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Purification of chloroplast elongation factor Tu from a somatic hybrid between Nicotiana tabacum and Nicotiana rustica
Plant Science Letter$ 32 (1983) 287--293
287
Elsevier Scientific Publishers Ireland Ltd.
P U R I F I C A T I O N OF C H L O R O P L A S T ELONGATION F A C T O R Tu F R O M A SOMATIC H Y B R I D BETWEEN N I C O T I A N A T A B A C U M A N D N I C O T I A N A R USTICA
S.M. ZHAO*, O. T1BONI and O. CIFERRI Department of Genetics and Microbiology, University of Pavia, 27100 Pavia (Italy)
(Received April 26th, 1983) (Accepted June 28th, 1983)
SUMMARY
Chloroplast elongation factor Tu (EF-TUch 1 has been purified from chloroplasts o f F2 plants of an interspecific somatic hybrid b e t w e e n Nicotiana tabacum and N. rustica. The protein has been purified approx. 90-fold with a recovery of approx. 15% o f t h e activity. By polyacrylamide gel electrophoresis, the molecular weight of EF-TUchl appears to be approx. 45 000. Similar values were found for the EF-Tu isolated from chloroplasts of the parent species. EF-TUchl from the Nicotiana hybrid was immunoprecipitated b y an antiserum raised against t h e spinach protein. Key words: Nicotiana -- Somatic hybrid -- Chloroplast -- Chloroplast elonga-
tion factor Tu INTRODUCTION
Chloroplasts contain soluble protein factors responsible for peptide chain elongation [1]. Such factors have been extensively purified from spinach chloroplasts [2] and partially characterized from intact cells o f Euglena gracilis [3,4]. To o u r knowledge no data are available in t h e case of other plants. We wish to report the procedure for the purification and t h e characterization of EF-TUcb1 present in chloroplasts of a somatic hybrid of Nicotiana as well as the t w o parents, N. tabacurn and N. rustica.
*Permanent address: Institute of Genetics, Academia Sinica, Bejing, China. Abbreviations: EF-Tueh1, chloroplast elongation factor Tu; PAGE, polyacrylamide gel electrophoresis. 0304-4211/83/$03.00
© 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
288 MATERIALS AND METHODS The somatic hybrid between N. tabacurn and N. rustica was prepared as reported [5]. The seeds of the F2 hybrid and of the parents were germinated and grown in a greenhouse. Leaves were harvested from 2-month-old plants. Chloroplasts, chloroplast extracts, and the ammonium sulfate fractions were prepared from leaves (approx. 2 kg, fresh wt.) following the procedure developed for spinach [6] by utilizing the buffers described by Rhodes et al. [ 7 ]. Preliminary experiments indicate$i that purification of tobacco EF-Tuchl by affinity chromatography on GDP-Sepharose using the rapid procedure developed for spinach chloroplasts [8 ] was unsatisfactory. Low yields of EF-TUchl and only partial purification of the protein were achieved following this procedure. It is possible that the presence of pigments in the ammonium sulfate fraction from tobacco chloroplasts interfered with the binding of tobacco EF-TUchl to GDP-Sepharose and/or its elution from the column. Therefore the ammonium sulfate fraction prepared as in [6] was adsorbed onto a 95 × 2.5 cm column packed with Sephadex G-100 in a buffer containing 50 mM Tris (pH 8), 10 mM Mg C12, 0.35 M NaC1, 1 mM dithiothreitol, 0.1 mM phenylmethylsulphonyl fluoride and 50 ~g/ml of soybean trypsin inhibitor. After adsorption of the sample, the same buffer was passed through the column at a flow rate of approx. 20 ml/h. The fractions containing EF-TUchl activity were localized by assaying their capacity to bind [H3]GDP (see later), pooled and precipitated with solid ammonium sulfate to give a 75% saturation at 0 °. After standing overnight in the cold, the protein precipitation was recovered by centrifuging (17 000 × g for 30 min) and dissolved in a small volume {less than 1 ml) of the above reported buffer. Adsorption and elution from GDPSepharose was performed as reported i~ the case of spinach [8]. Binding of [H3]GDP tO EF-TUchl, exchange of [H3]GDP with the GDP bound to EF-TUchl, separation of proteins by polyacrylamide gel electrophoresis {PAGE), and protein determination were performed as reported [6]. Escherichia coli EF-Tu and kirromycin were the generous gift of Dr. G. Chinali. The preparation and characterization of the antiserum against spinach EF-TUchl have been already described [9]. RESULTS AND DISCUSSION As depicted in Fig. 1, chromatography of the partially purified preparations on a Sephadex column not only allowed the removal of pigments {data not reported) but also of the majority of proteins including most of the ribulose-l,5-bisphosphate carboxylase. After this step, EF-TUcbl could be purified by affinity chromatography (Fig. 2). As outlined in Table I, the procedure allows a purification of approx. 90-fold with a recovery of approx. 15% of the activity present in the crude extracts. Confirmation that the activity recovered from GDP-Sepharose represents
289
o
v a Z
.x,
loj FRACTION NUMBER Fig. 1. Partial purification o f EF-TUchl o n Sephadex G-100. A p p r o x i m a t e l y 100 units of EF-TUch 1 ( 6 4 0 mg o f protein) were separated on a c o l u m n of Sephadex G-100 as described in Materials and Methods. Fractions o f 9 ml were collected. EF-TUch 1 activity was measured on 10-ul aliquots from the c o l u m n b y assaying the binding o f [H 3 ]GDP, (x- - - x). Protein was assayed spectrophotometrically, (* • ).
indeed EF-Tu was obtained by immunology since the purified preparation, as well as crude extracts from tobacco chloroplasts, were immunoprecipitated by a monospecific antiserum raised against spinach EF-TUch 1 (Fig. 3). Finally, binding of [H3]GDP to the purified EF-TUch 1 • GDP complex was
;~
~2~
2.
o x u 0 j Z
.1
I
I ! !
',
Z
o
\,~ \ k- ....
"%
FRACTION NUMBER Fig. 2. Purification o f EF-TUch 1 o n GDP-Sephamse. A p p r o x i m a t e l y 22 units o f partially purified EF-Tuch I (approx. 30 mg of protein) in 1 ml were m i x e d w i t h 0.6 ml of GDPSepharose and treated as described [8 ]. F o l l l o w i n g dialysis overnight, the GDP-Sepharose was packed in a small c o l u m n and w a s h e d w i t h approx. 150 ml o f the buffer described in Materials and Methods until the absorbation at 280 n m of the eluate reached baseline. One bed v o l u m e o f the same buffer containing 0.1 mM GDP then was added to the c o l u m n and, after adsorption, the f l o w was stopped for 1 h. The c o l u m n was then eluted w i t h 5 ml o f GDP containing buffer collecting 0 . 3 - m l fractions. EF-Tu activity o n 10-ul aliquots (x - - - x) and protein concentration (e o) were determined as reported in Fig. 1. As discussed in the text, protein could not be determined spectrophotometrically in the EF-Tu containing fractions~
290 TABLE I SUMMARY O F P U R I F I C A T I O N O F EF-TUch I F R O M NICOTIANA
THE SOMATIC HYBRID OF
One unit was defined as the amount of enzyme that catalyzes the binding of 1 × 10' c.p.m, of [HS]GDP (spec. act. 13 Ci/mmol)in 20 rain at 30°C. The values reported for fraction 4 are only estimates since protein could not be determined spectrophotometrically for the interference of the GDP present in the elution buffer. The amount of protein in each fraction before pooling was assessed from the intensity of the stained band after PAGE in comparison with the bands obtained by running known amounts of bovine serum albumin. Units of EF-TuchI activity were calculated taking in account the dilution in the assay mixture of the labelled GDP brought about by the unlabelled GDP present in the elution buffer. Fraction
Vol. (ml)
(1) Chloroplast extract 100 (2) Ammonium sulfate precipitate (from 1) 7.75 (3) Ammonium sulfate precipitate after chromatography on Sephadex G-100 1 (4) Pooled fractions after affinity chromatography on GDPSepharose 1.26
Total protein (rag)
Total act. (units)
Spec. act. (units/mg of protein)
Yield (%)
Purification (fold)
640
66
0.103
--
489
65
0.133
98
1.3
29.9
21
0.70
32
6.8
1.7
16
9.41
24
--
91
s t i m u l a t e d b y •~irromycin, an a n t i b i o t i c k n o w n t o p r o m o t e t h e e x c h a n g e b e t w e e n free G~)P and t h e GDP b o u n d in t h e E F - T u • GDP c o m p l e x [ 1 0 ] . At c o n c e n t r a t i o n o f 1, 0.1, 0.01, and 0 . 0 0 1 m g / m l , k i r r o m y c i n s t i m u l a t e d b y 426, 237, 205 and 143%, respectively, t h e e x c h a n g e b e t w e e n labelled GDP in s o l u t i o n and t h e u n l a b e l l e d GDP present in t h e c o m p l e x w i t h EF-Tu. As j u d g e d b y P A G E , t o b a c c o EF-TUch 1 appears t o be at least 90% p u r e (Fig. 4A). Indeed, o n l y o n e f a i n t b a n d o f c o n t a m i n a t i n g p r o t e i n is p r e s e n t with t h e M r o f t h e large s u b u n i t o f r i b u l o s e - l , 5 - b i s p h o s p h a t e carboxylase. The" m o l e c u l a r weight o f t o b a c c o EF-TUch I m a y b e e s t i m a t e d t o be a p p r o x . 45 000, a value close t o t h a t r e p o r t e d f o r t h e p r o t e i n isolated f r o m spinach c h l o r o p l a s t s [2] and f r o m s o m e bacteria such as E. coil [ 1 1 ] . A similar m o b i l i t y was f o u n d f o r t h e EF-TUchl p u r i f i e d f r o m the p a r e n t a l species (Fig. 4B), T h u s EF-TUchl, a p r o t e i n m o s t likely c o d e d in c h l o r o p l a s t D N A [ 1 2 ] , c a n n o t be u t i l i z e d as a m a r k e r f o r i d e n t i f y i n g t h e origin of t h e c y t o p l a s m in s o m a t i c hybrids. Nevertheless t h e results r e p o r t e d in this investigation s h o w t h a t t h e rapid p r o c e d u r e d e v e l o p e d t o p u r i f y EF-TUch 1 f r o m spinach [8] m a y be applied with slight m o d i f i c a t i o n s also t o the p r o t e i n p r e s e n t in t h e chloroplasts o f o t h e r plants. In addition, the
291
Fig. 3. Immunological characterizatiov of EF-TUchl. Thirty ul (30 ug of protein) of an antiserum against spinach EF-TUchI [ 9 ] were put in the central well. (1) Purified spinach EF-'ruch 1 (10 ug of protein); (2) purified tobacco EF-TUchl (8 ug of protein); (3) partially purified spinach EF-TUchl (0.75 mg of protein); (4) partially purified tobacco EF-Tuch 1 (0.95 mg of protein). After appearance of the precipitin lines (24 h at room temperature), the plate was washed extensively with physiological solution and then stained with Coomassie blue.
great similarity in structure and function between prokaryotic (and chloroplastic) EF-Tu and cytoplasmic EF-1 [13] would suggest that the procedure may be adopted also to purify the eukaryotic equivalent of EF-Tu. Finally the purification protocol may be utilized to isolate and identify rapidly any radioactively-labelled EF-TUchl synthesized when isolated chloroplasts are incubated in the light in the presence of labelled amino acids (unpublished results) thus allowing to establish if, in different plants, EF-TUchl is synthesized in the chloroplast.
$
I
~'ig. 4. Separation of EF-TUch I by PAGE. Standard SDS 10% PAGE was performed. (A) Track 1, ammonium sulfate precipitate from he crude extract (0.18 mg of protein); track 2, ammonium sulfate precipitate after Sephadex chromatography (0.12 mg of protein); .rack 3, fraction non-adsorbed on GDP-Sepharose (0.12 mg of protein); track 4, purified tobacco EF-TUchI from the GDP-Sepharose Lffinity column (13 ~g of protein); track 5, molecular weight markers (from top to bottom: phosphorylase, mol. wt. 94 000; bovine eroalbumin, tool wt. 68 000; ovalbumin, mol. wt. 45 000; carbonic anhydrase, mol. wt. 30 000; soybean trypsin inhibitor, tool. wt. .4 300). (B) Track 1, purified EF-Tuch1 from N. rustica (3 ~g of protein); track 2, from N. tabacum (8 ~g of protein); track 3, from he somatic hybrid (6 ~g of protein); track 4, E. coil EF-Tu (20 ug of protein); track 5, molecular weight markers as in (A)5. In racks 1--3, besides the band of E F - T u ~ , the most prominent contaminant shows a M r corresponding to that of the large subunit of
I
S
293 ACKNOWLEDGEMENTS
This work was supported by grants from Consiglio Nazionale delle Richerche (C.N.R.). One of us (S.M.Z.) acknowledges a fellowship awarded by C.N.R. under the scientific cooperation agreement between Academia Sinica and C.N.R.. Finally, the authors are grateful to G. Di Pasquale for skillful1 technical assistance. REFERENCES 1 0. Ciferri, 0. Tiboni, M.L. Munoz-Calvo and G. Camerino, in: L. Bogorad and J.H. Weil (Eds.), Nucleic Acids and Protein Synthesis in Plants, Plenum Publ. Co., 1977, p. 155. 2 0. Tiboni, G. Di Pasquale and 0. Ciferri, Eur. J. Biochem., 92 (1978) 471. 3 C.A. Breitenberger, M.V. Moore, D.W. Russell and L.L. Spremulli, Anal. Biochem., 99 (1979) 434. 4 L.L. Spremulli, Arch. Biochem. Biophys., 214 (1982) 734. 5 P.T. Wang, J.W. Chen, S.M. Zhao and J.X. Xu, Keyue Tongbao, 27 (1982) 429. 6 0. Ciferri and 0. Tiboni, in: M. Edelman, R.B. Hallick and N.H. Chua (Eds.), Methods in Chloroplast Molecular Biology, Elsevier Biomedical Press, 1982, p. 646. 7 P.R. Rhodes, Y.S. Zhu and S.D. Kung, Mol. Gen Genetics, 182 (1981) 106. 8 0. Tiboni and 0. Ciferri, FEBS Lett., 146 (1982) 197. 9 0. Tiboni, C.J. Leaver and 0. Ciferri, Plant Sci. Lett., 17 (1980) 159. 10 W. Worner and H. Wolf; FEBS Lett., 146 (1982) 322. 11 G.R. Jacobson and J.P. Rosenbush, Nature, 261 (1976) 23. 12 0. Ciferri, 0. Tiboni and G. Di Pasquale, in: 0. Ciferri and L. Dure (Eds.), Structure and function of plant genomes, 1983, Plenum Publ. Co., 1983, in press. 13 R. Amons, W. Pluijms, K. Roobol and W. Moller, FEBS Lett., 153 (1983) 37.
287
Elsevier Scientific Publishers Ireland Ltd.
P U R I F I C A T I O N OF C H L O R O P L A S T ELONGATION F A C T O R Tu F R O M A SOMATIC H Y B R I D BETWEEN N I C O T I A N A T A B A C U M A N D N I C O T I A N A R USTICA
S.M. ZHAO*, O. T1BONI and O. CIFERRI Department of Genetics and Microbiology, University of Pavia, 27100 Pavia (Italy)
(Received April 26th, 1983) (Accepted June 28th, 1983)
SUMMARY
Chloroplast elongation factor Tu (EF-TUch 1 has been purified from chloroplasts o f F2 plants of an interspecific somatic hybrid b e t w e e n Nicotiana tabacum and N. rustica. The protein has been purified approx. 90-fold with a recovery of approx. 15% o f t h e activity. By polyacrylamide gel electrophoresis, the molecular weight of EF-TUchl appears to be approx. 45 000. Similar values were found for the EF-Tu isolated from chloroplasts of the parent species. EF-TUchl from the Nicotiana hybrid was immunoprecipitated b y an antiserum raised against t h e spinach protein. Key words: Nicotiana -- Somatic hybrid -- Chloroplast -- Chloroplast elonga-
tion factor Tu INTRODUCTION
Chloroplasts contain soluble protein factors responsible for peptide chain elongation [1]. Such factors have been extensively purified from spinach chloroplasts [2] and partially characterized from intact cells o f Euglena gracilis [3,4]. To o u r knowledge no data are available in t h e case of other plants. We wish to report the procedure for the purification and t h e characterization of EF-TUcb1 present in chloroplasts of a somatic hybrid of Nicotiana as well as the t w o parents, N. tabacurn and N. rustica.
*Permanent address: Institute of Genetics, Academia Sinica, Bejing, China. Abbreviations: EF-Tueh1, chloroplast elongation factor Tu; PAGE, polyacrylamide gel electrophoresis. 0304-4211/83/$03.00
© 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
288 MATERIALS AND METHODS The somatic hybrid between N. tabacurn and N. rustica was prepared as reported [5]. The seeds of the F2 hybrid and of the parents were germinated and grown in a greenhouse. Leaves were harvested from 2-month-old plants. Chloroplasts, chloroplast extracts, and the ammonium sulfate fractions were prepared from leaves (approx. 2 kg, fresh wt.) following the procedure developed for spinach [6] by utilizing the buffers described by Rhodes et al. [ 7 ]. Preliminary experiments indicate$i that purification of tobacco EF-Tuchl by affinity chromatography on GDP-Sepharose using the rapid procedure developed for spinach chloroplasts [8 ] was unsatisfactory. Low yields of EF-TUchl and only partial purification of the protein were achieved following this procedure. It is possible that the presence of pigments in the ammonium sulfate fraction from tobacco chloroplasts interfered with the binding of tobacco EF-TUchl to GDP-Sepharose and/or its elution from the column. Therefore the ammonium sulfate fraction prepared as in [6] was adsorbed onto a 95 × 2.5 cm column packed with Sephadex G-100 in a buffer containing 50 mM Tris (pH 8), 10 mM Mg C12, 0.35 M NaC1, 1 mM dithiothreitol, 0.1 mM phenylmethylsulphonyl fluoride and 50 ~g/ml of soybean trypsin inhibitor. After adsorption of the sample, the same buffer was passed through the column at a flow rate of approx. 20 ml/h. The fractions containing EF-TUchl activity were localized by assaying their capacity to bind [H3]GDP (see later), pooled and precipitated with solid ammonium sulfate to give a 75% saturation at 0 °. After standing overnight in the cold, the protein precipitation was recovered by centrifuging (17 000 × g for 30 min) and dissolved in a small volume {less than 1 ml) of the above reported buffer. Adsorption and elution from GDPSepharose was performed as reported i~ the case of spinach [8]. Binding of [H3]GDP tO EF-TUchl, exchange of [H3]GDP with the GDP bound to EF-TUchl, separation of proteins by polyacrylamide gel electrophoresis {PAGE), and protein determination were performed as reported [6]. Escherichia coli EF-Tu and kirromycin were the generous gift of Dr. G. Chinali. The preparation and characterization of the antiserum against spinach EF-TUchl have been already described [9]. RESULTS AND DISCUSSION As depicted in Fig. 1, chromatography of the partially purified preparations on a Sephadex column not only allowed the removal of pigments {data not reported) but also of the majority of proteins including most of the ribulose-l,5-bisphosphate carboxylase. After this step, EF-TUcbl could be purified by affinity chromatography (Fig. 2). As outlined in Table I, the procedure allows a purification of approx. 90-fold with a recovery of approx. 15% of the activity present in the crude extracts. Confirmation that the activity recovered from GDP-Sepharose represents
289
o
v a Z
.x,
loj FRACTION NUMBER Fig. 1. Partial purification o f EF-TUchl o n Sephadex G-100. A p p r o x i m a t e l y 100 units of EF-TUch 1 ( 6 4 0 mg o f protein) were separated on a c o l u m n of Sephadex G-100 as described in Materials and Methods. Fractions o f 9 ml were collected. EF-TUch 1 activity was measured on 10-ul aliquots from the c o l u m n b y assaying the binding o f [H 3 ]GDP, (x- - - x). Protein was assayed spectrophotometrically, (* • ).
indeed EF-Tu was obtained by immunology since the purified preparation, as well as crude extracts from tobacco chloroplasts, were immunoprecipitated by a monospecific antiserum raised against spinach EF-TUch 1 (Fig. 3). Finally, binding of [H3]GDP to the purified EF-TUch 1 • GDP complex was
;~
~2~
2.
o x u 0 j Z
.1
I
I ! !
',
Z
o
\,~ \ k- ....
"%
FRACTION NUMBER Fig. 2. Purification o f EF-TUch 1 o n GDP-Sephamse. A p p r o x i m a t e l y 22 units o f partially purified EF-Tuch I (approx. 30 mg of protein) in 1 ml were m i x e d w i t h 0.6 ml of GDPSepharose and treated as described [8 ]. F o l l l o w i n g dialysis overnight, the GDP-Sepharose was packed in a small c o l u m n and w a s h e d w i t h approx. 150 ml o f the buffer described in Materials and Methods until the absorbation at 280 n m of the eluate reached baseline. One bed v o l u m e o f the same buffer containing 0.1 mM GDP then was added to the c o l u m n and, after adsorption, the f l o w was stopped for 1 h. The c o l u m n was then eluted w i t h 5 ml o f GDP containing buffer collecting 0 . 3 - m l fractions. EF-Tu activity o n 10-ul aliquots (x - - - x) and protein concentration (e o) were determined as reported in Fig. 1. As discussed in the text, protein could not be determined spectrophotometrically in the EF-Tu containing fractions~
290 TABLE I SUMMARY O F P U R I F I C A T I O N O F EF-TUch I F R O M NICOTIANA
THE SOMATIC HYBRID OF
One unit was defined as the amount of enzyme that catalyzes the binding of 1 × 10' c.p.m, of [HS]GDP (spec. act. 13 Ci/mmol)in 20 rain at 30°C. The values reported for fraction 4 are only estimates since protein could not be determined spectrophotometrically for the interference of the GDP present in the elution buffer. The amount of protein in each fraction before pooling was assessed from the intensity of the stained band after PAGE in comparison with the bands obtained by running known amounts of bovine serum albumin. Units of EF-TuchI activity were calculated taking in account the dilution in the assay mixture of the labelled GDP brought about by the unlabelled GDP present in the elution buffer. Fraction
Vol. (ml)
(1) Chloroplast extract 100 (2) Ammonium sulfate precipitate (from 1) 7.75 (3) Ammonium sulfate precipitate after chromatography on Sephadex G-100 1 (4) Pooled fractions after affinity chromatography on GDPSepharose 1.26
Total protein (rag)
Total act. (units)
Spec. act. (units/mg of protein)
Yield (%)
Purification (fold)
640
66
0.103
--
489
65
0.133
98
1.3
29.9
21
0.70
32
6.8
1.7
16
9.41
24
--
91
s t i m u l a t e d b y •~irromycin, an a n t i b i o t i c k n o w n t o p r o m o t e t h e e x c h a n g e b e t w e e n free G~)P and t h e GDP b o u n d in t h e E F - T u • GDP c o m p l e x [ 1 0 ] . At c o n c e n t r a t i o n o f 1, 0.1, 0.01, and 0 . 0 0 1 m g / m l , k i r r o m y c i n s t i m u l a t e d b y 426, 237, 205 and 143%, respectively, t h e e x c h a n g e b e t w e e n labelled GDP in s o l u t i o n and t h e u n l a b e l l e d GDP present in t h e c o m p l e x w i t h EF-Tu. As j u d g e d b y P A G E , t o b a c c o EF-TUch 1 appears t o be at least 90% p u r e (Fig. 4A). Indeed, o n l y o n e f a i n t b a n d o f c o n t a m i n a t i n g p r o t e i n is p r e s e n t with t h e M r o f t h e large s u b u n i t o f r i b u l o s e - l , 5 - b i s p h o s p h a t e carboxylase. The" m o l e c u l a r weight o f t o b a c c o EF-TUch I m a y b e e s t i m a t e d t o be a p p r o x . 45 000, a value close t o t h a t r e p o r t e d f o r t h e p r o t e i n isolated f r o m spinach c h l o r o p l a s t s [2] and f r o m s o m e bacteria such as E. coil [ 1 1 ] . A similar m o b i l i t y was f o u n d f o r t h e EF-TUchl p u r i f i e d f r o m the p a r e n t a l species (Fig. 4B), T h u s EF-TUchl, a p r o t e i n m o s t likely c o d e d in c h l o r o p l a s t D N A [ 1 2 ] , c a n n o t be u t i l i z e d as a m a r k e r f o r i d e n t i f y i n g t h e origin of t h e c y t o p l a s m in s o m a t i c hybrids. Nevertheless t h e results r e p o r t e d in this investigation s h o w t h a t t h e rapid p r o c e d u r e d e v e l o p e d t o p u r i f y EF-TUch 1 f r o m spinach [8] m a y be applied with slight m o d i f i c a t i o n s also t o the p r o t e i n p r e s e n t in t h e chloroplasts o f o t h e r plants. In addition, the
291
Fig. 3. Immunological characterizatiov of EF-TUchl. Thirty ul (30 ug of protein) of an antiserum against spinach EF-TUchI [ 9 ] were put in the central well. (1) Purified spinach EF-'ruch 1 (10 ug of protein); (2) purified tobacco EF-TUchl (8 ug of protein); (3) partially purified spinach EF-TUchl (0.75 mg of protein); (4) partially purified tobacco EF-Tuch 1 (0.95 mg of protein). After appearance of the precipitin lines (24 h at room temperature), the plate was washed extensively with physiological solution and then stained with Coomassie blue.
great similarity in structure and function between prokaryotic (and chloroplastic) EF-Tu and cytoplasmic EF-1 [13] would suggest that the procedure may be adopted also to purify the eukaryotic equivalent of EF-Tu. Finally the purification protocol may be utilized to isolate and identify rapidly any radioactively-labelled EF-TUchl synthesized when isolated chloroplasts are incubated in the light in the presence of labelled amino acids (unpublished results) thus allowing to establish if, in different plants, EF-TUchl is synthesized in the chloroplast.
$
I
~'ig. 4. Separation of EF-TUch I by PAGE. Standard SDS 10% PAGE was performed. (A) Track 1, ammonium sulfate precipitate from he crude extract (0.18 mg of protein); track 2, ammonium sulfate precipitate after Sephadex chromatography (0.12 mg of protein); .rack 3, fraction non-adsorbed on GDP-Sepharose (0.12 mg of protein); track 4, purified tobacco EF-TUchI from the GDP-Sepharose Lffinity column (13 ~g of protein); track 5, molecular weight markers (from top to bottom: phosphorylase, mol. wt. 94 000; bovine eroalbumin, tool wt. 68 000; ovalbumin, mol. wt. 45 000; carbonic anhydrase, mol. wt. 30 000; soybean trypsin inhibitor, tool. wt. .4 300). (B) Track 1, purified EF-Tuch1 from N. rustica (3 ~g of protein); track 2, from N. tabacum (8 ~g of protein); track 3, from he somatic hybrid (6 ~g of protein); track 4, E. coil EF-Tu (20 ug of protein); track 5, molecular weight markers as in (A)5. In racks 1--3, besides the band of E F - T u ~ , the most prominent contaminant shows a M r corresponding to that of the large subunit of
I
S
293 ACKNOWLEDGEMENTS
This work was supported by grants from Consiglio Nazionale delle Richerche (C.N.R.). One of us (S.M.Z.) acknowledges a fellowship awarded by C.N.R. under the scientific cooperation agreement between Academia Sinica and C.N.R.. Finally, the authors are grateful to G. Di Pasquale for skillful1 technical assistance. REFERENCES 1 0. Ciferri, 0. Tiboni, M.L. Munoz-Calvo and G. Camerino, in: L. Bogorad and J.H. Weil (Eds.), Nucleic Acids and Protein Synthesis in Plants, Plenum Publ. Co., 1977, p. 155. 2 0. Tiboni, G. Di Pasquale and 0. Ciferri, Eur. J. Biochem., 92 (1978) 471. 3 C.A. Breitenberger, M.V. Moore, D.W. Russell and L.L. Spremulli, Anal. Biochem., 99 (1979) 434. 4 L.L. Spremulli, Arch. Biochem. Biophys., 214 (1982) 734. 5 P.T. Wang, J.W. Chen, S.M. Zhao and J.X. Xu, Keyue Tongbao, 27 (1982) 429. 6 0. Ciferri and 0. Tiboni, in: M. Edelman, R.B. Hallick and N.H. Chua (Eds.), Methods in Chloroplast Molecular Biology, Elsevier Biomedical Press, 1982, p. 646. 7 P.R. Rhodes, Y.S. Zhu and S.D. Kung, Mol. Gen Genetics, 182 (1981) 106. 8 0. Tiboni and 0. Ciferri, FEBS Lett., 146 (1982) 197. 9 0. Tiboni, C.J. Leaver and 0. Ciferri, Plant Sci. Lett., 17 (1980) 159. 10 W. Worner and H. Wolf; FEBS Lett., 146 (1982) 322. 11 G.R. Jacobson and J.P. Rosenbush, Nature, 261 (1976) 23. 12 0. Ciferri, 0. Tiboni and G. Di Pasquale, in: 0. Ciferri and L. Dure (Eds.), Structure and function of plant genomes, 1983, Plenum Publ. Co., 1983, in press. 13 R. Amons, W. Pluijms, K. Roobol and W. Moller, FEBS Lett., 153 (1983) 37.