This modification is a result of at least two processes [5]. One of them is the deamidation of L and V [ 1]. The nature of the second is not yet ascertained. In the present work we have investigated the action of proteolytic enzymes of vetch DS [6] on L and V isolated from DS and GS. Comparison of the results is necessary to elucidate the significance of the modification of reserve proteins during germination. MATERIALS AND METHODS
To obtain the enzyme preparation the meal of vetch (Vicia sativa L.) seed cotyledons (harvest of 1972 or 1973) was extracted by 0.5 M NaC1 containing 1 ml/1 of 2-mercaptoethanol. The addition of mercaptoethanol considerably enhances the proteolytic activity in the range of pH 3.0 to 5.0 (these results were obtained using casein as substrate). To remove low-molecular-weight substances the extract was run through a Sephadex G-50 column and then Abbreviations: DS, dry seeds; GS, germinated seeds; L, legumin; TCA, trichloroacetic acid; V, vieilin.
310 diluted until a mesa: extract ratio of 1:20 (w/v) was reached. The final solution was 0.01 M in mercaptoethanol. L and V preparations were isolated from DS as described earlier [7]. Preparations obtained by the method of Danielson [8] were also investigated. The vetch seeds were germinated in the dark at 25 ° on wet filter paper for 8.5 days. L and V from GS were separated by zonal isoelectric precipitation on a Sephadex G-50 column [9]. L was further purified by gradient chromatography on DEAE~eUulose [7] and V by stepwise chromatography on CM-cellulose [10]. The methods of isolation lead to chromatographically and electrophoreticsaly homogeneous preparations [7, 8, 10]. According to ultracentrifugation and gel filtration (on Sephadex G-100) data none of the preparations contained admixtures of proteins of lower molecular weight. L and V of DS isolated by Danielson's method were contaminated by each other, but the content of admixtures did not exceed 10%. The sum of L and V of DS and GS practically devoid of non-reserve proteins was obtained by ssating-out with (NH4)2SO4 (50 to 90% saturation) and subsequent gel filtration on a Sephadex G-100 column. To determine the proteolytic activity 0.05 ml of 1% solution of substrate, 0.05 ml of phosphate-citrate buffer [11] of appropriate pH and 0.05 ml of enzyme preparation were incubated at 40 ° for 2 h. As demonstrated by preliminary experiments with casein as substrate under these conditions the increase of free NH2-groups in TCA filtrate is nearly linear with time. After the incubation 0.05 ml of 15% solution of TCA was added and the mixture was centrifuged. 0.05 ml of 7 mM N-ethylmaleimide was added to 0.1 ml of the supernatant to block the sulfhydryl groups of mercaptoethanol; the amount of free NH2-groups was determined with 2, 4, 6-trinitrobenzenesulfonic acid according to Langner et al. [12]. In control experiments the substrate was added after the incubation and addition of TCA. In calculating the concentration of NH2-groups the molar absorbance of trinitrophenyl derivatives of the amines was taken A340=13 500 (calculated according to the data of Langner et sa. [12] ). The activity determinations were carried out in the pH range 3.0--7.7. The values of the buffer's pH were corrected for the changes caused by the addition of substrate and enzyme preparation. With each of the substrates the pH-activity curve was determined 3--4 times each time a new enzyme preparation was used. The values of activity in parallel determinations varied markedly but the shape of pH-activity curves was reproducible. RESULTS At least 4 proteolytic enzymes were found in vetch DS extracts with casein as substrate [6]. Their pH optimums were at pH 3.8, 4.8, 5.7 and 7.2. All these enzymes were inactive with L and V of DS as substrates in the investigated range of pH (Fig.l). Several preparations of L and V of DS isolated by different methods and the sum of L and V from DS gave similar results. Both
311
.
_q
Caaei.n,pa 5.g . L, p~47.0
300
60
.~
E
"~,V,GS
"I
%% 200 E:ZL
/?,~"
/"
/ . zr 100 fl~/,/--
:£ z 3o I
2
3
5
7
pH
__ /------"
-- V,
j~
4
CaseLn,pN7.2
~
time (h)
8
Fig.1. Hydrolysis at different pH o f L and V from DS and GS by DS proteases. The m a x i m u m departure from 0 for V o f DS corresponds to the experimental value of extinction 0.012. Fig.2. Comparison o f hydrolysis velocities o f reserve proteins from GS and o f casein by DS proteases.
e n z y m e preparations from freshly harvested seeds and from those stored during a year were inactive. L and V from GS, however, are hydrolysed by s o m e o f the DS proteinases. With V as substrate t w o broad m a x i m a at pH 4 . 5 - - 5 . 0 and 5.5--6.2 were observed. With L there is o n e major m a x i m u m around pH 7.0 and a small but reproducible activity at pH 4 . 0 - - 4 . 5 ( F i g . l ) . At neutral pH the velocity o f hydrolysis o f L from GS is more than double the rate for casein. The initial rate o f hydrolysis o f V from GS at pH 5.9 also markedly exceeds that o f casein, but it declines rapidly while the rate o f casein hydrolysis is almost constant for 5 h (Fig.2). When proteases o f DS act u p o n the sum of L and V isolated from seeds germinated for 2.5 days, the pH-activiW curve characteristic o f V was obtain-
90 1
j / ~ 4 . 5 da95
a
5
7
pH
Fig.3. The hydrolysis o f the s u m of L and V isolated from seeds germinated during different periods by DS proteases.
312 ed, while after 4.5 days' germination a m-~imum in the neutral pH range characteristic of L is revealed as well (Fig.3). DISCUSSION The data on the action of DS proteases on seed reserve proteins are contradictory [13--19]. As a rule these data were obtained by studying globulins or their fractions which may contain n o t only the reserve proteins [20]. However, we have observed, some non-reserve proteins of DS are hydrolysed by DS proteases. Our data obtained with purified reserve proteins as substrates demonstrate that DS proteases do not act on the L and V from DS, b u t hydrolyse the same proteins which were isolated from GS. These results support previous reports on the modification of reserve proteins during germinatoin [1--5] and show that their availability to hydrolysis by DS proteases may be used as a criterion of modification of reserve proteins. According to this criterion the modification of V during germination begins earlier than the 2.5th day, and that of L between the 2.5th day. Evidently, factors modifying the reserve proteins are synthesized or activated during germination. Only after modification do the reserve proteins become available to the DS proteases. There are no appreciable changes of the molecular weight of reserve proteins during modification. This was demonstrated earlier [1] and confirmed in the present work when~hamcterizing L and V isolated from GS. Thus, preliminary dissociation as an initial step~in the hydrolysis of reserve proteins as postulated by several authors [21--24], is not always necessary. It cannot be asserted that the described way of hydrolysis is the only possible one. New proteolytic enzymes which can hydrolyse unmodified reserve proteins may be synthesized or activated during germination. Moreover, it can be assumed that the newly formed proteases act as modifying factors through limited proteolysis. ACKNOWLEDGEMENT
The authors express their sincere gratitude to Dr. E.M. Dobmskina for her help in the translation of this paper.
REFERENCES 1 A.D. Shutov and I.A. Vaintrsub, Fiziol. Rut., 20 (1973) 504. 2 J.M. Dechary, K.F. Talluto, W.J. Evans, W.B. Carney and A.M. Alt~hul, Nature, 190 (1961) 1125. 3 J. Dammant, N.J. Neueere and E.J. Conkerton, Plant Physiol., 44 (1969) 480. 4 J. Kloz, A. Turkova and E. Klozova, Biol. Flantarum Acad. Svi. Bohemoelov., 8 (1966) 164. 5 A.D. Shutov and Nguen Chong Tkhin, Fiziol. R u t , (in press).
313 6 T.N. Korolyova, M.V. Alekseeva, A.D. Shutov and LA. Vaintraub, Fiziol. Rast., 20 (1973) 769, 7 A.D. Shutov and I.A. Vaintraub, Biokhimiya, 31 (1966) 726. 8 C.E. Danielson, Acta Chem. Scan&, 4 (1950) 762. 9 A.D. Shutov and I.A. Vaintraub, Ukr. Biokhim. Zh., 37 (1965) 177. 10 V.S. Shvarz, A.D. Shutov and I.A. Vaintraub, Biol. Nauki, 11 N12 (1968) 99. 11 P.J. Elving, J.M. Markowitz and I. Rosenthal, Anal. Chem., 28 (1956) 1179. 12 J. Langner, S. Ansorge, P. Bohley, H. Kirschke and H. Hanson, Acta Biol. Med. Ger., 26 (1971) 935. 13 M. Mergentine and E.H. Wigand, Fruit Products J., 26 (1946) 72. 14 G. Tazawa and T. Hirokawa, J. Biochem., 43 (1956) 785. 15 S. Takagi, Nippon Nogei Kagaku Kaishi, 34 (1960) 357, quoted after CA, 58 (1963) 10456e. 16 S. Shinano and K. Fukushima, Agr. Biol. Chem., 33 (1969) 1236. 17 G.F.G. Morris, D.A. Thurman and D. Boulter, Phytochemistry, 9 (1970) 1707. 18 A.G.S. Angelo, R.L. Ory and H.G. Hanson, Phytochemistry, 9 (1970) 1933. 19 G.L. Guardiola and G.F. Sutcliffe, Ann. Botany (London), 35 (1971) 791. 20 I.A. Vaintraub and A.D. Shutov, Tr. Khim. Prir. Soedin., 7 (1968) 23. 21 V.L. Kretovich and T.I. Smirnova, Biokhim. Zerna i Khlebopecheniya, 6 (1960) 66. 22 V. Ghetie, Rev. Roum. Biochim., 3 (1963) 353. 23 N. Catsimpoolas, T.G. Campbell and E.M. Mayer, Plant Physiol., 43 (1968) 799. 24 M.A. Belozerskii, Dokl. Akad. Nauk SSSR, 199 (1971) 468.