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The binding and release of phosphate by a protein isolated from Escherichia coli
PRELIMINARY NOTES
369
anoxic than aerobic conditions, but further comparative study with N-ethylmaleimide is now under progress. Shionogi Research Laboratory, Shionogi and Co., Ltd., Fukushima-ku, Osaka (Japan)
YOlTI TITANI YUTAKA KATSUBE
i H. NISHIMURA, M. 1ViAYAMA,Y. KOMATSU, H. KATO, N. SHIMAOKAAND Y. TANAKA, J. Antibiot., 17 (1964) 148. 2 K. R. OARNALL, L. B. TOWNSEND AND R. K. ROBINS, Proc. Natl. Acad. Sci. U.S., 57 (1967) 548. 3 Y. NAKAGA~VA, H. KANO, Y. TSUKUDA AND H. I'{OYAMA, Tetrahedron Letters, 42 (1967) 41o5. 4 S. MATSUURA, O. SHIRATORI AND K. KATAGIRI, J. Antibiot., 17 (1964) 234. 5 Y. KOMATSU AND K. TANAKA, Agr. Biol. Chem., 32 (1968) lO21. 6 g. A. BRIDGES, Nature, 188 (196o) 415. 7 B. A. BRIDGES, J. Gen. Microbiol., 26 (1961) 467 . 8 H. MOROSON AND D. N. TENNEY, Radiation Res., 36 (1968) 418.
Received July I4th , 1969 Biochim. Biophys. Acta, 192 (1969) 367-369
BBA 21 269
The binding and release of phosphate by a protein isolated from Escherichia coli Amongst the proteins released from Gram-negative bacteria by osmotic shock 1, several have been shown to bind small molecules. Thus, a sulphate-binding protein has been isolated fr::m Salmonella typhimurium ~ and amino acid- and galactosebinding proteins from Escherichia coli 3-5. These proteins appear to be located in the periplasm ~,7 and are believed to be concerned in the transport of the molecules which they bind s. In the course of our study of phosphate transport in bacteria, we found that cold shock 1 abolished phosphate uptake in E. coli under conditions where viability was only slightly affected. We searched for, and found, a phosphate-binding protein in the shock fluid, This report describes the isolation of the phosphate-binding protein and some experiments on the release of phosphate from it by E. coli membrane preparations. E. coli AB33II (Reeves Met-) was grown to early stationary phase (300 Klett) at 37 ° in 0.05 M Tris-HC1 (pH 7.5) o.i % (NH4)2SO4-o.oi % MgS04. 7 H 2 0 - o . 4 % glucose-o. 1% yeast e x t r a c t - 2 o mM methionine medium. The cells were cold-shocked by the method of NEU AND CHOU 1, and the shock fluid was concentrated by ultrafiltration using an Amicon UMIo membrane (retention I-lO 4 daltons). Phosphatebinding protein from this fluid was purified by precipitation with (NH4)2SO 4 (0.8 0. 9 satn.) and by gel filtration on Sephadex G-I5O and G-Ioo. The final product travelled as a single component in polyacrylamide gel electrophoresis9. Phosphate-binding activity was tested either by the method of PARDEE et al. TM, or of PEARLMAN et al. n. Strong binding of phosphate to phosphate-binding protein was demonstrated by both methods, with a low dissociation constant of 0. 7 #M, which is in the range of similar values found for other binding proteins s. The reaction was optimal at pH 8.5. The binding was not affected by a variety of inhibitors and was only slightly augmented by added Mg2+ (Table I). Biochim. Biophys. Acta, 192 (1969) 369-371
PRELIMINARY NOTE.';
37 °
TABLE I THE
BINDING
OF
PHOSPHATE
13Y T H E
CONCENTRATED
]'J. CI)/I" SHOCI': F L U I D
The assay mixture (l ml), containing ~.o/~M a2Pb 5 ° mM Tris HCI (pH 8.5), shock fluid (2 lug protein), o.2 g of Sephadex G 25, and chloramphenicol, 25 /~g, was shaken at 2" overnight. Binding is given as B nmoles P bound per mg protein, using tile expression l~ :: (R 2 - R1)/cz,, where R 2 and R 1 are nmoles a'-'Pt in the excluded space in the presence of phosphate-binding protein and ultrafiltrate, respectively, c is the protein concentration (mg/ml) and v is the internal volume of the Sephadex (determined experimentally). Addition, s
t~ {,molc~ 1' per m z p r , t c i , )
~olle
[
lodoacetamide, N-ethyhnaleimide, p-chloromercuribenzoate (all IO II]Sf) Azide (t raM), -.5-dinitrophenol (2 raM) EDTA (io raM) 1((IO raM) Mg 2; (Io 1~115[)
.62
I.O 3
1.00
1.63 r.64 ~.64 '-74
\\"e n e x t a t t e n l p t e d t o find a s y s t e m c a p a b l e of c a u s i n g tile r e l e a s e of t h e b o u n d p h o s p h a t e , as t b i s s t e p is e s s e n t i a l if tile b i n d i n g p r o t e i n is i n v o l v e d in p h o s p h a t e t r a n s p o r t . T o m e a s u r e t h e r e l e a s e of p h o s p h a t e f r o m p h o s p h a t e - b i n d i n g p r o t e i n , we i n c u b a t e d p h o s p h a t e - b i n d i n g p r o t e i n ( c h a r g e d w i t h a2Pi) w i t h E. c o l t m e m b r a n e p r e p a r a t i o n s a n d s u b j e c t e d t h e m i x t u r e to u l t r a f i l t r a t i o n o n U M I o m e m b r a n e s . T h e r a d i o a c t i v i t y i n t h e u l t r a f i l t r a t e ( c o m p a r e d w i t h c o n t r o l s ) g a v e t h e m e a s u r e of Pi r e l e a s e . T h e c h e m i c a l i d e n t i t y of t h e a2p i n t h e f i l t r a t e w a s t e s t e d u s i n g a i n o d i f i e d ~2 p r o c e d u r e of S u ( ; I x o AN'D MIYOSH1 la. W e f o u n d ( T a b l e I I ) t h a t , in t h e p r e s e n c e of t h e c r u d e m e m b r a n e p r e p a r a t i o n , p h o s p h a t e b o u n d by, t h e p h o s p h a t e - b i n d i n g p r o t e i n w a s r e l e a s e d . R e m o v a l of t h e n l e n l b r a n e l i p i d s a b o l i s h e d t h e r e l e a s e . It c o u l d b e a r g u e d t h a t t h i s r e l e a s e w a s d u e to p r o t e o l y t i c a c t i o n a n d f r a g m e n t a t i o n of t h e p h o s p h a t e - b i n d i n g p r o t e i n i n t o s n l a l l e r p e p t i d e s c a p a b l e of p a s s i n g t h e filter m e m b r a n e . H o w e v e r , n o s u c h p e p t i d e s w e r e f o u n d in t h e u l t r a f i l t r a t e , n e i t h e r w a s its r a d i o a c t i v i t y e x c l u d e d f r o n t t h e i n t e r n a l s p a c e of S e p h a d e x G - I o . W e t h e r e f o r e c o n c l u d e t h a t t h e T,\ HLF. II THE
RELEASE
~, C R U D E
OF a 2 p i
FROM
l 7. COil" M E M B R A N E
THE
PHOSPHATE-I~IND1NG
PROTEIN
OF
1~. ('ldl' 1N T H E P R E S E N C E
OF
PREPARATION
The membrane fraction was precipitated by r.o M (Ntt4)2S() 4 froln a crude Ii. col! lysatet'L "['he phosphate-binding protein preparation (1o Fg protein per assay combined with a 2 p t - 4 4 o o o counts/rain) was subjected to ultrafiltration after 2-min incubation in Tris HCI (pH 8.5) alone (blank), or in the presence of 5 mg (wetwt.) of the membrane fraction (intact, or acetone extracted}.
t¢eactio~ mivlztr~
Buffer, phosphate-binding protein (b) As (a) + membranes (c) As (b), but membranes extracted with 80 ~}o a q . a c e t o n e
(a)
lCiochim. B i o p h y s . A c t a , I92 (t960) 369-371
Com~ts/mi~z i~ ultrafiltrate . . . . Total Identified as P~
8 ooo 20 ooo S ooo
Neleasc, count.~/mi~l i~ c.tce.~.~ o f (a) .... Total I d e n t i f i e d as t
8 I5o I () 3 0 0
Not determined
12 0 0 0
o
[ 1 150
o
PRELIMINARY NOTES
371
interaction of the membrane and the phosphate-binding protein-Pi caused a specific release of phosphate. The protein isolated by us is reminiscent of the other binding proteins. We are also aware that GAREN AND OTSUJ115 described a protein (R2) in E. coli, which is also known s to bind phosphate and which is released by osmotic shock (A. GAREN, personal communication). The function of R 2 is unknown but it is believed to be concerned in the regulation of the alkaline phosphatase system of E. coli. However, unlike the R 2 protein, the phosphate-binding protein isolated by us is present in the same amounts in both phosphate-starved and unstarved cells. These facts, together with the observed release of phosphate from the phosphate-binding protein during its interaction with membrane fragments, suggest that the phosphate-binding protein is distinct from R~ and may be involved specifically in phosphate transport in E. coli. One of the authors (N.M.) is an Australian National University Research Scholar.
Department of Biochemistry, John Curtin School of Medical Research, Australian National University, Canberra, A.C.T. (Australia)
N . MEDVECZKY H. ROSENBERG
I H. C. N E u AND J. C ~ o o , J. Bacteriol., 94 (1967) 1934. 2 A. B. PARDEE, J. Biol. Chem., 241 (1966) 5886. 3 J. R. PIPERNO AND D. L. OXENDER, J . Biol. Chem., 241 (1966) 5732. 4 0 . H. WILSON AND J. T. HOLDEN, J. Biol. Chem., 244 (1969) 2743. 5 Y. ANRAKU, J. Biol. Chem., 243 (1968) 3116. 6 P. K. NAKANE, G. E. NICHOALDS AND D. L. OXENDER, Science, 161 (1968) 182. 7 A. B. PARDEE AND K. WATANABE, J. Bacteriol., 96 (1968) lO49. 8 A. B. PAEDEE, Science, 162 (1968) 632. 9 B. J. DAVIS, Ann. N . Y . Acad. Sci., 121 (1964) 404 . io A. B. PARDEE, L. S. PRESTIDGE, M. B. WHIPPLE AND J. DREYFUSS, J. Biol. Chem., 241 (1966) 3962. i i W. H. PEARLMAN, I. F. F. FONG AND J. H. T o u , J. Biol. Chem., 244 (1969) 1373. 12 J. M. LA NAUZE AND H. ROSENBERG, Biochim. Biophys. Acta, 165 (1968) 438. 13 Y. SUGINO AND Y. MIYOSHI, J. Biol. Chem., 239 (1964) 2360. 14 G. B. Cox, A. M. SNOSWELL AND F. GIBSON, Biochim. Biophys. Acta, 153 (1968) I. 15 A. GAREN AND N. OTSUJI, J. Mol. Biol., 8 (1964) 841.
Received July I4th, 1969 Biochim. Biophys. Acta, 192 (1969) 369-371
369
anoxic than aerobic conditions, but further comparative study with N-ethylmaleimide is now under progress. Shionogi Research Laboratory, Shionogi and Co., Ltd., Fukushima-ku, Osaka (Japan)
YOlTI TITANI YUTAKA KATSUBE
i H. NISHIMURA, M. 1ViAYAMA,Y. KOMATSU, H. KATO, N. SHIMAOKAAND Y. TANAKA, J. Antibiot., 17 (1964) 148. 2 K. R. OARNALL, L. B. TOWNSEND AND R. K. ROBINS, Proc. Natl. Acad. Sci. U.S., 57 (1967) 548. 3 Y. NAKAGA~VA, H. KANO, Y. TSUKUDA AND H. I'{OYAMA, Tetrahedron Letters, 42 (1967) 41o5. 4 S. MATSUURA, O. SHIRATORI AND K. KATAGIRI, J. Antibiot., 17 (1964) 234. 5 Y. KOMATSU AND K. TANAKA, Agr. Biol. Chem., 32 (1968) lO21. 6 g. A. BRIDGES, Nature, 188 (196o) 415. 7 B. A. BRIDGES, J. Gen. Microbiol., 26 (1961) 467 . 8 H. MOROSON AND D. N. TENNEY, Radiation Res., 36 (1968) 418.
Received July I4th , 1969 Biochim. Biophys. Acta, 192 (1969) 367-369
BBA 21 269
The binding and release of phosphate by a protein isolated from Escherichia coli Amongst the proteins released from Gram-negative bacteria by osmotic shock 1, several have been shown to bind small molecules. Thus, a sulphate-binding protein has been isolated fr::m Salmonella typhimurium ~ and amino acid- and galactosebinding proteins from Escherichia coli 3-5. These proteins appear to be located in the periplasm ~,7 and are believed to be concerned in the transport of the molecules which they bind s. In the course of our study of phosphate transport in bacteria, we found that cold shock 1 abolished phosphate uptake in E. coli under conditions where viability was only slightly affected. We searched for, and found, a phosphate-binding protein in the shock fluid, This report describes the isolation of the phosphate-binding protein and some experiments on the release of phosphate from it by E. coli membrane preparations. E. coli AB33II (Reeves Met-) was grown to early stationary phase (300 Klett) at 37 ° in 0.05 M Tris-HC1 (pH 7.5) o.i % (NH4)2SO4-o.oi % MgS04. 7 H 2 0 - o . 4 % glucose-o. 1% yeast e x t r a c t - 2 o mM methionine medium. The cells were cold-shocked by the method of NEU AND CHOU 1, and the shock fluid was concentrated by ultrafiltration using an Amicon UMIo membrane (retention I-lO 4 daltons). Phosphatebinding protein from this fluid was purified by precipitation with (NH4)2SO 4 (0.8 0. 9 satn.) and by gel filtration on Sephadex G-I5O and G-Ioo. The final product travelled as a single component in polyacrylamide gel electrophoresis9. Phosphate-binding activity was tested either by the method of PARDEE et al. TM, or of PEARLMAN et al. n. Strong binding of phosphate to phosphate-binding protein was demonstrated by both methods, with a low dissociation constant of 0. 7 #M, which is in the range of similar values found for other binding proteins s. The reaction was optimal at pH 8.5. The binding was not affected by a variety of inhibitors and was only slightly augmented by added Mg2+ (Table I). Biochim. Biophys. Acta, 192 (1969) 369-371
PRELIMINARY NOTE.';
37 °
TABLE I THE
BINDING
OF
PHOSPHATE
13Y T H E
CONCENTRATED
]'J. CI)/I" SHOCI': F L U I D
The assay mixture (l ml), containing ~.o/~M a2Pb 5 ° mM Tris HCI (pH 8.5), shock fluid (2 lug protein), o.2 g of Sephadex G 25, and chloramphenicol, 25 /~g, was shaken at 2" overnight. Binding is given as B nmoles P bound per mg protein, using tile expression l~ :: (R 2 - R1)/cz,, where R 2 and R 1 are nmoles a'-'Pt in the excluded space in the presence of phosphate-binding protein and ultrafiltrate, respectively, c is the protein concentration (mg/ml) and v is the internal volume of the Sephadex (determined experimentally). Addition, s
t~ {,molc~ 1' per m z p r , t c i , )
~olle
[
lodoacetamide, N-ethyhnaleimide, p-chloromercuribenzoate (all IO II]Sf) Azide (t raM), -.5-dinitrophenol (2 raM) EDTA (io raM) 1((IO raM) Mg 2; (Io 1~115[)
.62
I.O 3
1.00
1.63 r.64 ~.64 '-74
\\"e n e x t a t t e n l p t e d t o find a s y s t e m c a p a b l e of c a u s i n g tile r e l e a s e of t h e b o u n d p h o s p h a t e , as t b i s s t e p is e s s e n t i a l if tile b i n d i n g p r o t e i n is i n v o l v e d in p h o s p h a t e t r a n s p o r t . T o m e a s u r e t h e r e l e a s e of p h o s p h a t e f r o m p h o s p h a t e - b i n d i n g p r o t e i n , we i n c u b a t e d p h o s p h a t e - b i n d i n g p r o t e i n ( c h a r g e d w i t h a2Pi) w i t h E. c o l t m e m b r a n e p r e p a r a t i o n s a n d s u b j e c t e d t h e m i x t u r e to u l t r a f i l t r a t i o n o n U M I o m e m b r a n e s . T h e r a d i o a c t i v i t y i n t h e u l t r a f i l t r a t e ( c o m p a r e d w i t h c o n t r o l s ) g a v e t h e m e a s u r e of Pi r e l e a s e . T h e c h e m i c a l i d e n t i t y of t h e a2p i n t h e f i l t r a t e w a s t e s t e d u s i n g a i n o d i f i e d ~2 p r o c e d u r e of S u ( ; I x o AN'D MIYOSH1 la. W e f o u n d ( T a b l e I I ) t h a t , in t h e p r e s e n c e of t h e c r u d e m e m b r a n e p r e p a r a t i o n , p h o s p h a t e b o u n d by, t h e p h o s p h a t e - b i n d i n g p r o t e i n w a s r e l e a s e d . R e m o v a l of t h e n l e n l b r a n e l i p i d s a b o l i s h e d t h e r e l e a s e . It c o u l d b e a r g u e d t h a t t h i s r e l e a s e w a s d u e to p r o t e o l y t i c a c t i o n a n d f r a g m e n t a t i o n of t h e p h o s p h a t e - b i n d i n g p r o t e i n i n t o s n l a l l e r p e p t i d e s c a p a b l e of p a s s i n g t h e filter m e m b r a n e . H o w e v e r , n o s u c h p e p t i d e s w e r e f o u n d in t h e u l t r a f i l t r a t e , n e i t h e r w a s its r a d i o a c t i v i t y e x c l u d e d f r o n t t h e i n t e r n a l s p a c e of S e p h a d e x G - I o . W e t h e r e f o r e c o n c l u d e t h a t t h e T,\ HLF. II THE
RELEASE
~, C R U D E
OF a 2 p i
FROM
l 7. COil" M E M B R A N E
THE
PHOSPHATE-I~IND1NG
PROTEIN
OF
1~. ('ldl' 1N T H E P R E S E N C E
OF
PREPARATION
The membrane fraction was precipitated by r.o M (Ntt4)2S() 4 froln a crude Ii. col! lysatet'L "['he phosphate-binding protein preparation (1o Fg protein per assay combined with a 2 p t - 4 4 o o o counts/rain) was subjected to ultrafiltration after 2-min incubation in Tris HCI (pH 8.5) alone (blank), or in the presence of 5 mg (wetwt.) of the membrane fraction (intact, or acetone extracted}.
t¢eactio~ mivlztr~
Buffer, phosphate-binding protein (b) As (a) + membranes (c) As (b), but membranes extracted with 80 ~}o a q . a c e t o n e
(a)
lCiochim. B i o p h y s . A c t a , I92 (t960) 369-371
Com~ts/mi~z i~ ultrafiltrate . . . . Total Identified as P~
8 ooo 20 ooo S ooo
Neleasc, count.~/mi~l i~ c.tce.~.~ o f (a) .... Total I d e n t i f i e d as t
8 I5o I () 3 0 0
Not determined
12 0 0 0
o
[ 1 150
o
PRELIMINARY NOTES
371
interaction of the membrane and the phosphate-binding protein-Pi caused a specific release of phosphate. The protein isolated by us is reminiscent of the other binding proteins. We are also aware that GAREN AND OTSUJ115 described a protein (R2) in E. coli, which is also known s to bind phosphate and which is released by osmotic shock (A. GAREN, personal communication). The function of R 2 is unknown but it is believed to be concerned in the regulation of the alkaline phosphatase system of E. coli. However, unlike the R 2 protein, the phosphate-binding protein isolated by us is present in the same amounts in both phosphate-starved and unstarved cells. These facts, together with the observed release of phosphate from the phosphate-binding protein during its interaction with membrane fragments, suggest that the phosphate-binding protein is distinct from R~ and may be involved specifically in phosphate transport in E. coli. One of the authors (N.M.) is an Australian National University Research Scholar.
Department of Biochemistry, John Curtin School of Medical Research, Australian National University, Canberra, A.C.T. (Australia)
N . MEDVECZKY H. ROSENBERG
I H. C. N E u AND J. C ~ o o , J. Bacteriol., 94 (1967) 1934. 2 A. B. PARDEE, J. Biol. Chem., 241 (1966) 5886. 3 J. R. PIPERNO AND D. L. OXENDER, J . Biol. Chem., 241 (1966) 5732. 4 0 . H. WILSON AND J. T. HOLDEN, J. Biol. Chem., 244 (1969) 2743. 5 Y. ANRAKU, J. Biol. Chem., 243 (1968) 3116. 6 P. K. NAKANE, G. E. NICHOALDS AND D. L. OXENDER, Science, 161 (1968) 182. 7 A. B. PARDEE AND K. WATANABE, J. Bacteriol., 96 (1968) lO49. 8 A. B. PAEDEE, Science, 162 (1968) 632. 9 B. J. DAVIS, Ann. N . Y . Acad. Sci., 121 (1964) 404 . io A. B. PARDEE, L. S. PRESTIDGE, M. B. WHIPPLE AND J. DREYFUSS, J. Biol. Chem., 241 (1966) 3962. i i W. H. PEARLMAN, I. F. F. FONG AND J. H. T o u , J. Biol. Chem., 244 (1969) 1373. 12 J. M. LA NAUZE AND H. ROSENBERG, Biochim. Biophys. Acta, 165 (1968) 438. 13 Y. SUGINO AND Y. MIYOSHI, J. Biol. Chem., 239 (1964) 2360. 14 G. B. Cox, A. M. SNOSWELL AND F. GIBSON, Biochim. Biophys. Acta, 153 (1968) I. 15 A. GAREN AND N. OTSUJI, J. Mol. Biol., 8 (1964) 841.
Received July I4th, 1969 Biochim. Biophys. Acta, 192 (1969) 369-371