companents were used: 9D3 (gpt), deprived of the enzyme 11 activity for MG bindiig and phosphorylation [“a]; 5624 (?&I, defective in the binding, but not in ~~osp~~o~~a~~~~ ofMG [.5,6]; P34621 dpM,H), lacking enzyme I and HP;, where enzyme 11 is able to bind MG witbout further pbosphorylation 171. AN the strains have the &XZ1 genetic background
ITS
Glucose, when added to bacterial cells, Bowers intracellular cyclic AMP levels due to inhibition of adenylate cyclase ac?ivity [I 1. Glucose, as well as its non-metabolizaie ana^logue methyl qjucoside is transported into Enterobacteria by means of the PEP-
Pinked phosphotransferase system 121. The MG transfer is followed by its phosphoryletion catalysed by the membrane component of the PT% the glucose specific enzyme HI, specified by the p2sG gene. In this reaction, the phosphate donor is a solubk phospho-factor 111protein, which is phosphorylated at the expense of phosphoenolpyruvate with participation of an enzyme 1 and a SAL1: protein HPr, specified by the prsl and ptsH gene, respectively. As a result, MG&phosph$e and pyruvate appear in the cell in a 1:1 ratio [3]. Therefore the MG uptake is accompa&d by: [i)
Bindihhg of subshale
by enzyme
HH;
(ii) Its tianslocation across the membrane with simultaneous phosphorylation; (iii) Equimolar formation of pyruvate. Here we report stud& on the role of each of the above-mentioned processes in the inhibition by MG of the A6 activity in intact cells of E. coli KP 2.
The following mutant strains defective in differefit Abbreviarions:
AC, ad,c;;ylatc cyclase (EC 4.6.1.1);
~6,
a-metl~yl~luco~~ranoside; ITS, pho~pphoeno~~yruvatc~epe~dent phosphotransfeerzse system (EC 2.7.3.99; CCCP, carbonykyanide 238
m-c~loropl~enylhydrazone G
171. Since the%@ substrate ATP could not enter the bacteria, we rendered the cells permeable by EDT’A treatment [S] and [31HjATP was added to the culture thus treated. After paper chromatography [9] radioactive cyclic [3H]Ai@? was determined. P-Galactosidase (EC 3.2.1.23) was induced by isopropylthio-fi-D-galactopyranosid:: over 5 min. 3. ResumllPs The results presented in table 1 indicate that MG considerably inhibits the AC! activity as well as j3-ga?actosidase synthesis in the wjld type cells. The mut .lts with different PTS lesions do not display such effects. The experiments show that for MG to exert a repressive eXect, it must botk be bound by its enzyme 11 as well as undergo transmembrane phosphorylation. Even being transported into the ~gl cell and phosphorylated in the cytoplasm, MG stti cannot repress the enzyme induction and the AC activity (table I), since it does not interact with the ‘bindmg’ site ofthe enzyme II [S]. The following experiments indicated that pyruvate generation during Me6 transport is also necessary for the inhibition of the AC activity.
Volume 103, number 2
FEBS XJx-IERS
July 1979
Table 1 effect on the adenylate cyclax
The a-methylghcoside
activivitfand
fi-galactosidase synthesis %621
CCCP W.OI mrw
P34621 o;fSl,H)
JD3 tit1
J624 @d)
0.m’)
N7.
-WCactMy (pm-I :yclic AMP.pg protein-’ .miX’, 37Qa
_
2.4 0.63
MC (0.5 mM)
0.75 0.8
l-18 1.3
-
1.52 1.48
&IS 0.62
@Calactosidase synthesis (units of activity.mg protein-* .min-‘, 37°C) b 4.53 7-21 10.85 3.25 18.38 17-37 2.18 9.26 19.31 3.84 10.33 9.37
_ MG (0.5 mM)
a The incubation mixhxe contained EDTA-treated cells in 0.12 M Tri--HC1 (PM S-C+Sj; 0.05 hf MgCf,; 1 mM cyclic AMP;.1 mM [“H]ATP. The cells were pretreated with CCCP for 3 min h me cells growing in I@9 salt medium, supplemented with 0.4% casamino acids, were induced by isopropylthio+?-D-galactopyranoside(1 m&l). CCCP and MC were added simultaneously with the inducer. Anaerobiosis was obtained by passing N, throu& the cell SUSpension 5 min before the addition of inducer
We inferred that intracellular accumulation of pyruvate and its subsequent oxidation would lead to the additional membrane energization.
The AC, changes in the presence of MB; were defined by assaying the i’4@]proline transport rates. The results obbtained (table 2) show that MC stimulates the [r?Jproline uptake by wild type cells and by the 5624 (@) mutant (which retained the
ability to phosphorylate MG); proline enter; the cell by active transport [5,6]. In the JD3 (spr) aad P34621 (ppfsI,,H) mutants, deprived of pyruvate ger. eration in the process of MG uptake, the addition of MC did not accelerate proline uptake. (30ntrol experiments revealed that lactate stimulates the amino acid transport in all the strainsused aswell (data are not shown). Similar changes in membrane potential ini7,uenced by
Table 2 Influence of cu-iethylglucoside on [ Tjproline bmol/g proteia at 37T) Strains
Initial rate (1 min)
Stimula-
accumulation
Steady-state (5 min)
tion J-621 Ws)‘ 3624 w,;r) JD3 &W) $34621 @NW) P
Stimuia-
tion
-
MG
(%)
-
MG
6)
3.11
4.85
56
4.81
6.66
39
1.64
2.49
5:
4.28
5.27
23
2.13
1.99
0
5.31
5.67
7
2.08
2.12
2
6.25
6.31
1
resuspended in M9 salt medium with chloramphenicol (SOrg/ ml). The concentiations were: MG, 0.5 mM; [‘4C]proltie, 0.013 m&g. Blanks,
WiShed Cells were
obtained with cells pretreated with 0.01 mM CCCP for 3 min, were subtracted d
239
Volwne 103, numb& 2 MC were registered using fluorescerice quenching of I -anilino8-~aph~a~en~s~~~obmate (data are not shown).
The necessity of the membrane hyperpolarization for the manifestation of MG repression is verified by the experiments denr ed in -iaXe 1. The AC in J621 cells is not affecter’ t?:- 36 in tbe presence of the uncoupler CCCP_ (Flat.. EDT&treated cells retain sufficient transmembrane potential M* [IO].) Concomitantly, P-galactosidase synthesis becomes resis-
(ii) Hypeqokatization of the membrane, pmbab8y a result of the oxidation of pyruvate, generated
as
by the PT reaction. Refferences 111 Peterkofsky, A. (1976) Adv. Cyclic Mucl. Res. 7. l-43. [2] Postma, P. IV_and Roseman, S. (I 976) Biochim.
tant to MG in the presence of CCCP or in anaerobic
Biophys. Acta 457,213-257. [3] Rornberg, H. IL.and Reeves, R. E. (1972) Biochem. J.
conditions. The discrepancy in the extent of the CCCP action upon the AC and /3-galactosidase activities
PI Curtis, S. 4. and Epstein, W. (1975) J. Bscteriol. 122,
may be the result of different treatment of the cells wed for these two enzyme assays (see footnotes for
1-wBourd, 6. I., ErBagaeva, R. S., Bolsbakova, T. N_ and
table 1). The results obtained other
authors,
denotkg
be ‘switched off’
correlate with the data
of
that ghacose repression could
by exposing bacteria to anaerobiosis
[l II-and that under these conditions the cyclic AMP levels are considerably increased [12]. The data reported suggest that changes in AC activity brought about by MG are caused by: (i) Conformational changes of the glucose enzyme I%
during the transmernbrane MG;
240
yhosphorylation
of
11$9-1P99.
Gezshanovich, V. N. (1975) Eur. J. Biochem. 53, 419-427. Mornbeg, PI. L. (1978) J. Cell Physiol. 89,54§-550.
Shulgina. M. Y_, Kalachev, J. \‘a. and Bourd, G. I. (1979) Biochemistry (En@. taansl.) 42, 1764-l 772.
Eeive, A. (1962) J. Biol. Chem. 9,2373-2380. and Hamilton, 1. R. (1972)
Khmdelwal, W. L. Biochem. Biophys. Schuldiner, S. and B4,5451--5441. Qkinaka,R. T-and 93,1644-1650.
AiCch.
151,75-84. Kabxk, H_ R. (1975) Biochemistry Dobrogosz, W. J. (1967) 9. Bacterial.
Wussefl, 2. V. and Yamazaki, H. (1978) Can. J. Microbiol. 24,629-631.
ITS
Glucose, when added to bacterial cells, Bowers intracellular cyclic AMP levels due to inhibition of adenylate cyclase ac?ivity [I 1. Glucose, as well as its non-metabolizaie ana^logue methyl qjucoside is transported into Enterobacteria by means of the PEP-
Pinked phosphotransferase system 121. The MG transfer is followed by its phosphoryletion catalysed by the membrane component of the PT% the glucose specific enzyme HI, specified by the p2sG gene. In this reaction, the phosphate donor is a solubk phospho-factor 111protein, which is phosphorylated at the expense of phosphoenolpyruvate with participation of an enzyme 1 and a SAL1: protein HPr, specified by the prsl and ptsH gene, respectively. As a result, MG&phosph$e and pyruvate appear in the cell in a 1:1 ratio [3]. Therefore the MG uptake is accompa&d by: [i)
Bindihhg of subshale
by enzyme
HH;
(ii) Its tianslocation across the membrane with simultaneous phosphorylation; (iii) Equimolar formation of pyruvate. Here we report stud& on the role of each of the above-mentioned processes in the inhibition by MG of the A6 activity in intact cells of E. coli KP 2.
The following mutant strains defective in differefit Abbreviarions:
AC, ad,c;;ylatc cyclase (EC 4.6.1.1);
~6,
a-metl~yl~luco~~ranoside; ITS, pho~pphoeno~~yruvatc~epe~dent phosphotransfeerzse system (EC 2.7.3.99; CCCP, carbonykyanide 238
m-c~loropl~enylhydrazone G
171. Since the%@ substrate ATP could not enter the bacteria, we rendered the cells permeable by EDT’A treatment [S] and [31HjATP was added to the culture thus treated. After paper chromatography [9] radioactive cyclic [3H]Ai@? was determined. P-Galactosidase (EC 3.2.1.23) was induced by isopropylthio-fi-D-galactopyranosid:: over 5 min. 3. ResumllPs The results presented in table 1 indicate that MG considerably inhibits the AC! activity as well as j3-ga?actosidase synthesis in the wjld type cells. The mut .lts with different PTS lesions do not display such effects. The experiments show that for MG to exert a repressive eXect, it must botk be bound by its enzyme 11 as well as undergo transmembrane phosphorylation. Even being transported into the ~gl cell and phosphorylated in the cytoplasm, MG stti cannot repress the enzyme induction and the AC activity (table I), since it does not interact with the ‘bindmg’ site ofthe enzyme II [S]. The following experiments indicated that pyruvate generation during Me6 transport is also necessary for the inhibition of the AC activity.
Volume 103, number 2
FEBS XJx-IERS
July 1979
Table 1 effect on the adenylate cyclax
The a-methylghcoside
activivitfand
fi-galactosidase synthesis %621
CCCP W.OI mrw
P34621 o;fSl,H)
JD3 tit1
J624 @d)
0.m’)
N7.
-WCactMy (pm-I :yclic AMP.pg protein-’ .miX’, 37Qa
_
2.4 0.63
MC (0.5 mM)
0.75 0.8
l-18 1.3
-
1.52 1.48
&IS 0.62
@Calactosidase synthesis (units of activity.mg protein-* .min-‘, 37°C) b 4.53 7-21 10.85 3.25 18.38 17-37 2.18 9.26 19.31 3.84 10.33 9.37
_ MG (0.5 mM)
a The incubation mixhxe contained EDTA-treated cells in 0.12 M Tri--HC1 (PM S-C+Sj; 0.05 hf MgCf,; 1 mM cyclic AMP;.1 mM [“H]ATP. The cells were pretreated with CCCP for 3 min h me cells growing in I@9 salt medium, supplemented with 0.4% casamino acids, were induced by isopropylthio+?-D-galactopyranoside(1 m&l). CCCP and MC were added simultaneously with the inducer. Anaerobiosis was obtained by passing N, throu& the cell SUSpension 5 min before the addition of inducer
We inferred that intracellular accumulation of pyruvate and its subsequent oxidation would lead to the additional membrane energization.
The AC, changes in the presence of MB; were defined by assaying the i’4@]proline transport rates. The results obbtained (table 2) show that MC stimulates the [r?Jproline uptake by wild type cells and by the 5624 (@) mutant (which retained the
ability to phosphorylate MG); proline enter; the cell by active transport [5,6]. In the JD3 (spr) aad P34621 (ppfsI,,H) mutants, deprived of pyruvate ger. eration in the process of MG uptake, the addition of MC did not accelerate proline uptake. (30ntrol experiments revealed that lactate stimulates the amino acid transport in all the strainsused aswell (data are not shown). Similar changes in membrane potential ini7,uenced by
Table 2 Influence of cu-iethylglucoside on [ Tjproline bmol/g proteia at 37T) Strains
Initial rate (1 min)
Stimula-
accumulation
Steady-state (5 min)
tion J-621 Ws)‘ 3624 w,;r) JD3 &W) $34621 @NW) P
Stimuia-
tion
-
MG
(%)
-
MG
6)
3.11
4.85
56
4.81
6.66
39
1.64
2.49
5:
4.28
5.27
23
2.13
1.99
0
5.31
5.67
7
2.08
2.12
2
6.25
6.31
1
resuspended in M9 salt medium with chloramphenicol (SOrg/ ml). The concentiations were: MG, 0.5 mM; [‘4C]proltie, 0.013 m&g. Blanks,
WiShed Cells were
obtained with cells pretreated with 0.01 mM CCCP for 3 min, were subtracted d
239
Volwne 103, numb& 2 MC were registered using fluorescerice quenching of I -anilino8-~aph~a~en~s~~~obmate (data are not shown).
The necessity of the membrane hyperpolarization for the manifestation of MG repression is verified by the experiments denr ed in -iaXe 1. The AC in J621 cells is not affecter’ t?:- 36 in tbe presence of the uncoupler CCCP_ (Flat.. EDT&treated cells retain sufficient transmembrane potential M* [IO].) Concomitantly, P-galactosidase synthesis becomes resis-
(ii) Hypeqokatization of the membrane, pmbab8y a result of the oxidation of pyruvate, generated
as
by the PT reaction. Refferences 111 Peterkofsky, A. (1976) Adv. Cyclic Mucl. Res. 7. l-43. [2] Postma, P. IV_and Roseman, S. (I 976) Biochim.
tant to MG in the presence of CCCP or in anaerobic
Biophys. Acta 457,213-257. [3] Rornberg, H. IL.and Reeves, R. E. (1972) Biochem. J.
conditions. The discrepancy in the extent of the CCCP action upon the AC and /3-galactosidase activities
PI Curtis, S. 4. and Epstein, W. (1975) J. Bscteriol. 122,
may be the result of different treatment of the cells wed for these two enzyme assays (see footnotes for
1-wBourd, 6. I., ErBagaeva, R. S., Bolsbakova, T. N_ and
table 1). The results obtained other
authors,
denotkg
be ‘switched off’
correlate with the data
of
that ghacose repression could
by exposing bacteria to anaerobiosis
[l II-and that under these conditions the cyclic AMP levels are considerably increased [12]. The data reported suggest that changes in AC activity brought about by MG are caused by: (i) Conformational changes of the glucose enzyme I%
during the transmernbrane MG;
240
yhosphorylation
of
11$9-1P99.
Gezshanovich, V. N. (1975) Eur. J. Biochem. 53, 419-427. Mornbeg, PI. L. (1978) J. Cell Physiol. 89,54§-550.
Shulgina. M. Y_, Kalachev, J. \‘a. and Bourd, G. I. (1979) Biochemistry (En@. taansl.) 42, 1764-l 772.
Eeive, A. (1962) J. Biol. Chem. 9,2373-2380. and Hamilton, 1. R. (1972)
Khmdelwal, W. L. Biochem. Biophys. Schuldiner, S. and B4,5451--5441. Qkinaka,R. T-and 93,1644-1650.
AiCch.
151,75-84. Kabxk, H_ R. (1975) Biochemistry Dobrogosz, W. J. (1967) 9. Bacterial.
Wussefl, 2. V. and Yamazaki, H. (1978) Can. J. Microbiol. 24,629-631.
