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Competitive inhibition of 3-ketosucrose formation by D-glucose
Vol. 11, No. 1, 1963
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
COkPEl'ITIV~ INHIBITION OF 3-KFXOSUCROSE FWMATION BY D-GLUCOSEl S. F'ukui2 and R. ki. Hochster Microbiology Research Institute, Research Branch, CanadaDepartment of Agriculture, Ottawa, Ontario, Canada
Received
February
28, 1963
A new, crystalline of the crown-gall
disaccharide was isolated
tumor-inducing
organism &obacteriun
sucrose as the carbon source (Fukui, & &.., a variety
of physical,
tumefaciens grown on It was characterized
of the new keto compoundto be:
Hexopyranosyl-3-ulose-B-D-fructofuranoside be used throughout this paper).
microbiological
1963).
from culture media
by
chemical and enzymatic techniques and evidence has been
presented to prove the structure
will
recently
a-D---
(the trivial
name Vketosucros@
In a continuing
study on someof the
aspects of 3-ketosucrose formation it was found that the above
conversion from sucrose was strongly
inhibited
It is the purpose of this comunication Materials
by D- but not by L-glucose.
to describe this effect.
and Methods
whereas D-glucose and other sugars used were camaercial preparations authentic
L-glucose was kindly
donated by Drs. H. S. Isbell
(National Bureau
of Standards, Washington) and W. Z. Iiassid ard E. F. Neufeld (University California).
of
Strain 86 of Axrobacterium tumefaciens was used in this study;
it was obtained originally
from Dr. A. C. Braun, Rockefeller
Institute,
New
York. The culture
mediumof McIntire
et -- al,
zinc ions and the phosphate concentration
lcontribution
(194Z)was used with the omission d was tripled
over the values given by
No. 555.
2National Research Council of Canada Fellow in collaboration Canada Department of Agriculture, 1961-1963. 50
with the
BIOCHEMICAL
Vol. 11, No. 1, 1963
these authors. ditions
mediumdid not fa3l
Cells of Ltumefaciens
below
the cell
out on the supernatant solutions.
shaker at
count reached 5-7 x109 cells
Cells were usually removed by centrifugation
per ml.
during the
6.8
were grown on a rotary
260 C for approximately 44 hours until
carried
RESEARCH COMMUNICATIONS
Sucrose (2%) was used as carbon source and under these con-
the pH of the culture
growth period.
AND BIOPHYSICAL
and all
analyses were
The mediumgenerally
contained 13-
16 mg of 3-ketosucrose per ml when cells were grown as stated above. 3-Ketosucrose reacted readily
in the N&on
(1944) modification
micro-Somogyi method at room temperature (230 C) where 95% of mati attained after
in 2 hours.
hydrolysis
difference
Sucrose itself
mixture (in 10 ml) were as follows:
g sucrose, 2.5 ml; 1.0 x phosphate buffer
MgSO4.7ii20, 0.1 ml; & tumefaciens resting distilled
Values were then obtained as a net
samples and the unhydrolyzed controls.
Componentsof the experimental reaction 1.17 x l&
water and inhibitor
color was
was determined by the samemethod but only
in 0.1 N/H2SO4for 30 min.
between the hydrolyzed
of the
cells,
(pH 7.0),
1.0 ml; 0.1 3
30 mg wet weight in 1.0 ml;
(where used), to volume.
Temperature of reaction,
26O C; time, 60 min. (except where indicated otherwise).
Results The first
striking
effect
observed was the inhibition
by D-glucose (0.2%
in the culture medium) of the conversion of sucrose (2%) to 3-ketosucrose when sucrose was used as carbon source in the presence of growing cells. of cell growth, in media containing
The rate
sucrose compared with others containing
both sucrose and D-glucose, was almost the same (8.6 and 8.9 mg/ml, respectively, after
44 hours), but the formation of 3-ketosucrose was completely suppressed
in the presence of D-glucose (e.g., with sucrose plus D-glucose), cells
it was calculated
cells/hour D-glucose
12.6 mg/ml with sucrose alone and < 0.2 mg/mJ.
From other experiments with intact
resting
that the rate of 3-ketosucrose formation/ml/.
wet
was 1.93 when sucrose was the substrate and 0.0 when sucrose and were
used
together.
51
BIOCHEMICAL
Vol. 11, No. 1, 1963
No inhibitory
effect
that:
RESEARCH COMMUNICATIONS
could be demonstrated when L-glucose was used in
The relevant
place of D-glucose.
AND BIOPHYSICAL
data are presented in Fig. 1A.
a) the rate of 3-ketosucrose formation was linear
hours with sucrose (S) as substrate, time (S+L
- G(0)) had no effect
glucose from the start ition
b) the addition
three
c) the presence of D-
(S + D - G(O)), however, resulted in a dramatic inhib-
had been allowed to start
(1934) plot of a typical the reaction
in the first
of L-glucose at zero
at aLl on this rate,
which could be achieved also by addition
the reaction
They show
of the D-glucose 30 min. after
(S + D - G (30)).
A Lineweaver-Burk
experiment in which D-glucose was used to inhibit
showed clearly
that the effect
observed was of the competitive
type (Fig. I&>.
,yeStD-G(O)
0
120
60
180
100
-10 ”
MINUTES
KP
200 1iS
300
fig. I. A) Effects of 5.5 x lo-3 5 D- and of L-glucose on the rate of conversion of sucrose to $ketosucrose in the presence of resting cells of & tumefaciena (4). B) tineweaver-Durk plot of the above reaction (1) and in the presence (2) of 2.8~ lo-3 H, D-glucose.
A
in the absence
comparison with other carbohydrates or carbohydrate-containing
stances as potential
inhibitors
is given in Table I. 52
sub-
All data in the Table
Vol. 11, No. 1, 1963
pertain
BIOCHEMICAL
to additions
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the potential
inhibitors
at zero time, i.e.
sametime at which the substrate (sucrose) was added. II-glucose for 30 min. prior to sucrose addition
Pre-incubation
did not change the effectiveness
Also, when the concentration each raised ten fold,
of the latter
effect
by
with 2:4-
significantly.
of D-galactose and of a-methyl-D-glucoside
a small inhibitory
with
increased the inhibition
5.5 x 10-3 g D-glucose to lOO,%. On the other hand, pre-incubation dinitrophenol
at the
was
of approx. 10% was observed.
Table 1 Effects
of somecarbohydrates and of 2:4-dinitrophenol
on
the conversion of sucrose to 3-ketosucrose by & tumefaciens Concentration (molar)
Compound D-Glucose D-Glucose L-Glucose D-Galactose a-Methyl-D-glucoside Phloridzin L)-*lose D-Fructose 2-Deoxy-D-glucose 2-Deoxy-D-ribose
5.5 5.5 1.1 5.5 5.5 1.0 6.6 2.9 6.1 6.2
2:4-Dinitrophenol 2 :4-Dinitronhenol
1.0 x 10-3 1.0 x lo-3
Tnhibitionx (per cent)
x 10-3 x 10-4 x 10-2 x 10-3 x 10-3 x 10-3 x 10-2 x lCY2 x lo-3 x10-3
0 0 0 28 0 0 0 0 32 s8=
xKu results pertain to a 6O-mi.n. reaction time except the one marked * which refers to an experimental time of 120 min.
The results
described above were obtained in studies with intact
!qhen the conversion of sucrose to 3-ketosucrose was carried free extract
of A,tumefaciens
(prepared by sonic oscillation
power output of 0.9 Amp. for 10 min.) no inhibition
53
out with a cellat 10 KC at a
could be demonstrated with
5.5 x 10-3 ,K u- g1ucose or with 1.0 x lC@3 E 2:4-dinitrophenol inhibitors.
ccl1~.
as the intended
BIOCHEMICAL
Vol. 11, No. 1, 1963
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Discussion The stereospecificity
of D-glucose as a competitive
inhibitor
of the
con-
version of sucrose to 3-ketosucrose shown above to occur only with intact but not with cell-free
preparations
mechanismof the D-glucose effect the bacterial 2:4&nitrophenol
cell.
cells
gives rise to speculation on the possible
with respect to the entry of sucrose into
The similarity
of the effect
compared to the action of
used under the sameexperimental conditions reinforces
view that the probable action of these inhibitors
the
is on the entry process.
ldork is now in progress which is designed to provide an
answer
to
this import-
ant question. References Fukui, S., Hochster, R. M., Durbin, R., Grebner, 8. IL, and Feingold, D. s., Bull. Hes. Count. of Israel, 11 A 4 (Hestrin Memorid Issue), 262 (1963). Lineweaver, H. and Burk, D., J. Am. Chem. Sot. j6, McIntire, F. C., Peterson, W. H., and Riker, A. J., (1942). Nelson, H., J. Viol.
Chem. s
37.5 (1944).
54
658 (1934). J. Biol.
Chem. I.&,
491
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
COkPEl'ITIV~ INHIBITION OF 3-KFXOSUCROSE FWMATION BY D-GLUCOSEl S. F'ukui2 and R. ki. Hochster Microbiology Research Institute, Research Branch, CanadaDepartment of Agriculture, Ottawa, Ontario, Canada
Received
February
28, 1963
A new, crystalline of the crown-gall
disaccharide was isolated
tumor-inducing
organism &obacteriun
sucrose as the carbon source (Fukui, & &.., a variety
of physical,
tumefaciens grown on It was characterized
of the new keto compoundto be:
Hexopyranosyl-3-ulose-B-D-fructofuranoside be used throughout this paper).
microbiological
1963).
from culture media
by
chemical and enzymatic techniques and evidence has been
presented to prove the structure
will
recently
a-D---
(the trivial
name Vketosucros@
In a continuing
study on someof the
aspects of 3-ketosucrose formation it was found that the above
conversion from sucrose was strongly
inhibited
It is the purpose of this comunication Materials
by D- but not by L-glucose.
to describe this effect.
and Methods
whereas D-glucose and other sugars used were camaercial preparations authentic
L-glucose was kindly
donated by Drs. H. S. Isbell
(National Bureau
of Standards, Washington) and W. Z. Iiassid ard E. F. Neufeld (University California).
of
Strain 86 of Axrobacterium tumefaciens was used in this study;
it was obtained originally
from Dr. A. C. Braun, Rockefeller
Institute,
New
York. The culture
mediumof McIntire
et -- al,
zinc ions and the phosphate concentration
lcontribution
(194Z)was used with the omission d was tripled
over the values given by
No. 555.
2National Research Council of Canada Fellow in collaboration Canada Department of Agriculture, 1961-1963. 50
with the
BIOCHEMICAL
Vol. 11, No. 1, 1963
these authors. ditions
mediumdid not fa3l
Cells of Ltumefaciens
below
the cell
out on the supernatant solutions.
shaker at
count reached 5-7 x109 cells
Cells were usually removed by centrifugation
per ml.
during the
6.8
were grown on a rotary
260 C for approximately 44 hours until
carried
RESEARCH COMMUNICATIONS
Sucrose (2%) was used as carbon source and under these con-
the pH of the culture
growth period.
AND BIOPHYSICAL
and all
analyses were
The mediumgenerally
contained 13-
16 mg of 3-ketosucrose per ml when cells were grown as stated above. 3-Ketosucrose reacted readily
in the N&on
(1944) modification
micro-Somogyi method at room temperature (230 C) where 95% of mati attained after
in 2 hours.
hydrolysis
difference
Sucrose itself
mixture (in 10 ml) were as follows:
g sucrose, 2.5 ml; 1.0 x phosphate buffer
MgSO4.7ii20, 0.1 ml; & tumefaciens resting distilled
Values were then obtained as a net
samples and the unhydrolyzed controls.
Componentsof the experimental reaction 1.17 x l&
water and inhibitor
color was
was determined by the samemethod but only
in 0.1 N/H2SO4for 30 min.
between the hydrolyzed
of the
cells,
(pH 7.0),
1.0 ml; 0.1 3
30 mg wet weight in 1.0 ml;
(where used), to volume.
Temperature of reaction,
26O C; time, 60 min. (except where indicated otherwise).
Results The first
striking
effect
observed was the inhibition
by D-glucose (0.2%
in the culture medium) of the conversion of sucrose (2%) to 3-ketosucrose when sucrose was used as carbon source in the presence of growing cells. of cell growth, in media containing
The rate
sucrose compared with others containing
both sucrose and D-glucose, was almost the same (8.6 and 8.9 mg/ml, respectively, after
44 hours), but the formation of 3-ketosucrose was completely suppressed
in the presence of D-glucose (e.g., with sucrose plus D-glucose), cells
it was calculated
cells/hour D-glucose
12.6 mg/ml with sucrose alone and < 0.2 mg/mJ.
From other experiments with intact
resting
that the rate of 3-ketosucrose formation/ml/.
wet
was 1.93 when sucrose was the substrate and 0.0 when sucrose and were
used
together.
51
BIOCHEMICAL
Vol. 11, No. 1, 1963
No inhibitory
effect
that:
RESEARCH COMMUNICATIONS
could be demonstrated when L-glucose was used in
The relevant
place of D-glucose.
AND BIOPHYSICAL
data are presented in Fig. 1A.
a) the rate of 3-ketosucrose formation was linear
hours with sucrose (S) as substrate, time (S+L
- G(0)) had no effect
glucose from the start ition
b) the addition
three
c) the presence of D-
(S + D - G(O)), however, resulted in a dramatic inhib-
had been allowed to start
(1934) plot of a typical the reaction
in the first
of L-glucose at zero
at aLl on this rate,
which could be achieved also by addition
the reaction
They show
of the D-glucose 30 min. after
(S + D - G (30)).
A Lineweaver-Burk
experiment in which D-glucose was used to inhibit
showed clearly
that the effect
observed was of the competitive
type (Fig. I&>.
,yeStD-G(O)
0
120
60
180
100
-10 ”
MINUTES
KP
200 1iS
300
fig. I. A) Effects of 5.5 x lo-3 5 D- and of L-glucose on the rate of conversion of sucrose to $ketosucrose in the presence of resting cells of & tumefaciena (4). B) tineweaver-Durk plot of the above reaction (1) and in the presence (2) of 2.8~ lo-3 H, D-glucose.
A
in the absence
comparison with other carbohydrates or carbohydrate-containing
stances as potential
inhibitors
is given in Table I. 52
sub-
All data in the Table
Vol. 11, No. 1, 1963
pertain
BIOCHEMICAL
to additions
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the potential
inhibitors
at zero time, i.e.
sametime at which the substrate (sucrose) was added. II-glucose for 30 min. prior to sucrose addition
Pre-incubation
did not change the effectiveness
Also, when the concentration each raised ten fold,
of the latter
effect
by
with 2:4-
significantly.
of D-galactose and of a-methyl-D-glucoside
a small inhibitory
with
increased the inhibition
5.5 x 10-3 g D-glucose to lOO,%. On the other hand, pre-incubation dinitrophenol
at the
was
of approx. 10% was observed.
Table 1 Effects
of somecarbohydrates and of 2:4-dinitrophenol
on
the conversion of sucrose to 3-ketosucrose by & tumefaciens Concentration (molar)
Compound D-Glucose D-Glucose L-Glucose D-Galactose a-Methyl-D-glucoside Phloridzin L)-*lose D-Fructose 2-Deoxy-D-glucose 2-Deoxy-D-ribose
5.5 5.5 1.1 5.5 5.5 1.0 6.6 2.9 6.1 6.2
2:4-Dinitrophenol 2 :4-Dinitronhenol
1.0 x 10-3 1.0 x lo-3
Tnhibitionx (per cent)
x 10-3 x 10-4 x 10-2 x 10-3 x 10-3 x 10-3 x 10-2 x lCY2 x lo-3 x10-3
0 0 0 28 0 0 0 0 32 s8=
xKu results pertain to a 6O-mi.n. reaction time except the one marked * which refers to an experimental time of 120 min.
The results
described above were obtained in studies with intact
!qhen the conversion of sucrose to 3-ketosucrose was carried free extract
of A,tumefaciens
(prepared by sonic oscillation
power output of 0.9 Amp. for 10 min.) no inhibition
53
out with a cellat 10 KC at a
could be demonstrated with
5.5 x 10-3 ,K u- g1ucose or with 1.0 x lC@3 E 2:4-dinitrophenol inhibitors.
ccl1~.
as the intended
BIOCHEMICAL
Vol. 11, No. 1, 1963
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Discussion The stereospecificity
of D-glucose as a competitive
inhibitor
of the
con-
version of sucrose to 3-ketosucrose shown above to occur only with intact but not with cell-free
preparations
mechanismof the D-glucose effect the bacterial 2:4&nitrophenol
cell.
cells
gives rise to speculation on the possible
with respect to the entry of sucrose into
The similarity
of the effect
compared to the action of
used under the sameexperimental conditions reinforces
view that the probable action of these inhibitors
the
is on the entry process.
ldork is now in progress which is designed to provide an
answer
to
this import-
ant question. References Fukui, S., Hochster, R. M., Durbin, R., Grebner, 8. IL, and Feingold, D. s., Bull. Hes. Count. of Israel, 11 A 4 (Hestrin Memorid Issue), 262 (1963). Lineweaver, H. and Burk, D., J. Am. Chem. Sot. j6, McIntire, F. C., Peterson, W. H., and Riker, A. J., (1942). Nelson, H., J. Viol.
Chem. s
37.5 (1944).
54
658 (1934). J. Biol.
Chem. I.&,
491