- Email: [email protected]
An oviducal enzyme isolated by affinity chromatography which acts upon the vitelline envelope of Bufo arenarum coelomic oocytes
289 Biochimica et Biophysica Acta, 526 (1978) 289--292
© Elsevier/North-Holland Biomedical Press
BBA Report
BBA 61340 AN OVIDUCAL ENZYME ISOLATED BY AFFINITY CHROMATOGRAPHY WHICH ACTS UPON THE VITELLINE ENVELOPE OF B U F O A R E N A R UM COELOMIC OOCYTES
D O R A C. MICELI*, SILVIA N. F E R N A N D E Z and ENRIQUE J. DEL PINO Institutode Biolog~a,Facultad de Bioqm'mica, Qu[mica y Farmacia, UniversidadNacional de Tucumdn, San Miguel de Tucumdn (Argentina)
(ReceivedMarch 10th, 1978)
Summary A trypsin4ike oviducal proteinase acting upon the vitelline envelope of B u f o arenarum coelomic oocytes has been purified to apparent homogeneity by gel filtration on Sephadex G-200 and by affinity chromatography on a column of Sepharose 4-B containing covalently bound concanavalin A (Con A). The biologically active molecule migrated as a single band of protein upon SDS polyacrylamide gel electrophoresis.
During ovulation, amphibian oocytes are released from the ovary into the body cavity,thus receiving the name of body cavity or coelomic oocytes. Although m a n y attempts have been made to fertilizesuch oocytes [I--3], littleor no succes has been obtained unless the viteUine envelope has been previously altered, whether mechanically or by means of proteolytic enzymes [4, 5]. This would suggest that the vitellinecoat would act as a barrier to fertilization [4]. Previous results obtained in our laboratories have shown that Bufo arenarum coelomic oocytes can be fertilizedafter they have passed through the portion of the oviduct k n o w n as pars recta (PR) [6]. Recently it has been definitivelyconfirmed that coelomic oocytes can be fertilized successfully when they are treated with P R extract. In addition, we have partially isolated the active product of the crude extract, which resulted to be in a proteolytic-likeenzyme acting upon the vitellineenvelope [7]. Preliminary experiments designed to establish the mechanism of action of this protein during fertilization,indicate that the same can interact with concanavalin A (Con A), a metalloprotein isolated from the jack bean, *This w o r k is p a r t o f a d o c t o r a l t h e s i s t o be s u b m i t t e d in f u l f i l l m e n t of t h e r e q u i r e m e n t s f o r t h e D o c t o r in B i o c h e m i s t r y d e g r e e a t t h e U n i v e r s i d a d N a c i o n a l de T u c u m ~ n CArgentina).
290 Canavalia ensiformis [8]. These results suggest that affinity chromatography on immobilized Con A might be usable in the purification of this oviducal fertilization factor. In the present paper we report the purification of this molecule by affinity chromatography on Con A-Sepharose. The procedure for the preparation of the PR extract has been described elsewhere [9]. The vitelline coat ofB. arenarum coelomic oocytes is not attacked by sperm lysin. However, if the same oocytes are pretreated with a crude PR extract ("conditioned oocytes"), the vitelline envelope becomes sensible to sperm lysin. The bioassay used to estimate the activity of the active molecule during their purification is based on the above described principle. The bioassay was performed in plastic culture plates, with 0.5-ml wells at 25°C. 15 coelomic oocyte batches were conditioned in 0.5 ml of the fractions to be assayed over 30 min, being then transferred to 0.3 ml 10% Ringer containing 0.01 M Tris-HC1 (pH 7.6) to which 2 ug/ml of sperm lysin prepared according to the method of Cabada [9] had been added. After 3 min the culture plates were observed under the stereoscopic microscope. Lysis degree of the vitelline envelope was compared with that of controls. Results were reported as 0 (no apparent egg alteration) to 4+ (enlargement and softening of the vitelline envelope accompanied with wrinkling of the egg surface and a decrease of egg tension). Results are expressed from 0 (no lysis) to 4+ (complete lysis). Con A (Sigma Chemical Co., St. Louis, Mo.) was conjugated to Sepharose 4-B (Pharmacia Fine Chemicals, Uppsala, Sweden) according to the procedure described by Cuatrecasas [ 10]. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed as described by Laemmli [11] using Coomassie Blue stain. Protein content was determined by the procedure of Lowry et al. [i2] with bovine serum albumin as a standard. The active protein was isolated from the crude extract by two steps: gel filtration and affinity chromatography. The PR crude extract was directly applied to a column containing Sephadex G-200 (Pharmacia Fine Chemicals, Uppsala, Sweden) equilibrated and eluted with Ringer containing 0.01 M TrisHC1 (pH 7.4). Column dimensions were 50 × 2.5 cm and the void volume (V0) was determined with Dextran Blue as a marker. Each 1.7-ml fraction was checked for adsorption at 280 nm and for biological activity. The active material from the Sephadex G-200 column was pooled and then applied to a column (0.7 × 6 cm) of Sepharose-bound Con A which had been equilibrated with Ringer containing 0.01 M Tris-HC1 and 0.001 M each MgC12 and MnC12 (pH 7.4). The column was washed with this solution until 280 nm absorbance had fallen to a value below 0.05. The eluant was collected in 1-ml fractions at the rate of 1.5 ml/min. The bound material was then eluted with 0.2 M a-methyl-D-mannoside (Sigma Chemical Co., St. Louis, Mo.) (Fig. 1). In control column the specific sugar was added to the sample before chromatography (Fig. 2). The protein content of the pooled material of the void volume was nearly the same as that of the control column, indicating that only a very small fraction of the total protein had adsorbed. Identical results were obtained when the crude extract was directly chromatographed on the plant lectin column. SDS polyacrylamide gel electrophoresis of the PR crude extract was performed and the results are shown in Fig. 3. The Coomassie Blue stain revealed 11 distinct bands. On the other hand, when examined by SDS gel
291
4!
4I
03-
03
-
,.
IL 0
10 20 Fraction number
30
0
'0 2'0 Fraction number
Fig, I . A f f i n i t y c h r o m a t o g r a p h y o f t h e a c t i v e f r a c t i o n e l u t e d f r o m S e p h a d e x G - 2 0 0 o n a c o l u m n o f C o n A b o u n d t o S e p h a r o s c 4-B. T h e c o l u m n w a s w a s h e d w i t h R i n g e r c o n t a i n i n g 0 . 0 1 M Tris-HCl a n d 0 . 0 0 1 M e a c h MgCI 2 a n d MnCI 2 ( p H 7 . 4 ) t o r e m o v e m a t e r i a l n o t b o u n d t o t h e C o n A f o l l o w e d b y elut i o n w i t h 0.2 M a - D - m a n n o s i d e . T h e a r r o w d e n o t e s t h e p o i n t at w h i c h t h e specific sugax w a s a d d e d to the c o l u m n . F r a c t i o n s w e r e a s s a y e d f o r a c t i v i t y (o----o) a n d f o r a b s o r b a n c e a t 2 8 0 n m ( c c). F i ~ 2. C o n A - S e p h a r o s e c o n t r o l c o l u m n . 0.2 M a - m e t h y l - D - m a n n o s i d e ~¢as a d d e d t o t h e s a m p l e b e f o r e c h r o m a t o g r a p h y . C h r o m a t o g r a p h i c c o n d i t i o n s a n d s y m b o l s are as in Fig. 1.
.
.
.
.
.
Fig. 3. SDS p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s o f PR e x t r a c t . Gel A, c r u d e P R e x t r a c t ( 2 0 0 Dg o f p r o t e i n ) ; Gel B, p r o t e i n e l u t e d f r o m t h e C o n A - S e p h a r o s e c o l u m n ( 3 0 ~ g o f p r o t e i n ) . E l e c t r o p h o r e s i s w a s c a r r i e d o u t o n 7.5% p o l y a c r y l a m i d e gels c o n t a i n i n g 0 . 1 % s o d i u m d o d e c y l sulfate. T h e gels w e r e r u n in T r i s - g l y c i n e b u f f e r ( p H 8 . 3 ) a n d s t a i n e d w i t h C o o m a s s l e Blue. D i r e c t i o n o f t h e m i g r a t i o n is f r o m t o p ( - ) t o b o t t o m (+),
electrophoresis the material isolated from the Con A-Sepharose column exhibited only a single protein band (Fig. 3). The data reported in this paper demonstrated that affinity chromatography using Con A covalently linked to agarose appears to be an useful tool
292 for the isolation of this proteolytic-like enzyme. This technique allows the recovery of high yields of the material and seems to possess a high degree of specificity. Inherent advantages of this method of purification are the rapidity and readiness of a potential single-step procedure. Con A affinity chromatography provides preliminar information on the molecular identity of this enzyme. Our data indicate that it is a glycoprotein since the active molecule binds to Con A-Sepharose and can be eluted with the appropriate specific sugar haptene. It has been shown that any glycoprotein possessing multiple a-D-mannopyranosyl or a-D-glucopyranosyl end groups or internal 2-O-linked a-D-mannopyranosyl residues will bind to Con A [13]. Lectins exhibit a high degree of discrimination between complex carbohydrates, can discriminate between stereoisomers and may give information about the type and position of substitution [14]. However, it should be noted that the reaction of lectins with oligosaccharides is highly complex depending not only on the type of monosaccharide present but also on their sequence and on the nature of the glycosidic linkages involved [15]. Thus, although the present study allows discrimination about this oviducal enzyme on the basis of its lectin interaction properties, further characterization of the carbohydrate content of this glycoprotein is necessary for the best knowledge of its function. Such experiments are presently in progress, and will provide additional information regarding the role of the female duct in the fertilization process. We are grateful to Dr. Francisco D. Barbieri for many suggestions and stimulating discussions and to Dr. Arnaldo H. Legname and Marcelo O. Cabada for critically evaluating this work. Appreciation is expressed to Mr. Eduardo Rothe for his assistance in the preparation of the manuscript. This work was supported in part by grants from the Consejo Nacional de Investigaciones Cientificas y T~cnicas (Argentina) (CONICET), The Population Council, New York (awarded to the CONICET), and the FundaciSn Lucio Cherny (Argentina). E.J. del Pino was supported by a scholarship from the CONICET. References 1 2 3 4 5 6
Rugh, R. (1935) J. Exp. Zool. 71, 163 Elinson, R.P. (1971) J. Exp. Zool. 176, 415 Wolf, D.P. and H e d r i c k , J.L. (1971) Dev. Biol. 25, 360 Elinson, R.P. (1973) J. Exp. Zool. 183, 291 Katagiri, Ch. (1974) J. Embryol. Exp. Morphol. 3 1 , 5 7 3 Barbicri, F.D. (1971) In R e c i e n t e s A d e l a n t o s en Biolog~a (Mej~a, R.H. and Moguilevsky, J.A., eds.), p. 402, Bona S.C.A, Buenos Aires 7 Miceli, D.C., Fernandez, S.N., Raisman, J.S. and Barbicri, F.D. (1978) J. Embryo1. Exp. Morphol., s u b m i t t e d for p u b l i c a t i o n
8 9 10 11 12 13 14 15
Sumner, J.B. and Howell, S.F. (1936) J. Bacteriol. 32, 227 Cabada, M.O., Mariano, M.I. a n d Raisman, J.S. (1977) J. Exp. Zool., in the press Cuatreeasas, P. (1970) J. Biol. Chem. 245, 3059 Laernmli, U.K. (1970) Nature 227, 680 Lowry, O.H, Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265--275 Goldstein, LJ., Reichert, C.M., Misaki, A. and Gorin, A.J. (1973) Biochim. B i o p h y s . A c t a 3 1 7 , 5 0 0 Sharon, N. and Lis, H. (1972) Science 177, 949 Kornfield, R. and Ferris, C. (1975) J. Biol. Chem. 250, 2614
© Elsevier/North-Holland Biomedical Press
BBA Report
BBA 61340 AN OVIDUCAL ENZYME ISOLATED BY AFFINITY CHROMATOGRAPHY WHICH ACTS UPON THE VITELLINE ENVELOPE OF B U F O A R E N A R UM COELOMIC OOCYTES
D O R A C. MICELI*, SILVIA N. F E R N A N D E Z and ENRIQUE J. DEL PINO Institutode Biolog~a,Facultad de Bioqm'mica, Qu[mica y Farmacia, UniversidadNacional de Tucumdn, San Miguel de Tucumdn (Argentina)
(ReceivedMarch 10th, 1978)
Summary A trypsin4ike oviducal proteinase acting upon the vitelline envelope of B u f o arenarum coelomic oocytes has been purified to apparent homogeneity by gel filtration on Sephadex G-200 and by affinity chromatography on a column of Sepharose 4-B containing covalently bound concanavalin A (Con A). The biologically active molecule migrated as a single band of protein upon SDS polyacrylamide gel electrophoresis.
During ovulation, amphibian oocytes are released from the ovary into the body cavity,thus receiving the name of body cavity or coelomic oocytes. Although m a n y attempts have been made to fertilizesuch oocytes [I--3], littleor no succes has been obtained unless the viteUine envelope has been previously altered, whether mechanically or by means of proteolytic enzymes [4, 5]. This would suggest that the vitellinecoat would act as a barrier to fertilization [4]. Previous results obtained in our laboratories have shown that Bufo arenarum coelomic oocytes can be fertilizedafter they have passed through the portion of the oviduct k n o w n as pars recta (PR) [6]. Recently it has been definitivelyconfirmed that coelomic oocytes can be fertilized successfully when they are treated with P R extract. In addition, we have partially isolated the active product of the crude extract, which resulted to be in a proteolytic-likeenzyme acting upon the vitellineenvelope [7]. Preliminary experiments designed to establish the mechanism of action of this protein during fertilization,indicate that the same can interact with concanavalin A (Con A), a metalloprotein isolated from the jack bean, *This w o r k is p a r t o f a d o c t o r a l t h e s i s t o be s u b m i t t e d in f u l f i l l m e n t of t h e r e q u i r e m e n t s f o r t h e D o c t o r in B i o c h e m i s t r y d e g r e e a t t h e U n i v e r s i d a d N a c i o n a l de T u c u m ~ n CArgentina).
290 Canavalia ensiformis [8]. These results suggest that affinity chromatography on immobilized Con A might be usable in the purification of this oviducal fertilization factor. In the present paper we report the purification of this molecule by affinity chromatography on Con A-Sepharose. The procedure for the preparation of the PR extract has been described elsewhere [9]. The vitelline coat ofB. arenarum coelomic oocytes is not attacked by sperm lysin. However, if the same oocytes are pretreated with a crude PR extract ("conditioned oocytes"), the vitelline envelope becomes sensible to sperm lysin. The bioassay used to estimate the activity of the active molecule during their purification is based on the above described principle. The bioassay was performed in plastic culture plates, with 0.5-ml wells at 25°C. 15 coelomic oocyte batches were conditioned in 0.5 ml of the fractions to be assayed over 30 min, being then transferred to 0.3 ml 10% Ringer containing 0.01 M Tris-HC1 (pH 7.6) to which 2 ug/ml of sperm lysin prepared according to the method of Cabada [9] had been added. After 3 min the culture plates were observed under the stereoscopic microscope. Lysis degree of the vitelline envelope was compared with that of controls. Results were reported as 0 (no apparent egg alteration) to 4+ (enlargement and softening of the vitelline envelope accompanied with wrinkling of the egg surface and a decrease of egg tension). Results are expressed from 0 (no lysis) to 4+ (complete lysis). Con A (Sigma Chemical Co., St. Louis, Mo.) was conjugated to Sepharose 4-B (Pharmacia Fine Chemicals, Uppsala, Sweden) according to the procedure described by Cuatrecasas [ 10]. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed as described by Laemmli [11] using Coomassie Blue stain. Protein content was determined by the procedure of Lowry et al. [i2] with bovine serum albumin as a standard. The active protein was isolated from the crude extract by two steps: gel filtration and affinity chromatography. The PR crude extract was directly applied to a column containing Sephadex G-200 (Pharmacia Fine Chemicals, Uppsala, Sweden) equilibrated and eluted with Ringer containing 0.01 M TrisHC1 (pH 7.4). Column dimensions were 50 × 2.5 cm and the void volume (V0) was determined with Dextran Blue as a marker. Each 1.7-ml fraction was checked for adsorption at 280 nm and for biological activity. The active material from the Sephadex G-200 column was pooled and then applied to a column (0.7 × 6 cm) of Sepharose-bound Con A which had been equilibrated with Ringer containing 0.01 M Tris-HC1 and 0.001 M each MgC12 and MnC12 (pH 7.4). The column was washed with this solution until 280 nm absorbance had fallen to a value below 0.05. The eluant was collected in 1-ml fractions at the rate of 1.5 ml/min. The bound material was then eluted with 0.2 M a-methyl-D-mannoside (Sigma Chemical Co., St. Louis, Mo.) (Fig. 1). In control column the specific sugar was added to the sample before chromatography (Fig. 2). The protein content of the pooled material of the void volume was nearly the same as that of the control column, indicating that only a very small fraction of the total protein had adsorbed. Identical results were obtained when the crude extract was directly chromatographed on the plant lectin column. SDS polyacrylamide gel electrophoresis of the PR crude extract was performed and the results are shown in Fig. 3. The Coomassie Blue stain revealed 11 distinct bands. On the other hand, when examined by SDS gel
291
4!
4I
03-
03
-
,.
IL 0
10 20 Fraction number
30
0
'0 2'0 Fraction number
Fig, I . A f f i n i t y c h r o m a t o g r a p h y o f t h e a c t i v e f r a c t i o n e l u t e d f r o m S e p h a d e x G - 2 0 0 o n a c o l u m n o f C o n A b o u n d t o S e p h a r o s c 4-B. T h e c o l u m n w a s w a s h e d w i t h R i n g e r c o n t a i n i n g 0 . 0 1 M Tris-HCl a n d 0 . 0 0 1 M e a c h MgCI 2 a n d MnCI 2 ( p H 7 . 4 ) t o r e m o v e m a t e r i a l n o t b o u n d t o t h e C o n A f o l l o w e d b y elut i o n w i t h 0.2 M a - D - m a n n o s i d e . T h e a r r o w d e n o t e s t h e p o i n t at w h i c h t h e specific sugax w a s a d d e d to the c o l u m n . F r a c t i o n s w e r e a s s a y e d f o r a c t i v i t y (o----o) a n d f o r a b s o r b a n c e a t 2 8 0 n m ( c c). F i ~ 2. C o n A - S e p h a r o s e c o n t r o l c o l u m n . 0.2 M a - m e t h y l - D - m a n n o s i d e ~¢as a d d e d t o t h e s a m p l e b e f o r e c h r o m a t o g r a p h y . C h r o m a t o g r a p h i c c o n d i t i o n s a n d s y m b o l s are as in Fig. 1.
.
.
.
.
.
Fig. 3. SDS p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s o f PR e x t r a c t . Gel A, c r u d e P R e x t r a c t ( 2 0 0 Dg o f p r o t e i n ) ; Gel B, p r o t e i n e l u t e d f r o m t h e C o n A - S e p h a r o s e c o l u m n ( 3 0 ~ g o f p r o t e i n ) . E l e c t r o p h o r e s i s w a s c a r r i e d o u t o n 7.5% p o l y a c r y l a m i d e gels c o n t a i n i n g 0 . 1 % s o d i u m d o d e c y l sulfate. T h e gels w e r e r u n in T r i s - g l y c i n e b u f f e r ( p H 8 . 3 ) a n d s t a i n e d w i t h C o o m a s s l e Blue. D i r e c t i o n o f t h e m i g r a t i o n is f r o m t o p ( - ) t o b o t t o m (+),
electrophoresis the material isolated from the Con A-Sepharose column exhibited only a single protein band (Fig. 3). The data reported in this paper demonstrated that affinity chromatography using Con A covalently linked to agarose appears to be an useful tool
292 for the isolation of this proteolytic-like enzyme. This technique allows the recovery of high yields of the material and seems to possess a high degree of specificity. Inherent advantages of this method of purification are the rapidity and readiness of a potential single-step procedure. Con A affinity chromatography provides preliminar information on the molecular identity of this enzyme. Our data indicate that it is a glycoprotein since the active molecule binds to Con A-Sepharose and can be eluted with the appropriate specific sugar haptene. It has been shown that any glycoprotein possessing multiple a-D-mannopyranosyl or a-D-glucopyranosyl end groups or internal 2-O-linked a-D-mannopyranosyl residues will bind to Con A [13]. Lectins exhibit a high degree of discrimination between complex carbohydrates, can discriminate between stereoisomers and may give information about the type and position of substitution [14]. However, it should be noted that the reaction of lectins with oligosaccharides is highly complex depending not only on the type of monosaccharide present but also on their sequence and on the nature of the glycosidic linkages involved [15]. Thus, although the present study allows discrimination about this oviducal enzyme on the basis of its lectin interaction properties, further characterization of the carbohydrate content of this glycoprotein is necessary for the best knowledge of its function. Such experiments are presently in progress, and will provide additional information regarding the role of the female duct in the fertilization process. We are grateful to Dr. Francisco D. Barbieri for many suggestions and stimulating discussions and to Dr. Arnaldo H. Legname and Marcelo O. Cabada for critically evaluating this work. Appreciation is expressed to Mr. Eduardo Rothe for his assistance in the preparation of the manuscript. This work was supported in part by grants from the Consejo Nacional de Investigaciones Cientificas y T~cnicas (Argentina) (CONICET), The Population Council, New York (awarded to the CONICET), and the FundaciSn Lucio Cherny (Argentina). E.J. del Pino was supported by a scholarship from the CONICET. References 1 2 3 4 5 6
Rugh, R. (1935) J. Exp. Zool. 71, 163 Elinson, R.P. (1971) J. Exp. Zool. 176, 415 Wolf, D.P. and H e d r i c k , J.L. (1971) Dev. Biol. 25, 360 Elinson, R.P. (1973) J. Exp. Zool. 183, 291 Katagiri, Ch. (1974) J. Embryol. Exp. Morphol. 3 1 , 5 7 3 Barbicri, F.D. (1971) In R e c i e n t e s A d e l a n t o s en Biolog~a (Mej~a, R.H. and Moguilevsky, J.A., eds.), p. 402, Bona S.C.A, Buenos Aires 7 Miceli, D.C., Fernandez, S.N., Raisman, J.S. and Barbicri, F.D. (1978) J. Embryo1. Exp. Morphol., s u b m i t t e d for p u b l i c a t i o n
8 9 10 11 12 13 14 15
Sumner, J.B. and Howell, S.F. (1936) J. Bacteriol. 32, 227 Cabada, M.O., Mariano, M.I. a n d Raisman, J.S. (1977) J. Exp. Zool., in the press Cuatreeasas, P. (1970) J. Biol. Chem. 245, 3059 Laernmli, U.K. (1970) Nature 227, 680 Lowry, O.H, Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265--275 Goldstein, LJ., Reichert, C.M., Misaki, A. and Gorin, A.J. (1973) Biochim. B i o p h y s . A c t a 3 1 7 , 5 0 0 Sharon, N. and Lis, H. (1972) Science 177, 949 Kornfield, R. and Ferris, C. (1975) J. Biol. Chem. 250, 2614