609
Biochimica et Biophysica Acta, 474 (1977) 609---618 © Elsevier/North-Holland Biomedical Press
BBA 98841
A SPECIFIC POLYADENYLASE FROM ESCHERICHIA COLI KI2
D. ANTONIADESand O. ANTONOGLOU Biochemistry Dept., Theagenion Medical Institute, Thessaloniki (Greece) (Received July 9th, 1976) (Revised manuscript received September 23rd, 1976)
Summary A polyadenylase, degrading specifically poly(A) sequences was isolated from Escherichia coli K12. The enzyme was purified about 850 times to practically electrophoretic homogeneity. It was free of poly(A) polymerase activity, as well as of the well known E. coli RNAases I and II. It is stimulated by bivalent cations like Mg2+ and Mn 2+ and splits poly(A) to 3'-AMP and therefore it can be considered as an exonuclease. The enzyme does not degrade any other ribohomopolymer or RNA.
Introduction It is well known that eukaryotic mRNAs (with the exception of histones) contain poly(A} sequences at their 3'-OH terminus [1--4]. Enzymes synthesizing such poly(A) sequences (poly(A) polymerases) have been described in these cells [5--7] as well as enzymes degrading specifically polyadenylic acid (polyadenylases). The latter were found localized mainly in mitochondria [8]. In the case of prokaryotic cells, strong enzymatic activity responsible for poly(A) synthesis from ATP has been found [9], despite the generally accepted idea that their mRNA does not contain this ribohomopolymer [10]. However, several authors have reported the existence of poly(A) sequences in E. coli messenger RNA [11,12]. An indication of the presence of poly(A) in prokaryotic mRNA is the existence of poly(A)-degrading enzyme in E. coil (PR7 RNAase-, pnp) [13,14]. In studying the poly(A) polymerase of E. coil K12, we observed a nuclease degrading the newly synthesized poly(A). Here we report the purification and characterization of this enzyme. The presence in E. coil of high poly(A)-synthesizing and -degrading enzymatic activities cannot be evidence that these activities are related to the existence of poly(A) sequences in bacterial mRNA. It is possible that the synthe-
610
sized poly(A) is used in a different way in prokaryotic cells, possibly as storage for adenylate [1]. In any case the existence of a complete enzymatic system for synthesis and degradation of this h o m o p o l y m e r supports the idea that it plays a role in microbial metabolism. Materials and Methods Materials DEAE-cellulose (capacity 0.85 mequiv./g) and ECTEOLA-cellulose (capacity 0.3 mequiv./g) were Sigma products. P-cellulose P l l (capacity 7.4 mequiv./g) was a Whatman product. Sephadex G-75, G-100, G-150 and poly(U) Sepharose 4B (capacity 150/zg RNA/ml swollen gel) were products of Pharmacia. Radioactive ATP (3H, 20 Ci/mM) was purchased from the Radiochemical Centre Amersham, U.K. Acrylamide and N,N~-methylene-bis-acrylamide were products of BDH, U.K. They were recrystallized in chloroform and acetone respectively before use. Bovine ribonuclease and DNAase I, were products of Schwartz-Mann. The non-radioactive ribohomopolymers used in this work were purchased from P.L. Biochemicals. Bovine serum albumin was a product of Koch-Light Laboratories, U.K. Methods E. coli K~2, cultured up to the stationary phase in a minimal medium (containing 1.0 gr NH4C1, 1.0 gr (NH4)2SO4, 41 mg Mg SO4 • 7H20, 1.56 mg Fe (NH4)2(SO4)2 • 6H20, 1.68 mg citric acid and 10 g glucose per 1 of 0.1 M phosphate buffer pH 7.0) was used for the isolation of the enzyme, as well as for poly(A) polymerase, transfer and ribosomal RNAs. E. coli t R N A was prepared according to Ehrenstein [15] using isopropanol fractionation for the final purification. E. coli r R N A was prepared essentially b y the method of Takanami [16] with the only difference that Sephadex G-200 was n o t used. Heterogenous nuclear R N A (HnRNA) was prepared from rat liver according to Hemminiki [17]. Rat liver DNA was prepared according to Antonoglou and Georgatsos with a detergent/phenol method [18]. DNA was denatured b y heating it at 100°C for 10 min and cooling rapidly. Radioactive poly(A) was synthesized in vitro using E. coli poly(A) polymerase. The poly(A) polymerase fraction from the ECTEOLA column (see Results), after gel filtration on Sephadex G-150 and subsequent concentration with polyethylene glycol, was used for this synthesis. The conditions for the incubation were the same as those used for the enzyme assay, except that the final volume was 10 ml. After 40 min of incubation at 37°C the mixture was diluted with water to bring the KC1 concentration to 0.1 M and EDTA was added to a final concentration of 1 mM. The synthesized poly(A) was extracted according to Perry et al. [19] with the only difference that the pH of the solution was brought to 7.5, then dissolved in buffer, treated with DNAase and RNAase A, as described b y Green et al. [20], and purified on a poly(U) Sepharose column, according to Wagner et al. [21].
611
Poly(A ) polymerase assay The standard assay mixture contained in a total volume of 55/M: 2.5 ttmol Tris • HC1 buffer pH 9.0, 1 pmol MgCI2, 40 pmol KC1, 0.1 /~mol ATP (unlabelled), 0.25 pCi [3H]ATP and 2.5 pg of enzyme preparation protein. Samples were incubated for 40 min at 37°C and the acid-insoluble material was measured in a Packard liquid scintillation spectrometer according to Bollum [22].
Assay for nuclease The assay mixture contained in a total volume of 60 pl: 2.5 gmol Tris • HCI buffer pH 8.0, 1.5 ttmol of MgCI2, 20/~mol KC1, 10 pl of radioactive poly(A) (6.2 pg, 10000 cpm) and varying quantities of enzyme preparations. After an incubation period of 40 min at 37 ° C, 50/~1 were loaded on paper filters and the trichloroacetic acid-insoluble radioactive material was counted according to BoUum [22]. The difference between the original radioactivity and that on the filters indicated that the poly(A) degraded into acid-soluble material. A unit of enzyme is defined as the quantity which liberates 1 pg of trichloroacetic acid-soluble material in a minute under the conditions described above. Specific activity is defined as the units of enzyme per mg of protein. Protein was measured by the method of Lowry et al. [23]. The absorbance at 280 nm was used for protein estimation during the ion-exchange chromatography. Polyacrylamide gel electrophoresis under nondenaturing conditions was performed according to Davis [24] and under denaturing conditions according to Laemmli [25] as modified by Schwartz and Roeder [26]. The enzyme elution from the gel was performed according to Schwartz and Roeder [26]. Results and Discussion
Enzyme isolation 15 g of wet bacteria were suspended in an equal volume of extraction buffer (0.01 M MgC12/0.2 M KC1/0.1 mM EDTA/0.1 mM dithiothreitol/10% (v/v) glycerol/0.05 M Tris • HC1 pH 7.5) and disrupted with a Branson sonifier B12 (8 bursts of 15 s each) at setting 8. After centrifugation at 16000 × g for 10 min, the pellet was suspended in 2 vol. of extraction buffer and sonicated again (4 bursts of 15 s each). The homogenate was centrifuged at 16000 × g and the two combined supernatants were centrifuged at 105000 × g for 1 h. The supernatant obtained was loaded on to a 2 X 15 cm DEAECellulose column, previously equilibrated with standard buffer (0.5 mM EDTA/0.5 mM dithiothreitol/10% (v/v) glycerol/0.05 M Tris • HCI pH 8.3). After the elution of non-retained material, a linear 0--0.5 M ammonium sulphate gradient in standard buffer was applied to the column (Fig. 1A). The polyadenylase activity was eluted at a salt concentration of about 0.2 M, followed by poly(A) polymerase activity, which partly overlaps that of polyadenylase.
612
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F i g . I . P u r l f i c a t i o n o f p o l y a d e n y l a s e . ( A ) D E A E - c e l l u l o s e c h r o m a t o g r a p h y of E. coli e x t r a c t . A 2 × 15 c m c o l u m n w a s u s e d a n d t h e e n z y m a t i c a c t i v i t y e l u t e d b y a l i n e a r a m m o n i u m s u l f a t e g r a d i e n t ( 0 - - 0 . 5 M) in s t a n d a r d b u f f e r . 70 m l of each b u f f e r were used. (B) E C T E O L A - c e l l u l o s e c h r o m a t o g r a p h y of nuclease f r a c t i o n s f r o m t h e D E A E - c e l l u l o s e c o l u m n . A 1 . 5 X 2 0 c m c o l u m n w a s u s e d w i t h t h e s a m e g r a d i e n t as t h a t u s e d f o r D E A E c h r o m a t o g r a p h y a p p l i e d . ( C ) P-cellulose c h r o m a t o g r a p h y o f n u c l e a s e f r a c t i o n s f r o m t h e E C T E O L A c o l u m n . A 1 . 5 X 6 c m c o l u m n w a s u s e d a n d t h e e l u t i o n w a s a c h i e v e d b y a 0---0.5 M a m m o n i u m s u l f a t e g r a d i e n t in s t a n d a r d b u f f e r ( 4 5 + 4 5 m l ) , F r a c t i o n s o f 3.5 m l w e r e c o l l e c t e d i n all t h r e e procedures. , A28 0 ; o o, n u c l e a s e a c t i v i t y ; • --, p o l y ( A ) p o l y m e r a s e a c t i v i t y .
T h e f r a c t i o n s c o n t a i n i n g t h e n u c l e a s e activity w e r e p o o l e d , c o n c e n t r a t e d in p o l y e t h y l e n e g l y c o l , d i a l y z e d in standard b u f f e r and c h r o m a t o g r a p h e d o n a 1.5 × 20 c m E C T E O L A - c e l l u l o s e c o l u m n . T h e s a m e gradient as t h a t used in t h e
613
previous column was applied. As indicated in Fig. 1B, the nuclease activity was eluted at an a m m o n i u m sulfate concentration of about 0.2 M, again followed by the polymerase activity. A third chromatography on a P-cellulose column separated the nuclease activity from that of polymerase, giving at the same time a high degree of purification of the nuclease (Fig. 1C). As one can observe in this figure, polymerase activity, as well as non-specific nuclease activity were not retained on the column, while the polyadenylase activity was eluted at an a m m o n i u m sulfate concentration of about 0.15 M. This fraction was concentrated with polyethylene glycol and used for the study of the enzyme. Gel electrophoresis patterns of various enzyme fractions under nondenaturing conditions are shown in Fig. 2. The overall purification was about 850-fold and the enzyme was free of any polymerase activity. Gel electrophoresis of the pure enzyme preparation under denaturing conditions, in the presence of dodecyl sulfate,revealed that there is only one protein band (Fig. 3). This protein moves about the same distance as bovine serum
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G Fig. 2. Acrylamide gel electrophoresls of t h e e n z y m e p r e p a r a t i o n obtained a t various steps of purification. A 7.5% acrylamlde gel in pH 8.3 was used. Electrophoresis o f the samples was carried out a t 3 mA per t u b e f o r 90 rain a n d t h e gels w e r e stained with amido black. (A) DEAE-cellulose c h r o m a t o g r a p h y (peak fraction); (B) ECTEOLA-cellulose c h r o m a t o g r a p h y ( c o n c e n t r a t e d e n z y m e fraction), (C) P-cellulose chrom a t o g r a p h y ( c o n c e n t ~ t t e d e n z y m e fraction).
614
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Fig. 3. Disc e l e c t r o p h o r e s l s m 5% a c r y l a m l d e p H 8.9. E l e c t r o p h o r e s l s was p e r f o r m e d f o r 6 h w i t h a curr e n t of 2 m A ] t u b e . ( A ) P u r i f i e d e n z y m e P r e p a r a t i o n ( 2 5 pg of P r o t e i n ) . E l e e t r o p b o r e s i s was p e r f o r m e d u n d e r n o n d e n a t u r m g c o n d i t i o n s a c c o r d i n g to S c h w a r t z a n d R o e d e r [ 2 6 ] . T h e e n z y m e was e l u t e d as d e s c r i b e d b y t h e s e a u t h o r s a n d a s s a y e d as d e s c r i b e d in M e t h o d s . (B) P u r i f i e d e n z y m e p r e p a r a t i o n (25 pg 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 p e r f o r m e d u n d e r d e n a t u r i n g c o n d i t i o n s [ 2 6 ] . (C) A l b u m i n ( 5 0 ~g). ElectrophoresLs was p e r f o r m e d u n d e r d e n a t u r i n g c o n d l t t o n s .
albumin (Fig. 3), suggesting that the molecular weight of this protein is a b o u t 6 7 0 0 0 . This is also supported by gel filtration through Sephadex G-75, where the enzyme protein is eluted very close to albumin (results n o t shown). To clarify whether the protein shown in the gel is the enzyme itself, after gel electrophoresis under nondenaturing conditions, slices of a b o u t 1.5 mm were cut, the protein removed according to Schwartz and Roeder [26], and assayed for enzyme activity. The results indicate that the enzymatic activity coincides with the protein (Fig. 3). This fact strongly supports the idea that the protein shown on acrylamide electrophoresis represents the enzyme. A summary of the purification procedure is presented in Table I.
Properties o f the enzyme As one can see in Fig. 4, the enzyme has an o p t i m u m range of activity around pH 8.0. The bivalent cation requirements of the enzyme are presented in Fig. 5. The enzyme is activated in the presence of either Mn 2÷ or Mg 2÷, the optimal concentration being 2.3 mM for Mn 2÷ and 3.0 mM for Mg 2÷. The optimal ionic strength, on the other hand, is at a concentration of 0.2 M KC1 (Fig. 6). The enzyme remains stable when stored for several months a t - - 2 0 ° C , while it is inactivated after heating for 5 min at 50 ° C. The specificity of the enzyme
615
TABLE I PURIFICATION
OF POLYADENYLASE
Purifwahon step
Protein (mg/ml)
E. colz e x t r a c t DEAE
60
chromatography ECTEOLA chromatography P-cellulose chromatography C o n c e n t r a t e d Pcellulose fraction
F R O M E. COLI K I 2 . Spec. act. (units/mg)
Total umts
Purification factor
Yield (%)
0.25
600
1
100
3.23
2.50
448
10
77.00
0.25
64.00
336
256
56.00
0,03
180.00
264
720
44.00
0.183
211.00
310
844
51.60
was determined by a number of experiments, described below. Various non-radioactive substrates were incubated with enzyme under the same conditions used for the enzyme assay, with the exception that the final volume was 1 ml. After an incubation period of 40 min the trichloroacetic acidsoluble material was measured at 260 nm. The results are presented in Table II. It is clear that the enzyme, practically, degrades only poly(A). The significant degradation of H n R N A could be explained by the well known existence of poly(A) sequences in its molecule. This method of measuring the products was, however, inadequate to check endonucleases degrading a substrate to rather large oligonucleotides. Disk electrophoresis, according to MacLeod [ 27 ], with acryalmide gels and gel filtration on Sephadex G-100 were then used to exclude the possible formation of a few nicks at various substrates.
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Biochimica et Biophysica Acta, 474 (1977) 609---618 © Elsevier/North-Holland Biomedical Press
BBA 98841
A SPECIFIC POLYADENYLASE FROM ESCHERICHIA COLI KI2
D. ANTONIADESand O. ANTONOGLOU Biochemistry Dept., Theagenion Medical Institute, Thessaloniki (Greece) (Received July 9th, 1976) (Revised manuscript received September 23rd, 1976)
Summary A polyadenylase, degrading specifically poly(A) sequences was isolated from Escherichia coli K12. The enzyme was purified about 850 times to practically electrophoretic homogeneity. It was free of poly(A) polymerase activity, as well as of the well known E. coli RNAases I and II. It is stimulated by bivalent cations like Mg2+ and Mn 2+ and splits poly(A) to 3'-AMP and therefore it can be considered as an exonuclease. The enzyme does not degrade any other ribohomopolymer or RNA.
Introduction It is well known that eukaryotic mRNAs (with the exception of histones) contain poly(A} sequences at their 3'-OH terminus [1--4]. Enzymes synthesizing such poly(A) sequences (poly(A) polymerases) have been described in these cells [5--7] as well as enzymes degrading specifically polyadenylic acid (polyadenylases). The latter were found localized mainly in mitochondria [8]. In the case of prokaryotic cells, strong enzymatic activity responsible for poly(A) synthesis from ATP has been found [9], despite the generally accepted idea that their mRNA does not contain this ribohomopolymer [10]. However, several authors have reported the existence of poly(A) sequences in E. coli messenger RNA [11,12]. An indication of the presence of poly(A) in prokaryotic mRNA is the existence of poly(A)-degrading enzyme in E. coil (PR7 RNAase-, pnp) [13,14]. In studying the poly(A) polymerase of E. coil K12, we observed a nuclease degrading the newly synthesized poly(A). Here we report the purification and characterization of this enzyme. The presence in E. coil of high poly(A)-synthesizing and -degrading enzymatic activities cannot be evidence that these activities are related to the existence of poly(A) sequences in bacterial mRNA. It is possible that the synthe-
610
sized poly(A) is used in a different way in prokaryotic cells, possibly as storage for adenylate [1]. In any case the existence of a complete enzymatic system for synthesis and degradation of this h o m o p o l y m e r supports the idea that it plays a role in microbial metabolism. Materials and Methods Materials DEAE-cellulose (capacity 0.85 mequiv./g) and ECTEOLA-cellulose (capacity 0.3 mequiv./g) were Sigma products. P-cellulose P l l (capacity 7.4 mequiv./g) was a Whatman product. Sephadex G-75, G-100, G-150 and poly(U) Sepharose 4B (capacity 150/zg RNA/ml swollen gel) were products of Pharmacia. Radioactive ATP (3H, 20 Ci/mM) was purchased from the Radiochemical Centre Amersham, U.K. Acrylamide and N,N~-methylene-bis-acrylamide were products of BDH, U.K. They were recrystallized in chloroform and acetone respectively before use. Bovine ribonuclease and DNAase I, were products of Schwartz-Mann. The non-radioactive ribohomopolymers used in this work were purchased from P.L. Biochemicals. Bovine serum albumin was a product of Koch-Light Laboratories, U.K. Methods E. coli K~2, cultured up to the stationary phase in a minimal medium (containing 1.0 gr NH4C1, 1.0 gr (NH4)2SO4, 41 mg Mg SO4 • 7H20, 1.56 mg Fe (NH4)2(SO4)2 • 6H20, 1.68 mg citric acid and 10 g glucose per 1 of 0.1 M phosphate buffer pH 7.0) was used for the isolation of the enzyme, as well as for poly(A) polymerase, transfer and ribosomal RNAs. E. coli t R N A was prepared according to Ehrenstein [15] using isopropanol fractionation for the final purification. E. coli r R N A was prepared essentially b y the method of Takanami [16] with the only difference that Sephadex G-200 was n o t used. Heterogenous nuclear R N A (HnRNA) was prepared from rat liver according to Hemminiki [17]. Rat liver DNA was prepared according to Antonoglou and Georgatsos with a detergent/phenol method [18]. DNA was denatured b y heating it at 100°C for 10 min and cooling rapidly. Radioactive poly(A) was synthesized in vitro using E. coli poly(A) polymerase. The poly(A) polymerase fraction from the ECTEOLA column (see Results), after gel filtration on Sephadex G-150 and subsequent concentration with polyethylene glycol, was used for this synthesis. The conditions for the incubation were the same as those used for the enzyme assay, except that the final volume was 10 ml. After 40 min of incubation at 37°C the mixture was diluted with water to bring the KC1 concentration to 0.1 M and EDTA was added to a final concentration of 1 mM. The synthesized poly(A) was extracted according to Perry et al. [19] with the only difference that the pH of the solution was brought to 7.5, then dissolved in buffer, treated with DNAase and RNAase A, as described b y Green et al. [20], and purified on a poly(U) Sepharose column, according to Wagner et al. [21].
611
Poly(A ) polymerase assay The standard assay mixture contained in a total volume of 55/M: 2.5 ttmol Tris • HC1 buffer pH 9.0, 1 pmol MgCI2, 40 pmol KC1, 0.1 /~mol ATP (unlabelled), 0.25 pCi [3H]ATP and 2.5 pg of enzyme preparation protein. Samples were incubated for 40 min at 37°C and the acid-insoluble material was measured in a Packard liquid scintillation spectrometer according to Bollum [22].
Assay for nuclease The assay mixture contained in a total volume of 60 pl: 2.5 gmol Tris • HCI buffer pH 8.0, 1.5 ttmol of MgCI2, 20/~mol KC1, 10 pl of radioactive poly(A) (6.2 pg, 10000 cpm) and varying quantities of enzyme preparations. After an incubation period of 40 min at 37 ° C, 50/~1 were loaded on paper filters and the trichloroacetic acid-insoluble radioactive material was counted according to BoUum [22]. The difference between the original radioactivity and that on the filters indicated that the poly(A) degraded into acid-soluble material. A unit of enzyme is defined as the quantity which liberates 1 pg of trichloroacetic acid-soluble material in a minute under the conditions described above. Specific activity is defined as the units of enzyme per mg of protein. Protein was measured by the method of Lowry et al. [23]. The absorbance at 280 nm was used for protein estimation during the ion-exchange chromatography. Polyacrylamide gel electrophoresis under nondenaturing conditions was performed according to Davis [24] and under denaturing conditions according to Laemmli [25] as modified by Schwartz and Roeder [26]. The enzyme elution from the gel was performed according to Schwartz and Roeder [26]. Results and Discussion
Enzyme isolation 15 g of wet bacteria were suspended in an equal volume of extraction buffer (0.01 M MgC12/0.2 M KC1/0.1 mM EDTA/0.1 mM dithiothreitol/10% (v/v) glycerol/0.05 M Tris • HC1 pH 7.5) and disrupted with a Branson sonifier B12 (8 bursts of 15 s each) at setting 8. After centrifugation at 16000 × g for 10 min, the pellet was suspended in 2 vol. of extraction buffer and sonicated again (4 bursts of 15 s each). The homogenate was centrifuged at 16000 × g and the two combined supernatants were centrifuged at 105000 × g for 1 h. The supernatant obtained was loaded on to a 2 X 15 cm DEAECellulose column, previously equilibrated with standard buffer (0.5 mM EDTA/0.5 mM dithiothreitol/10% (v/v) glycerol/0.05 M Tris • HCI pH 8.3). After the elution of non-retained material, a linear 0--0.5 M ammonium sulphate gradient in standard buffer was applied to the column (Fig. 1A). The polyadenylase activity was eluted at a salt concentration of about 0.2 M, followed by poly(A) polymerase activity, which partly overlaps that of polyadenylase.
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F i g . I . P u r l f i c a t i o n o f p o l y a d e n y l a s e . ( A ) D E A E - c e l l u l o s e c h r o m a t o g r a p h y of E. coli e x t r a c t . A 2 × 15 c m c o l u m n w a s u s e d a n d t h e e n z y m a t i c a c t i v i t y e l u t e d b y a l i n e a r a m m o n i u m s u l f a t e g r a d i e n t ( 0 - - 0 . 5 M) in s t a n d a r d b u f f e r . 70 m l of each b u f f e r were used. (B) E C T E O L A - c e l l u l o s e c h r o m a t o g r a p h y of nuclease f r a c t i o n s f r o m t h e D E A E - c e l l u l o s e c o l u m n . A 1 . 5 X 2 0 c m c o l u m n w a s u s e d w i t h t h e s a m e g r a d i e n t as t h a t u s e d f o r D E A E c h r o m a t o g r a p h y a p p l i e d . ( C ) P-cellulose c h r o m a t o g r a p h y o f n u c l e a s e f r a c t i o n s f r o m t h e E C T E O L A c o l u m n . A 1 . 5 X 6 c m c o l u m n w a s u s e d a n d t h e e l u t i o n w a s a c h i e v e d b y a 0---0.5 M a m m o n i u m s u l f a t e g r a d i e n t in s t a n d a r d b u f f e r ( 4 5 + 4 5 m l ) , F r a c t i o n s o f 3.5 m l w e r e c o l l e c t e d i n all t h r e e procedures. , A28 0 ; o o, n u c l e a s e a c t i v i t y ; • --, p o l y ( A ) p o l y m e r a s e a c t i v i t y .
T h e f r a c t i o n s c o n t a i n i n g t h e n u c l e a s e activity w e r e p o o l e d , c o n c e n t r a t e d in p o l y e t h y l e n e g l y c o l , d i a l y z e d in standard b u f f e r and c h r o m a t o g r a p h e d o n a 1.5 × 20 c m E C T E O L A - c e l l u l o s e c o l u m n . T h e s a m e gradient as t h a t used in t h e
613
previous column was applied. As indicated in Fig. 1B, the nuclease activity was eluted at an a m m o n i u m sulfate concentration of about 0.2 M, again followed by the polymerase activity. A third chromatography on a P-cellulose column separated the nuclease activity from that of polymerase, giving at the same time a high degree of purification of the nuclease (Fig. 1C). As one can observe in this figure, polymerase activity, as well as non-specific nuclease activity were not retained on the column, while the polyadenylase activity was eluted at an a m m o n i u m sulfate concentration of about 0.15 M. This fraction was concentrated with polyethylene glycol and used for the study of the enzyme. Gel electrophoresis patterns of various enzyme fractions under nondenaturing conditions are shown in Fig. 2. The overall purification was about 850-fold and the enzyme was free of any polymerase activity. Gel electrophoresis of the pure enzyme preparation under denaturing conditions, in the presence of dodecyl sulfate,revealed that there is only one protein band (Fig. 3). This protein moves about the same distance as bovine serum
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G Fig. 2. Acrylamide gel electrophoresls of t h e e n z y m e p r e p a r a t i o n obtained a t various steps of purification. A 7.5% acrylamlde gel in pH 8.3 was used. Electrophoresis o f the samples was carried out a t 3 mA per t u b e f o r 90 rain a n d t h e gels w e r e stained with amido black. (A) DEAE-cellulose c h r o m a t o g r a p h y (peak fraction); (B) ECTEOLA-cellulose c h r o m a t o g r a p h y ( c o n c e n t r a t e d e n z y m e fraction), (C) P-cellulose chrom a t o g r a p h y ( c o n c e n t ~ t t e d e n z y m e fraction).
614
100 tO
A m f._
50 "i3
< '5 n.
0
II I I I
B
i
C
o
2
,i
Fig. 3. Disc e l e c t r o p h o r e s l s m 5% a c r y l a m l d e p H 8.9. E l e c t r o p h o r e s l s was p e r f o r m e d f o r 6 h w i t h a curr e n t of 2 m A ] t u b e . ( A ) P u r i f i e d e n z y m e P r e p a r a t i o n ( 2 5 pg of P r o t e i n ) . E l e e t r o p b o r e s i s was p e r f o r m e d u n d e r n o n d e n a t u r m g c o n d i t i o n s a c c o r d i n g to S c h w a r t z a n d R o e d e r [ 2 6 ] . T h e e n z y m e was e l u t e d as d e s c r i b e d b y t h e s e a u t h o r s a n d a s s a y e d as d e s c r i b e d in M e t h o d s . (B) P u r i f i e d e n z y m e p r e p a r a t i o n (25 pg 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 p e r f o r m e d u n d e r d e n a t u r i n g c o n d i t i o n s [ 2 6 ] . (C) A l b u m i n ( 5 0 ~g). ElectrophoresLs was p e r f o r m e d u n d e r d e n a t u r i n g c o n d l t t o n s .
albumin (Fig. 3), suggesting that the molecular weight of this protein is a b o u t 6 7 0 0 0 . This is also supported by gel filtration through Sephadex G-75, where the enzyme protein is eluted very close to albumin (results n o t shown). To clarify whether the protein shown in the gel is the enzyme itself, after gel electrophoresis under nondenaturing conditions, slices of a b o u t 1.5 mm were cut, the protein removed according to Schwartz and Roeder [26], and assayed for enzyme activity. The results indicate that the enzymatic activity coincides with the protein (Fig. 3). This fact strongly supports the idea that the protein shown on acrylamide electrophoresis represents the enzyme. A summary of the purification procedure is presented in Table I.
Properties o f the enzyme As one can see in Fig. 4, the enzyme has an o p t i m u m range of activity around pH 8.0. The bivalent cation requirements of the enzyme are presented in Fig. 5. The enzyme is activated in the presence of either Mn 2÷ or Mg 2÷, the optimal concentration being 2.3 mM for Mn 2÷ and 3.0 mM for Mg 2÷. The optimal ionic strength, on the other hand, is at a concentration of 0.2 M KC1 (Fig. 6). The enzyme remains stable when stored for several months a t - - 2 0 ° C , while it is inactivated after heating for 5 min at 50 ° C. The specificity of the enzyme
615
TABLE I PURIFICATION
OF POLYADENYLASE
Purifwahon step
Protein (mg/ml)
E. colz e x t r a c t DEAE
60
chromatography ECTEOLA chromatography P-cellulose chromatography C o n c e n t r a t e d Pcellulose fraction
F R O M E. COLI K I 2 . Spec. act. (units/mg)
Total umts
Purification factor
Yield (%)
0.25
600
1
100
3.23
2.50
448
10
77.00
0.25
64.00
336
256
56.00
0,03
180.00
264
720
44.00
0.183
211.00
310
844
51.60
was determined by a number of experiments, described below. Various non-radioactive substrates were incubated with enzyme under the same conditions used for the enzyme assay, with the exception that the final volume was 1 ml. After an incubation period of 40 min the trichloroacetic acidsoluble material was measured at 260 nm. The results are presented in Table II. It is clear that the enzyme, practically, degrades only poly(A). The significant degradation of H n R N A could be explained by the well known existence of poly(A) sequences in its molecule. This method of measuring the products was, however, inadequate to check endonucleases degrading a substrate to rather large oligonucleotides. Disk electrophoresis, according to MacLeod [ 27 ], with acryalmide gels and gel filtration on Sephadex G-100 were then used to exclude the possible formation of a few nicks at various substrates.
8O A
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6o
0 L
v "U
40
a
20
0
4
5
6
7
8
9
10
11
pH Fig. 4, p H vs. a c t i v i t y c u r v e o f p o l y a d e n y ] a s e . T h e c o n d i t i o n s o f i n c u b a t i o n w e r e t h e s a m e w i t h t h o s e d e s c r i b e d u n d e r n u c l e a s e assay in M e t h o d s .
616
A
70
6O
v E O "O o
L
60
50
40
~J
40
"0
