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A radioisotope incorporation method for studying the transport and utilization of peptides by Escherichia coli
FEM$ latttn ! (l~/?) 91-94 © CoErtt#tt Federationof FauopmnMletobiololkalSocieties Ih~ by Eimv~lNorth-ltol~amJ1NonmllcalI ~
A RADIOISOTOPE INCORPORATION METHOD F O R STUDYING THE T R A N S P O R T AND UTILIZATION O F PEPTIDES BY E $ C H E R I C H I A C O L ! J.W. PAYNE and G. BELL Del~rtm¢~ of Botany. $¢ltmce lalboratorics, Univer~ty of Durham, South Road. Durham Dill 3LE, U K.
Received22 December1976
Most microorganismsutilize peptide~ nutritionally at sotnces of amino acids for protein synthesis [ l - 4 ] . In Escherkq~ coli, which lacks extracellular peptidases, peptides are tra.,nported intact and hydrolysed intracellularly. Present ideas on the peptide permeases of E. coil derive mainly from experiments in which amino acid auxotroph$ have been grow, on defined peptides [ I - 6 ] . However, this indirect way of studying transport is of limited melulneu in that it requires long incubation periods [8-20 hJ and relatively large amounts of peptide$ to produce measurable growth resonances. "I]fispaper d~:ribea a new approach, using radioactive amino acids, that allows a rapid and sensitive study to be made of peptide transport and utilization, and in addition, provides peptide binding affmities for the transport systems. A preliminary account of some of this material has been presented
171. 20 l d m d d , and metkeds B,wterk/stm/ns used were: E. co/l K12 (X478) (thi, ieu B, met E, pro t~, put E, tip, lys A, ara, xyl, lac Z, rod, str, ton A, tsx F) and its oli$opeptide permeasc defkknt strain (opp-I) (M 1229), obtained from Dr M. laccmino |8];and E. ¢o//KI2 (ASOI3) (leu-, lys-. tlu-, thi-) and its opp- mutant [4]. ~ : P~k:l~ wereobtain4H/from Sigma (L~don) Ltd., and Cydo Cl~nd¢~ Ltd. Lysine
i~ptid~ wore .yntlun/z~l 16]. pllil~olbe (S.A. I ¢i/mm,~) and I h q k ~ : ~ (S.A. 0.2 Ci/mmol) were ptuehamd horn the Radioehamkal Centre, Amenham.
PCS solubilizer was obtained from Hopkin & Williams Ltd., and PPO (2,3-diphenyloxazole)Oom Koch Light Labs. Ltd. Other general chen~cals were of laborato~ reagent grade. Radioactive co.,~nting was done using ~ modified Bray's reagent (prepared from scintillation grade, naphthalene (I00 g), PPO (5 g), methanol (I00 ml), PCS solubilizer (20 ml) and dioxan to I litre) w~.h a Becknmn LS-2OOBliquid scintillation counter. Growth o f bacteria was at 37°C in E medium [8] supplemented with glucose (0.5%), amino acids (each 0.2 raM), thiamine (0.02 m~ml), and adenosine (0.025 mg~ml) as required. Test procedure" Lysine, and strain X478 are used a ; examples, but a similar procedure is applicable to other amino acids and double auxotrophic strains. Tile lysine concentration in the medium was lowered to 0.04 mM so that it limited growth at about 5 × 10s cells/ml (E66o = 0.4, Bausch & Lomb Spectronic 20). When this final absorbance was reached, incubation was continued for I h to ensure complete lysine depletion. The starved cells were collected by eentrifugation (approximately 8000 X g; I 0 m~n) using a bench centrifuge, washed with E medium minus all supplements, and resuspended at 37°C in double strength complete growth medium without lysine and proline to give E ~ o = 0.I. Aliquots (0.5 ml) of this suspension were added to aqueous solution~ (0.5 ml) held at 37°C and containing: a lysine peptide at various concentrations (typically 0.02 /JM-0.02 raM), and fixed concentrations of unlabelled proline (0,01 raM)and radioactive proline (0.005 mM, that is, approximately 2 X 106 cpm with [3H]countins efficiency of i 8%); controls lacked a ly~ae murce, or had free lysine at high concentration (0.1 mM). Samples were incubated for 5 min to achieve
adeq,ate protein synthesis, 20% w/v TCA (2.0 ml) was a'Jded, and after 15 mm at room temperature they "wereboiled for 15 rain. Using a filter manifold° precipitated material was collected on a Millipore filter (type HA, 0.45/JM) :rod washed th,ee times with 10% w/v TCA (30 m0 and once with water (3.0 ml). The filters were air dried and dissolved in 5 ml of scintillant for counting of radioactiv: proline incorporated into protein.
3. Results Figure I shows a double reciprocal plot for 5 man in :orporation of radioactive proline as a function of di'ysine com:enttation, in kinetic experiments, using fL~"example I ;~Mdilystne, proline incorporation i3 :teased props rtionally with time up to 20 man. The in,creep: on abscissa, corresponding to the dilysine (:(,nccntr:.tion that allows half maximal r3te of protein ~) ~thesi:~,~ves a measure of dilysine affinity for the
peptide permeate. The same value was obtained using ten times the standard proliue concentration. ~milar plots were obtained with other ~ptides using different strains and starvation conditions, and the pemtease affinities determined Ire liven lit Table 1 (each the range of 2 - 6 experiments). Parent and opp- mutants, which show eqmd growth respome to dipeptides (*x. periments I--4). had siniiiar dipeptide p e r m e ~ affinities (table 1, experiments 1--5). in contrast, the inability of opp- strains to grow on certain ollsopeptidet (expedments I - 4 ) , for example trflysine, was reflected in a lO-20,fold lower affinity (table !, experiments 13-16), although trllyune did actually enter oppmutants sufficiently fast to permet protein eynthcaa. Recent repom [13,14] that cetlain Olil;opepttdes (for example trileucine) can support growth of opp- strain: is corroborated by the similar pe,~measeaffinities found with this peptide for parent and opp- strains (table I, experiment 18, 19).
4. iYmctmion 4
m
/
x// 3 -
"T, c~ 2
/
_
/
/
o
1
-2
/
0
,.
2
I
4
i
6
I
8
I
]0
...J
]2
I/Oilysine (:one (IjM "~) Fig. 1. Iktermination (,¢ dtl~.,3tide permeaseaffinity of Escheri..llia coil KI2 (X478) for diiy$tne. Ordinate indicat~ [ 3HlProi,neincorpozatedinto proteinin $ manby t ml of ~il
suspensionwith Ee~O= 0.12.
Using the present procedure, peptide transport and utilization by E. coil can be demonstrated with s 2-mit incubation, and with less than 0.1% of the peptide required for a [rowth assay, it is important to keep the bacterial ct3ncentration relatively low, and the peptide c ncentration sufficiently ~ that it does not chanse sif,niflcantly durinl[ the incubation. Instead of starved cells, auxotropha that had been washed free of required amino acids could pomibly be used in the pr~gnt procedure. Any radioactive amino acid that is incorporated into protein without siF~nificantcatabolism can be used to monitor peptido dependent synthelds, although different amino acids wili'occur with different frequencies in the synthesize, proteins. The proteins synthesized durinl; the incu. bation will b~. only a few of the total complenwnt ma, durlns the cell cycle. Radioisotope lnootvoration resulting from protein turnover is nellliltbie ¢ompmed with the pepttde-dependent ln¢oworation. 8¢¢au~ of the intense activity of the i n t r ~ l l u h t r Imptidatgs of E. coil, peptid* hydrolysis is not rate Itmaleq~for protc !n syntheMt [9- I t ]. Thus, at low pc~ptM, centrations, tramport becomes the step that limit* the rate of protein syntluefls, allowinll the lmnnetm affinities for p0pttdcmto be mmmured; them afflnJtim
TABLE 1 [tel)tide p e l ~
affinities det~mtne6 ~)y radioisotop', incorporatio~ using amino acid auxotroph$ of Escheria coil
Sami~
Peptide
T ............. 2 3 4 5 6 7 8 9 I0 II !2 !3 14, 15 16 !7 18 19 20 21
iys-ly$" iys4y$ lyIPlys lys4ys lys4ys ku.mct ku-met met-lieu met-lcu leu4rp leudrp lyHyHys lyS,4ylPly$ lys4yf,-lys lys4ys4y$ lys-lys4ys lys-lys-ly~4y| ku-ku.ku ku-lcu4cu trp.ssp-n~t-phe Nil 2 trp-asp-met-Fh~ NH2
Strain
X478 X478 M1229 AS013 AS013 o p p X478 X478 X478 )[478 X478 X478 X478 )[478 M1229 AS013 AS013 oppX478 X478 M1229 X478 ](4"/8
(table I) a~e analosous to the limit concentrations described by ,~nes l l 2 ) . Re.~ults with the o p p - strains SUll~t that all olii;opeptid~, rather than just some [13,14), may m e more than one transport system in E. colt ahhouf, h they may have widely different affinities, We found that trilysiJte could support 8rowth o f o p p - strains when suppli~.d at I O - 2 0 times the concentration used for the parent.
Financial support for this ~ork was provided by the Science Research Counci! and the Royal Society.
Itdemmcm | I I Paym, J.W. ~ 181-244,
Gtlvaql, C. (1971) Adv. En~ymol. 35,
Amino acid used for Starvation
Incorporation
Pelfiltease affinity (x 10 -~ M)
iys lys lys lys lys lea met leu met trp leo lys lys ly~ lys lys lys lea leu met trp
leu pro pro leu l~u pro pro pro pro pro px'J prc, leu pro leu leu pro pro pro pro pro ....
7-i1 10-ll 8-10 7-10 6-11 7.-11 6-8 8-11 8-10 3-.5 3-4 1-2 2-4 15-20 1-2 h)-20 4-5 3-10 10-12 67- }25 61-90
[2] Payne, J.W. (1975) in Peptide Transport in l~rot©in Nutrition (Matthews, D.M. and Payne, J.W. eds) North-Holland and American Elsevier, pp. 284-364. 131 Payne, J.W. (19"/6) Adv. Microbial Physiol. 13, 55-113. [4] Barak, Z. and Gilvarg, C. (1975) in Biomembranes (Eisen. berg, H., Katchalski-Katzit, E. and Manson, L.A., eds), vol. 7, pp. 167-218,Plenum. 15] Gilvarg C. and IC~,tchalski, E. (1965) J. Biol. Chem., 240, 3093-3098. [6] Pay,e, J.W. (1968) J. B/ol. Chem. 243, 3395-3403. 171 Payne, J.W. (1977) In Peptide Transport and Hydrolysis, Clba Soc. Syrup. No. 50, in press. [8] De FeUce, M., Guardiola, J., Lamb.~rti, A. and laccarino, M. (1973) J. Bacteriol. 116, 751-756. 191 Payne. J.W. (1972) L Gen. Miczot,ioi. 71,267-279. [10l Slmmonds, S., Szeto, K,W. and l']:ttedck, C,G. (1976) Biochemistry 15,261-270. [1 ! ] Ikll, G. ~nd Payne, J.W. (1977) J. Gen. MicrobioL in pTets. [12l Ames. G.F (1964)/Uch. Biochem. Biophys. 104,1-18, 113] Bank. Z and GUvarI,C. (1975) J. Bacteriol 122, 1200-1207. [14J Naider0 F. and Ikck~r° J.M. (1975) J. kctertol, t22° 1208-1215.
A RADIOISOTOPE INCORPORATION METHOD F O R STUDYING THE T R A N S P O R T AND UTILIZATION O F PEPTIDES BY E $ C H E R I C H I A C O L ! J.W. PAYNE and G. BELL Del~rtm¢~ of Botany. $¢ltmce lalboratorics, Univer~ty of Durham, South Road. Durham Dill 3LE, U K.
Received22 December1976
Most microorganismsutilize peptide~ nutritionally at sotnces of amino acids for protein synthesis [ l - 4 ] . In Escherkq~ coli, which lacks extracellular peptidases, peptides are tra.,nported intact and hydrolysed intracellularly. Present ideas on the peptide permeases of E. coil derive mainly from experiments in which amino acid auxotroph$ have been grow, on defined peptides [ I - 6 ] . However, this indirect way of studying transport is of limited melulneu in that it requires long incubation periods [8-20 hJ and relatively large amounts of peptide$ to produce measurable growth resonances. "I]fispaper d~:ribea a new approach, using radioactive amino acids, that allows a rapid and sensitive study to be made of peptide transport and utilization, and in addition, provides peptide binding affmities for the transport systems. A preliminary account of some of this material has been presented
171. 20 l d m d d , and metkeds B,wterk/stm/ns used were: E. co/l K12 (X478) (thi, ieu B, met E, pro t~, put E, tip, lys A, ara, xyl, lac Z, rod, str, ton A, tsx F) and its oli$opeptide permeasc defkknt strain (opp-I) (M 1229), obtained from Dr M. laccmino |8];and E. ¢o//KI2 (ASOI3) (leu-, lys-. tlu-, thi-) and its opp- mutant [4]. ~ : P~k:l~ wereobtain4H/from Sigma (L~don) Ltd., and Cydo Cl~nd¢~ Ltd. Lysine
i~ptid~ wore .yntlun/z~l 16]. pllil~olbe (S.A. I ¢i/mm,~) and I h q k ~ : ~ (S.A. 0.2 Ci/mmol) were ptuehamd horn the Radioehamkal Centre, Amenham.
PCS solubilizer was obtained from Hopkin & Williams Ltd., and PPO (2,3-diphenyloxazole)Oom Koch Light Labs. Ltd. Other general chen~cals were of laborato~ reagent grade. Radioactive co.,~nting was done using ~ modified Bray's reagent (prepared from scintillation grade, naphthalene (I00 g), PPO (5 g), methanol (I00 ml), PCS solubilizer (20 ml) and dioxan to I litre) w~.h a Becknmn LS-2OOBliquid scintillation counter. Growth o f bacteria was at 37°C in E medium [8] supplemented with glucose (0.5%), amino acids (each 0.2 raM), thiamine (0.02 m~ml), and adenosine (0.025 mg~ml) as required. Test procedure" Lysine, and strain X478 are used a ; examples, but a similar procedure is applicable to other amino acids and double auxotrophic strains. Tile lysine concentration in the medium was lowered to 0.04 mM so that it limited growth at about 5 × 10s cells/ml (E66o = 0.4, Bausch & Lomb Spectronic 20). When this final absorbance was reached, incubation was continued for I h to ensure complete lysine depletion. The starved cells were collected by eentrifugation (approximately 8000 X g; I 0 m~n) using a bench centrifuge, washed with E medium minus all supplements, and resuspended at 37°C in double strength complete growth medium without lysine and proline to give E ~ o = 0.I. Aliquots (0.5 ml) of this suspension were added to aqueous solution~ (0.5 ml) held at 37°C and containing: a lysine peptide at various concentrations (typically 0.02 /JM-0.02 raM), and fixed concentrations of unlabelled proline (0,01 raM)and radioactive proline (0.005 mM, that is, approximately 2 X 106 cpm with [3H]countins efficiency of i 8%); controls lacked a ly~ae murce, or had free lysine at high concentration (0.1 mM). Samples were incubated for 5 min to achieve
adeq,ate protein synthesis, 20% w/v TCA (2.0 ml) was a'Jded, and after 15 mm at room temperature they "wereboiled for 15 rain. Using a filter manifold° precipitated material was collected on a Millipore filter (type HA, 0.45/JM) :rod washed th,ee times with 10% w/v TCA (30 m0 and once with water (3.0 ml). The filters were air dried and dissolved in 5 ml of scintillant for counting of radioactiv: proline incorporated into protein.
3. Results Figure I shows a double reciprocal plot for 5 man in :orporation of radioactive proline as a function of di'ysine com:enttation, in kinetic experiments, using fL~"example I ;~Mdilystne, proline incorporation i3 :teased props rtionally with time up to 20 man. The in,creep: on abscissa, corresponding to the dilysine (:(,nccntr:.tion that allows half maximal r3te of protein ~) ~thesi:~,~ves a measure of dilysine affinity for the
peptide permeate. The same value was obtained using ten times the standard proliue concentration. ~milar plots were obtained with other ~ptides using different strains and starvation conditions, and the pemtease affinities determined Ire liven lit Table 1 (each the range of 2 - 6 experiments). Parent and opp- mutants, which show eqmd growth respome to dipeptides (*x. periments I--4). had siniiiar dipeptide p e r m e ~ affinities (table 1, experiments 1--5). in contrast, the inability of opp- strains to grow on certain ollsopeptidet (expedments I - 4 ) , for example trflysine, was reflected in a lO-20,fold lower affinity (table !, experiments 13-16), although trllyune did actually enter oppmutants sufficiently fast to permet protein eynthcaa. Recent repom [13,14] that cetlain Olil;opepttdes (for example trileucine) can support growth of opp- strain: is corroborated by the similar pe,~measeaffinities found with this peptide for parent and opp- strains (table I, experiment 18, 19).
4. iYmctmion 4
m
/
x// 3 -
"T, c~ 2
/
_
/
/
o
1
-2
/
0
,.
2
I
4
i
6
I
8
I
]0
...J
]2
I/Oilysine (:one (IjM "~) Fig. 1. Iktermination (,¢ dtl~.,3tide permeaseaffinity of Escheri..llia coil KI2 (X478) for diiy$tne. Ordinate indicat~ [ 3HlProi,neincorpozatedinto proteinin $ manby t ml of ~il
suspensionwith Ee~O= 0.12.
Using the present procedure, peptide transport and utilization by E. coil can be demonstrated with s 2-mit incubation, and with less than 0.1% of the peptide required for a [rowth assay, it is important to keep the bacterial ct3ncentration relatively low, and the peptide c ncentration sufficiently ~ that it does not chanse sif,niflcantly durinl[ the incubation. Instead of starved cells, auxotropha that had been washed free of required amino acids could pomibly be used in the pr~gnt procedure. Any radioactive amino acid that is incorporated into protein without siF~nificantcatabolism can be used to monitor peptido dependent synthelds, although different amino acids wili'occur with different frequencies in the synthesize, proteins. The proteins synthesized durinl; the incu. bation will b~. only a few of the total complenwnt ma, durlns the cell cycle. Radioisotope lnootvoration resulting from protein turnover is nellliltbie ¢ompmed with the pepttde-dependent ln¢oworation. 8¢¢au~ of the intense activity of the i n t r ~ l l u h t r Imptidatgs of E. coil, peptid* hydrolysis is not rate Itmaleq~for protc !n syntheMt [9- I t ]. Thus, at low pc~ptM, centrations, tramport becomes the step that limit* the rate of protein syntluefls, allowinll the lmnnetm affinities for p0pttdcmto be mmmured; them afflnJtim
TABLE 1 [tel)tide p e l ~
affinities det~mtne6 ~)y radioisotop', incorporatio~ using amino acid auxotroph$ of Escheria coil
Sami~
Peptide
T ............. 2 3 4 5 6 7 8 9 I0 II !2 !3 14, 15 16 !7 18 19 20 21
iys-ly$" iys4y$ lyIPlys lys4ys lys4ys ku.mct ku-met met-lieu met-lcu leu4rp leudrp lyHyHys lyS,4ylPly$ lys4yf,-lys lys4ys4y$ lys-lys4ys lys-lys-ly~4y| ku-ku.ku ku-lcu4cu trp.ssp-n~t-phe Nil 2 trp-asp-met-Fh~ NH2
Strain
X478 X478 M1229 AS013 AS013 o p p X478 X478 X478 )[478 X478 X478 X478 )[478 M1229 AS013 AS013 oppX478 X478 M1229 X478 ](4"/8
(table I) a~e analosous to the limit concentrations described by ,~nes l l 2 ) . Re.~ults with the o p p - strains SUll~t that all olii;opeptid~, rather than just some [13,14), may m e more than one transport system in E. colt ahhouf, h they may have widely different affinities, We found that trilysiJte could support 8rowth o f o p p - strains when suppli~.d at I O - 2 0 times the concentration used for the parent.
Financial support for this ~ork was provided by the Science Research Counci! and the Royal Society.
Itdemmcm | I I Paym, J.W. ~ 181-244,
Gtlvaql, C. (1971) Adv. En~ymol. 35,
Amino acid used for Starvation
Incorporation
Pelfiltease affinity (x 10 -~ M)
iys lys lys lys lys lea met leu met trp leo lys lys ly~ lys lys lys lea leu met trp
leu pro pro leu l~u pro pro pro pro pro px'J prc, leu pro leu leu pro pro pro pro pro ....
7-i1 10-ll 8-10 7-10 6-11 7.-11 6-8 8-11 8-10 3-.5 3-4 1-2 2-4 15-20 1-2 h)-20 4-5 3-10 10-12 67- }25 61-90
[2] Payne, J.W. (1975) in Peptide Transport in l~rot©in Nutrition (Matthews, D.M. and Payne, J.W. eds) North-Holland and American Elsevier, pp. 284-364. 131 Payne, J.W. (19"/6) Adv. Microbial Physiol. 13, 55-113. [4] Barak, Z. and Gilvarg, C. (1975) in Biomembranes (Eisen. berg, H., Katchalski-Katzit, E. and Manson, L.A., eds), vol. 7, pp. 167-218,Plenum. 15] Gilvarg C. and IC~,tchalski, E. (1965) J. Biol. Chem., 240, 3093-3098. [6] Pay,e, J.W. (1968) J. B/ol. Chem. 243, 3395-3403. 171 Payne, J.W. (1977) In Peptide Transport and Hydrolysis, Clba Soc. Syrup. No. 50, in press. [8] De FeUce, M., Guardiola, J., Lamb.~rti, A. and laccarino, M. (1973) J. Bacteriol. 116, 751-756. 191 Payne. J.W. (1972) L Gen. Miczot,ioi. 71,267-279. [10l Slmmonds, S., Szeto, K,W. and l']:ttedck, C,G. (1976) Biochemistry 15,261-270. [1 ! ] Ikll, G. ~nd Payne, J.W. (1977) J. Gen. MicrobioL in pTets. [12l Ames. G.F (1964)/Uch. Biochem. Biophys. 104,1-18, 113] Bank. Z and GUvarI,C. (1975) J. Bacteriol 122, 1200-1207. [14J Naider0 F. and Ikck~r° J.M. (1975) J. kctertol, t22° 1208-1215.