Published October 1, 1968
Stimulation of the LabeUar
Sugar Receptor of the
Fleshfly by Mono- and Disaccharides
H I R O M I C H I M O R I T A and AKIO SHIRAISHI
From the Department of Biology,Faculty of Science, Kyushu University,Fukuoka,Japan.
Dr. Shiraishi's present address is Willis H. Booth Computing Center, California Institute of
Technology,Pasadena, California91109
INTRODUCTION
Among the taste receptors, the sugar receptor has the unique property t h a t
it is stimulated by uncharged molecules. This is quite remarkable, since almost all types of structures on the cell m e m b r a n e (muscle end plates, neuron
synapses, etc., as well as salt taste receptors) are stimulated only by charged
molecules or ions. H o w can uncharged molecules produce electrical changes
in t h e receptor membrane? This is one of the most interesting problems in
m e m b r a n e physiology. One approach to this problem is to study the relative
stimulating effectiveness of different sugars and to evaluate the relation between the molecular structure of the sugar and its stimulating effectiveness.
T h e final goal of this approach would be to discover the molecular structure
of the site on the receptor m e m b r a n e with which sugar molecules combine.
In fact, m u c h work thus oriented has been done in both vertebrates and invertebrates. These studies have been based on two groups of experiments.
One estimates the threshold concentration of sugars for a definite behavioral
response, including psychophysical experiments in man. Accordingly, the
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The Journal of General Physiology
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ABSTRACT Responses of the labellar sugar receptor of the fleshfly, Boettcherisca peregrina, were studied over a wide range of concentrations of several sugars
(sucrose, maltose, glucose, fructose, and mannose) in single solutions and in
mixtures. The results suggest (a) that the receptor sites are not completely differentiated for glucose and for fructose combination, (b) that the receptor site
is composed of two subunits. Such suggestions are based on the classical model,
where the response is proportional to the number of the sites, two subunits of
each site being simultaneously occupied with one molecule of disaccharides or
two molecules of monosaccharides. It is shown, however, that an allosteric
model gives a somewhat better interpretation of the experimental results.
Published October 1, 1968
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sugars are tested at their very dilute concentrations. T h e other compares
magnitudes of neural response of the receptor to a fixed concentration of
different sugars. However, the order of effectiveness is not necessarily the
same at different concentrations. Therefore, the effectiveness must be compared by recording the response of single receptors to sugars over a wide
range of concentrations. T h a t is, we have to investigate the response magnitude of the receptor vs. the concentration of each sugar. Such a study will
give us another aspect of information; i.e., the mode of complex formation
between the receptor site and the sugar molecules.
T h e labellar sugar receptor of m a n y species of flies is the best material for
quantitative studies. First, we can easily record its sensory activities from the
sidewall of the labellar chemosensory hair (Morita, 1959). Second, it has
been ascertained in this receptor that the receptor potential can be considered
proportional to the impulse frequency (Morita and Yamashita, 1966). This
implies that we can quantitatively, though not in their absolute values, discuss the displacement of the receptor m e m b r a n e potential and the receptor
m e m b r a n e current by measuring the impulse frequency, since they are also
proportional to the recorded receptor potential. Third, there is a phase which
can be regarded as stationary in the sensory adaptation curve of the impulse
(in the blowfly, Steinhardt, Morita, and Hodgson, 1966). We can deal with
the response magnitude in this phase in the same way as with the rate of
enzyme reaction, where a steady state is assumed in the process of formation
of the enzyme-substrate complex. Last, but not least, we can obtain quantitatively reproducible responses in this receptor if the stimulus duration is
kept below 0.5 sec (Steinhardt et al., 1966). This method of short stimulation
was introduced by Evans and Mellon, and was applied successfully by t h e m
to the labellar water receptor of the blowfly (1962 a) and to the salt receptor
(1962 b).
Morita, Hidaka, and Shiraishi (1966) showed that the results obtained in
the sugar receptor of the fleshfly could be explained by assuming that the
response magnitude is proportional to the n u m b e r of the receptor sites, each
of which is occupied by one molecule of sucrose. Such an assumption is the
basis of Beidler's taste theory (Beidler, 1954), and was found to hold for the
salt receptor of the rat (Beidler, 1954) and the labellar salt receptor (Evans
and Mellon, 1962 b; but see Gillary, 1966). As to the receptor site, Evans
(1963) has claimed that there are at least two different types in a single sugar
receptor of the blowfly, one being the glucose-combining site and the other
the fructose-combining one.
In the present work we have tried to clarify the properties of the receptor
site of the labe]lar sugar receptor of the fleshfly, investigating the responses
to solutions of sugars and mixtures of different sugars over wide ranges of
concentration.
Published October 1, 1968
H. MOR~TA ANn A. SHIRAISm Mono- and Disaccharides and Sugar Receptor
MATERIAL
~6 t v" Y.
AND METHODS
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T h e fleshfly, Boettcherisca peregrina, was used throughout this work. T h e larvae were
raised on minced horse meat, and the imagos were raised in the same way, but fed
also with 5 % sucrose solution. Imagos between 3 and 6 days old were used in the
experiments.
T h e recording and stimulation systems were almost the same as described elsewhere (Morita, 1959). An isolated proboscis was mounted on a piece of platinum
wire which was inserted into the proboscis through the cut end and served as an
indifferent electrode. A long hair on the marginal zone of the labeUum was selected,
and the sidewall of the hair was cracked with a microneedle about 50 # from the tip
by supporting the hair with the tip (about 15 p in diameter) of a capillary electrode
on the opposite side. Then, the cracking needle was replaced with a second capillary
with a tip diameter of about 30 p, its tip having been previously dipped briefly into the
same electrolyte solution as that in the capillary electrode. When the tip of the
capillary electrode was brought into contact with the surface of the solution in the
second capillary, the solution began to move from the electrode to the second
capillary. Thus, the solution near ~.he surface was renewed continuously and condensation by evaporation at the electrode tip was prevented. Sensory activity was
recorded from the cracked part of the hair which was kept in contact with this continuously renewed surface, and the receptor responded at least for 2 hr in a quantitatively reproducible manner unless the receptor was injured during the cracking
procedure. T h e electrolyte solution used for the capillary electrode was Waterhouse's
saline (Buck, 1953).
Stimuli contained in a third capillary whose tip diameter was 50-100 p were applied to the receptor at the hair tip. Movement of this capillary was controlled by a
small electromagnet, which was supplied with electric current by an electronic
stimulator. T h e duration of stimulation never exceeded 0.5 see. Intervals between
stimuli were adjusted with various stimulus strengths. For example, in sucrose stimulation, the intervals were 1.5, 3, and 5 min after stimulation by solutions below 0.1,
0.1-0.2, and above 0.2 M, respectively. All experiments were done at ambient temperatures of 25°C 4- 0.2°C, and at relative humidities of 60-70 %. T h e sensory impulses picked up from the cracked part on the sidewall were fed into an oscilloscope
through an amplifier with grid leak of 10u ohm and of low grid current (below 10-12
amp) in its head stage. T h e y were then photographed on running oscillographic paper,
and the impulses were counted for 0.15-0.3 see from 0.15 see after the beginning of
the stimulus, as a measure of the magnitude of the stationary response. I n the present
paper this value will be referred to as the magnitude of response.
All sugars used were of special grade of Wako Chemical Industries, Ltd., J a p a n
(D-form for monosaeeharides), except for D-fructose, which was made by the British
Drug Houses, Ltd., England. T h e specification attached to the fructose sample showed
that the specific rotation [a]~° was --89 to --92 and that contamination from Dglucose was less than 1%.
The sugars were dissolved in distilled and deionized water for experimental use.
Unless otherwise stated, concentrations of sugars are expressed in molarity. This was
Published October 1, 1968
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for convenience in preparing solutions over a wide range of concentrations. We plotted concentrations principally on logarithmic scales, so that the difference between
molarity and molality (and thermodynamic activity, too) is relatively small.
RESULTS
Responses to Single Sugars
C O M P A R I S O N O F SUCROSE= G L U C O S E , A N D F R U C T O S E
Fig. 1 shows one of
the examples in which the responses of a single sugar receptor to sucrose,
5
i
20
x
x2°
.---~:2.~
. . . . . . . . . .
19
bJ
O~
_j
B
a.
-
.,--=il,/-/
o GLucosE
14.
0
it=
0
0,5
MOLAR
1.0
CONCENTRATION
FmuPa~ I. Comparison of response-concentration relation in stimulations by sucrose,
glucose, and fructose. Number attached to each symbol indicates the order of stimulation.
glucose, and fructose were studied over a range of concentration between
0.01 and 1.0 M. T h e numbers attached to the circles represent the order
of stimulation. As the numbers show, stimulations were given in ascending
order as to the concentration of the sugar used. T h e receptor was stimulated
by 0.2 M sucrose at times to check the reproducibility of the magnitude of the
response. Such procedures were routine for other experiments in the present
work.
T h e results of Fig. 1 show that the order of stimulating effectiveness was
sucrose > fructose > glucose below 0.3 ~, but that it changed to sucrose >
glucose > fructose above 0.3 M. Sucrose was the most effective at all concentrations and in all preparations. Between fructose and glucose the concentration at which the order changed varied with the preparation, ranging
from 0.1 to 0.4 M, but the reversal was observed in all preparations.
Table I shows a comparison of the responses to sucrose, glucose, and fruc-
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lal
Published October 1, 1968
H . MORITA AND A. SmRAISHI Mono- and Disaccharides and Sugar Receptor
563
tose at their m a x i m u m values experimentally obtained at high concentrations. These values would not be the same as the m a x i m u m response each
sugar could produce. T h e values were obtained, however, from a series of
experiments, in which the concentration range extended above 1.0 M and the
response-concentration curve showed a practically horizontal line at high concentrations. Therefore, as rough estimations of the m a x i m u m response, relative
to that to sucrose, we m a y take averages of 0.71 and 0.46 in responses to glucose and fructose, respectively.
Hassett, Dethier, and Gans (1950) studied the relative sensitivity of the
blowfly to the three sugars, comparing individual ascending tarsal thresholds
to the sugars for behavioral response. Their results indicated that fructose
TABLE
I
C O M P A R I S O N OF M A X I M A L R E S P O N S E S T O
S U C R O S E , G L U C O S E , AND F R U C T O S E
Sucrose
Glucose
Fructose
0.26
0.73
0.33
0.4
0.52
0.56
0.45
0.4O
0.53
275
274
273
218
216
215
214
213
208
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.78
0.73
Average
1.0
0.71
0.69
0.46
was the most effective (twice as effective as sucrose), sucrose next, and glucose
least. T h e y used different experimental methods, species of fly, and receptor
locations, b u t their results correspond to ours at low stimulus concentrations.
Discrepancy between the two works is obvious when the relative sensitivities
to sucrose and fructose are under consideration. W e considered impurity in
our fructose as one of the causes for the discrepancy, and used D-fructose
(extra pure for injection) made by E. Merck (Germany) and found no difference in the results for the two fructose samples. However, there is also the
possibility of the same sort of impurity existing in the fructose m a d e b y E.
Merck.
TENTATIVE MODEL For the case in which the response magnitude is
proportional to the n u m b e r of sties, each of which is occupied by one stimulus
molecule, we can use an equation similar to the Michaelis-Menten equation
which describes enzyme reactions. Lineweaver and Burk (1934) modified
this equation and introduced two types of plots, giving straight-line relation-
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preparation
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ships. Fig. 2 corresponds to the second type of the Lineweaver-Burk plot, and
is the same as that which Beidler (1954) first applied to the chemoreceptor.
A straight-line relationship was obtained for sucrose, as observed by Morita
et al. (1966), but not for glucose. This means that the response to glucose is
not proportional to the n u m b e r of 1 : 1 complexes as formed between the glucose
molecule and the receptor site. We, therefore, examined a model of a 2:1
I
•
GLUCOSE
.
X
0
0.5
MOLAL
1.0
CONCENTRATION ( C |
FIGURE2. Beidler's (or Lineweaver-Burk) plot in stimulations by glucose and sucrose.
Deviation from a straight line in stimulation by glucose shows that the response cannot
be described as Beidler's (or the Michaelis-Menten) type.
complex
simplest
When
into two
formation between stimulus molecules and the receptor site as the
after that of a 1 : 1 complex.
the 2:1 complex is formed, the reaction m a y generally be divided
steps as
,,t + S ~ - A S
(1)
A --b AS.-~-A2S,
(2)
and
where A, ,.7, AS, and A~S represent a molecule of stimulus substance, the receptor site, and the 1 : 1 and 2 : 1 complexes, respectively. Allowing a, nl , and
n2 to be the concentration or number of A, AS, and A~S, respectively, and s the
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.
Published October 1, 1968
H. MORITAAND A. Sr~RAISm Mono-and Disaccharidesand SugarReceptor
565
sum of the numbers of S, AS, and A2S, we m a y assume the existence of constants,/(1 and/(2 (not necessarily equal to dissociation constants), in a steady
state, as
K1 = a ( s - n x - n2)/nl,
(3)
K~ = anx/n2.
(4)
and
If the magnitude of response, r, is proportional to the number of the complexes,
A2S, as formed, the following equation is obtained:
r/r., = 1/(i + XUa + XllC2/a~),
(5)
r/r,• = 1/(1 q- a/c q- ct/c~).
(6)
This equation shows that the curve representing the value of r/r,, plotted
against c is determined b y the value of a; that is, the curves representing
equation (5) with the same value of 1£2/1£1 are the same in shape when plotted
against a on a logarithmic scale, even if the values of K1 are different. Thus,
the test of the experimental results with equation (5) is facilitated to a great
extent.
Disaccharides Before going into the results with monosaccharides, let us
describe the results with sucrose, when the 1:1 complex model was considered to hold as shown b y Fig. 2. In this case, the response is proportional
to nx, and n2 is zero because K2 = co. Accordingly, the relative response is
expressed as
r/r,, = 1/(1 .-[--K1/a).
(7)
T h e theoretical curve calculated from equation (7) is compared in Fig. 3
with experimental values obtained in a single sugar receptor. In this and the
two following figures, the experimental values were reduced by 2 - 5 % to get
the best fit at high sugar concentrations.
As seen in Fig. 3, the theoretical curve fits the experimental values fairly
well over the range 0.001 to 1.5 M sucrose. Slight deviation from the curve is
seen at concentrations from 0.003 to 0.03 M, and m a y be significant as shown
also in Fig. 4 by plots of results with six preparations. T h e value of the
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where r,, is the maximal response resulting from all the sites being occupied
b y two molecules of the stimulant, so that the value of r/r,~ m a y be called
the relative response. Introducing the "relative concentration," c = a/K~,
and a constant, a = K,./Kx, we can rewrite equation (5) as
Published October 1, 1968
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c o n s t a n t , K1, v a r i e d w i t h p r e p a r a t i o n s , b u t , if the response is expressed b y e q u a tion (7), all e x p e r i m e n t a l values should b e r e p r e s e n t e d b y a single c u r v e plotted a g a i n s t t h e r e l a t i v e c o n c e n t r a t i o n , e v e n w i t h different KI values in differe n t p r e p a r a t i o n s . As Fig. 4 shows, t h e results of six different p r e p a r a t i o n s
(filled circles) c a n b e t h o u g h t of as expressed b y a single t h e o r e t i c a l curve,
1.0
W
El
Z
0
fit.
El
UJ
r¢
•
I
'
'
I ....
I
I
i
'
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,
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~
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,
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"
0.5
hi
>
_
~
0.001
,
~
o:Y
1
0.002
I
0.005
0.01
0,02
,
,
KI
II,.,,J
0.05
0.1
0.2
0.5
i
1,0
2,0
MOLAR CONCENTRATION OF SUCROSE
FIGUR~ 3. Responses relative to the maximum vs. concentration of sucrose plotted
on a logarithmic scale (in a single receptor). Open circles represent experimental values
and the continuous line represents the theoretical curve calculated by the equation
(response of Beidler's or Michaelis-Menten type) :
r/r~ = 1/(1 + Kx/C)
where r is the magnitude of response; rm, the maximum response when all the sites are
occupied each by one molecule of sucrose; K1, a constant corresponding to the dissociation constant of a 1 : 1 complex between the site and sucrose molecule; C, molar concentration of sucrose. The maximum responses experimentally obtained are estimated
as 95% of the true maximum which the receptor could reach, so that the best fit is obtained at high concentrations of sucrose.
b u t , h e r e also, d e v i a t i o n f r o m t h e c u r v e is o b v i o u s o v e r the r a n g e f r o m 0.05
to 0.5 in the relative c o n c e n t r a t i o n .
Fig. 5 shows t h e results w i t h m a l t o s e chosen as a n o t h e r d i s a c c h a r i d e a n d
t r e a t e d in the s a m e w a y as in Fig. 4. Fig. 5 includes t h e results w i t h five different p r e p a r a t i o n s . T h e results w i t h the i n d i v i d u a l p r e p a r a t i o n s w e r e f o u n d
to b e expressed b y t h e t h e o r e t i c a l curves c a l c u l a t e d f r o m e q u a t i o n (7), a n d
t h e r e w a s n o t the d e v i a t i o n seen w i t h sucrose.
I n t h e results s h o w n in Figs. 4 a n d 5, the v a l u e of Kx for sucrose was 0.06 M,
r a n g i n g f r o m 0.05 to 0.14 M (with six p r e p a r a t i o n s ) ; t h a t for m a l t o s e was
0.1 g, r a n g i n g f r o m 0.05 to 0.15 M (with 5 p r e p a r a t i o n s ) (see T a b l e I I ) .
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,J
ttl
tic
Published October 1, 1968
H. MOIRITA AND A. SI-IIRAISttI Mono- and Disaccharidesand Sugar Receptor
1.0
I
' ' I .... I
I
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I,. 'i''1
....
*'.. I.•
567
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RELATIVE
'I
0.5
I
CONCENTRATION
I
, , [iiirl
2
5
OF
I
tO
20
SUCROSE
Monosaccharides Figs. 6 and 7 show tests of the results with fructose and
glucose, respectively, by equation (6). As seen in these figures, the experimental values (filled circles) are in good agreement with the theoretical
curves (continuous curves) of the 2:1 complex model. Fig. 6 includes the
results with nine different preparations, and the value of a (= K~/K1) for
f,o
"
•
•
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e•
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0.
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receptors,
eoe
I
0.5
, , lil*,l
0.5
i
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2
,
, I ....
5
i
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20
CONCENTRATION OF MALTOSE
T h e same as in Fig. 4, but in stimulation by maltose, with five different
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Fmum~ 4. T h e same type of test as in Fig. 3, but with experimental values (filled circles) in six different receptors. Relative concentration is C/Kx, where C is molar concentration. Continuous line represents the theoretical curve calculated with the same equation used in Fig. 3. Experimental values were reduced as in Fig. 3.
Published October 1, 1968
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the theoretical curve was 4. Fitting the theoretical curve with the experimental values obtained from one preparation, the value of/(1 for this preparation was determined by reading the concentration corresponding to unity in
the relative concentration. T h e values of K1 thus obtained for individual
preparations ranged from 0.014 to 0.095 M and were averaged as 0.015 M.
Accordingly, the average of K2 values was 0.06 ~ ( K 2 / K ~ = a = 4). Similarly, for glucose (Fig. 7) tested on 10 different preparations, a was unity,
and the averaged value of KI was 0.1 M, ranging from 0.08 to 0.12 M.
1.0
',
....
i
,
I
....
i
~ *
LU
(D
Z
•
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oe~e
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• o•
•
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0.5
o
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0.5
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~ I ,,,,I
2
5
10
I
20
,
, I .... I
50
100
RELATIVE CONCENTRATION OF FRUCTOSE
FmURE 6. Comparison of experimental results (filled circles) in nine receptors stimulated by fructose with the theoretical curve (continuous line) calculated from the equation:
r/r,,
= 1/(1 +
a / c --k ot/c2),
where c = C / K 1 (relative concentration), a t = K ~ / K 1 ) = 4.0 (see equations 3 and 4
in the text as to K1 and K s ) , t h e others the same as in Fig. 3.
T h e r e was a distinct difference between the results with mono- and disaccharides. While the results with disaccharides with the individual preparations were all expressed by the same theoretical curve plotted against the
relative concentration, those with monosaccharides were not. This is because
the value of a varied with preparations. Nevertheless, the results with different preparations were on the whole expressed by a single theoretical curve
for each sugar as shown in Figs. 6 and 7. This means that there was a mean
in the value of a for each monosaccharide, and this value varied r a n d o m l y
about the mean with different preparations.
In T a b l e II are summarized the values of K1 a n d / f 2 estimated as above.
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D(/)
hi
IV
Published October 1, 1968
Mono- and Disaccharidesand Sugar Receptor
H. MORITA AND A. SmRAISm
569
T h e table shows that fructose is the most effective in the sense that it has the
highest affinity for the receptor site.
Interactions between Different Sugars
It has been shown above that stimulations b y disaccharides and b y monosaccharides are explained by the 1 : 1 and 2 : 1 complex models, respectively.
1.0
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RELATIVE
_~
",:1,,,,I
0.5
I
I
,
2
,
I ,,,,f
5
CONCENTRATION
I
I0
20
F t o u a s 7. T h e same as in
Fig. 6, but with 10 receptors
stimulated by glucose. T h e
value of a used for calculation
of the theoretical curve is 1.0.
OF GLUCOSE
TABLE
II
A P P A R E N T K1, Ks V A L U E S F O R T H E S U G A R S
TO COMPLEX WITH THE RECEPTOR SITE
Sugars
KI
Range of KI
Sucrose
0.06
0.05-0.14
Maltose
0.1
0.015
0.1
0.05-0.15
0.014-0.025
0.08-0.12
M
Fructose
Glucose
K~
M
0.06
0.1
For the meaning of Kt and K2, see equations (3) a n d (4) in the text.
T h e structure or nature of the receptor site will be described below while the
interaction between different sugars stimulating the same receptor is also
investigated.
"GLUCOSE AND FRUCTOSECOMBININGSITES" Evans (1963) has postulated
that there are at least two types of combining sites on the m e m b r a n e of one
sugar receptor, one for glucose and the other for fructose. H e has also claimed
that sucrose acts predominantly at the "fructose site" (Evans, 1961). If these
sites are strictly specific for each substrate (i.e. sucrose combines only with the
fructose site), there would be little interaction between sucrose and glucose
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w
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w h e n the two sugars are given in the same solution. O n e of the e x p e r i m e n t a l
results is shown in Fig. 8, w h e r e the responses to plain sucrose solutions are
c o m p a r e d w i t h the responses to m i x t u r e s of 1 M glucose a n d various c o n c e n trations of sucrose. C o n c e n t r a t i o n s of sucrose in the plain a n d m i x e d solutions
are p l o t t e d on the X axis. T h e results show t h a t the response to the m i x t u r e of 1
M glucose a n d one of the various c o n c e n t r a t i o n s of sucrose could b e r e g a r d e d
as slightly additive o n l y at a low c o n c e n t r a t i o n of sucrose. T h e response to t h e
same c o n c e n t r a t i o n of sucrose was h i g h e r in the plain solution t h a n in the
30
3
24
14
13
5.10fl3
u~
~J
3
Oil
8 9
T
20
4
_ _
~
,,KI'7
6
00
hi
~O
J
I1.
F/
• iM GLUCOSE+SUCROSE
LJ
o COINCIDENCE OF o AND •
IO
I/
l/
6 r
x
2,19
19 I
I
0
I
i
I
IM GLUCOSE
I
I
I
0.5
MOLAR
CONCENTRATION
I
]
LO
OF
SUCROSE
FIGUP,~8. Effects of I Mglucose on stimulation of a single receptor by sucrose. Numbers
attached to symbols represent the order of stimulations (the same in the following
figures). The broken line shows the average of responses to 1 ~ glucose. Values on
X axis represent concentrations of sucrose both in plain solutions and in mixtures with
1 ~ glucose.
m i x t u r e at h i g h c o n c e n t r a t i o n s of sucrose. T h e r e f o r e , we c a n n o t c o n c l u d e t h a t
the sucrose m o l e c u l e c o m b i n e s only with one t y p e of site, q u i t e i n d e p e n d e n t l y
of the ,'glucose site." O n the c o n t r a r y , we will h a v e to assume fairly strong
c o m p e t i t i o n b e t w e e n sucrose a n d glucose for the same r e c e p t o r site.
A l m o s t the same e x t e n t of i n t e r a c t i o n was observed b e t w e e n sucrose a n d
fructose (Fig. 9). C o m p a r e d with the inhibition by glucose shown in Fig. 8,
t h a t b y fructose was n o stronger. T h e s e results show t h a t sucrose molecules
c o m b i n e with the glucose site as well as with the fructose site, if t h e r e is a n y
d i f f e r e n t i a t i o n a m o n g the r e c e p t o r sites.
I n t e r a c t i o n b e t w e e n glucose a n d fructose should give us i n f o r m a t i o n a b o u t
the differentiation b e t w e e n the glucose a n d fructose sites. T h e results of the
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(J
,,i
15
Published October 1, 1968
I--I. MORITAAND A. Sn~RAzsm Mono- and Disaccharidesand Sugar Receptor
57 z
25
20
rj
LU
O3
oJ
I0
~8
Ig
Xl2
I
2
=E
0
X17
0 SUCROSE ONLY
I
t
•
I M FRUCTOSE 4- SUCROSE
o
COINCIDENCE
x
I M FRUCTOSE
t
I
0
,
OF o A N D I
[
I
I
I
I
I,
1.0
0.5
FIGURE 9.
T h e same as in Fig. 8, but with interaction between fructose and sucrose.
experiment as they affect this problem are shown by Fig. 10. T h e responses
to the mixtures of 0.05 M glucose and various concentrations of fructose were
higher, apparently by an a m o u n t of the response to 0.05 ~ glucose, than those
to plain fructose solutions over the entire range of fructose concentrations
tested. Accordingly, the glucose site might be assumed to be differentiated
from the fructose site. However, the lack of significant difference between the
response to the mixtures of 1 ~ glucose with fructose (half-filled circles) and
.
ro
i,i
o3
.
.
-
/
•
IM GLUCOSE
~"
e
! M GLUCOSE + FRUCTOSE
L
/
N
(~
u)
.
14
~
-
29
16
IO
LIJ
CO
--I
3
O.
X15
//,4/v'-
0 . 0 5 M GLUCOSE + FRUCTOSE
X FRUCTOSE ONLY
&k
A O.OSM GLUCOSE
~X:
0
is
.
o
,
i
0
~
i
I
i
t
~a
A--
---A---
,
B
0.5
MOLAR
CONCENq'RATION
I
1.0
OF
FRUCTOSE
FIGum~ 10. Interaction between glucose and fructose, Concentration of glucose in
mixtures was fixed at 1.0 or 0.05 z~. Averages of responses to 1.0 and 0.05 M glucose are
represented by broken and dot and dash lines, respectively.
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MOLAR CONCENTRATION OF SUCROSE
Published October 1, 1968
572
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
• VOLUME
52
• I968
that to plain 1 M glucose (filled circles) suggests that glucose molecules can
occupy the fructose site fairly accurately, competing with fructose molecules.
This means that the differentiation is poor, if it exists at all. T h e apparent
additivity mentioned with the mixture of 0.05 M glucose and fructose m a y be
explained by assuming that the complex type, such as the fructose-glucosereceptor site, can be formed (see Fructose effects).
MANNOSE EFFECTS Mannose has been known as a unique monosaccharide. In spite of a very weak stimulating effect, it is a strong competitive
inhibitor for fructose stimulation according to the behavioral study on the
blowfly by Dethier, Evans, and Rhoades (1956). T h e results shown in Fig. 11
L i
i
, i i ' ~,,i
1.0
,
, , i, " , i
4
'
' ' (~.
8
14
W
0.5
• CONTROL(0.4M FRUCTOSE)
o 0.4M FRUCTOSE+MANNOSE /
x MANNOSE ONLY ,=,~........//=,"~/
I
0.01
,
,
,
i ,,,,,t
I
0.05 0.1
t
t
I ,,,,I
0.5
=
I.(3
,
,
t/
5.0
MOLAR CONCENTRATION OF MANNOSE
FIGURE l 1. Effectsof various concentrations of mannose on stlmu]ation by a fixed
concentration of 0.4 Mfructose, where response was taken as unity.
verify their conclusion. Here, again, the response to plain mannose solutions
cannot be explained by the 1:1 complex model, since, in that case, the response-intensity curve should cover a concentration range of 1 to 103 for zero
to the m a x i m u m response (see Figs. 3-5), whereas the curve in the mannose
response covers a concentration range of less than 1 to l0 S (see also Figs. 12
and 13). Furthermore, mannose has a definite effect on the response to fructose at concentrations at which mannose does not have any stimulating effect
by itself. This suggests that a mannose molecule, at these concentrations,
occupies one of the two units for fructose molecules in a receptor site and blocks
the response.
T h e results of the same type of experiments for glucose and sucrose are
shown in Figs. 12 and 13. At concentrations (around 0.05 M) of no stimulating
effect by itself, mannose apparently had a synergistic effect on the responses to
glucose and sucrose. Also in the results shown in Figs. 12 and 13 an inhibition
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m
Z
Published October 1, 1968
Mono-and Disaccharides and Sugar Receptor
H . MORITA AND A . SHIRAISHI
I
'
'
~
I ''"1
,
'
'
I 'L"I
'
'
'
573
I
4
1,0
5
9
12
•
13
liJ
O~
Z
O
O.
,,, 0.5
. CONmOLtO.SM GLUCOSE~
>
o 0.6 M GLUCOSE+ MANNOSE
J'~
x MANNOSE ONLY
~
O:::
¢.....~.j|
]
1
/ x
~=9'2V'x
o
. . I . . ' .~ . .. ~ ., . .
0.05 0.1
I
0.01
t
,
I ....
0.5
l
,
I.O
5.0
by mannose is observed, but it was weak compared with the effect on fructose
and occurred at concentrations at which mannose could stimulate by itself.
FRUCTOSE EFFECTS When the results presented above are considered,
we might imagine the simplest picture of sugar stimulation as follows. The
receptor site with which one sucrose molecule combines is comprised of two
subunits. For excitation the receptor site has to be simultaneously occupied
'
I
1.0
w
z
oB.
w
it.
'
'
'
I ....
I
'
'
' I ''"I
'
~
'
~ I
dP
23
o.5
>_
• CONTROL( 0.2 M SUCROSE)
SUCROSE + MANNOSE
x MANNOSE ON
.._i
hi
I
O.OI
,
,
I I ....
0.05
~_..----r'~-,
O.I
.
t ....
0.5
19 ~o 2t
I
,
,
I.O
,
I
5.0
MOLAR CONCENTRATION OF MANNOSE
FIGuP-E 13. E f f e c t s of v a r i o u s c o n c e n t r a t i o n s o f m a n n o s e o n s t i m u l a t i o n b y a fixed
c o n c e n t r a t i o n o f 0.2 M sucrose, w h e r e r e s p o n s e w a s t a k e n as u n i t y .
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MOLAR CONCENTRATION OF MANNOSE
FzGUP.~ 12. Effects of various concentrations of marmose on s t i m u l a t i o n b y a fixed
c o n c e n t r a t i o n of 0 . 6 M glucose, w h e r e r e s p o n s e w a s t a k e n as u n i t y .
Published October 1, 1968
574
THE
JOURNAL
OF G E N E R A L
PHYSIOLOGY
" VOLUME
52
- I968
at the two subunits. Disaccharides could fill the two units with one molecule,
but monosaccharides would have to fill t h e m with two molecules.
A problem here is the behavior of fructose. As shown in Table I I fructose is
considered to have the highest affinity (even if the value of Ks is taken into
account) among the sugars tested. Nevertheless, its competitive effect on
stimulation by other sugars was very weak (see Figs. 9 and 10), and its stimulating effect was deeply depressed by low concentrations of mannose (Fig. 11 ).
Another example of such a property of fructose is shown by experiments in
which the receptor was stimulated by mixtures of 0.1 g sucrose and various
20
O O.I M SUCROSE +
FRUCTOSE
x 0.1 M SUCROSE
~'~I M
(.)
12
1515
13 ~ •
~ 0.5 M FRUCTOSE
i7
18
I
(5
I0
~7
XIo
G
: - . . - . 9 . _ -.~.. - -
14
(/)
._I
0.
I
I
I
I
I
I
t
2
5
4
5
6
MOLAR CONCENTRATION OF FRUCTOSE
FIGURE 14. Effectsof various concentrations of fructose on stimulation by a fixod concentration of 0.1 ~ sucrose. Averages of responses to 0.1 M sucrose and the maximal
response to fructose experimentally obtained are represented by broken and dot and
dash lines, respectively.
concentrations of fructose, up to 6 M (Fig. 14). F r o m the values of K1 (shown
in Table II) and the concentration ratio, sucrose molecules should occupy
less than 1/240 of the total of the receptor sites in the mixture of 0.1 M sucrose
and 6 M fructose, and the response to the mixture should be almost the same
as the m a x i m u m response to fructose, provided that a sucrose moleucle never
shares one of the sites with another molecule. T h e response was not reduced
significantly, however, compared with that to single solutions of 0.1 u sucrose.
One explanation for such a result is simply to assume that one receptor site
can be shared by each of the sucrose and fructose molecules. If such a heterogeneous complex is more effective in excitation than the 2:1 complex of
fructose and the receptor site, the inhibitory effect of fructose on stimulation
by other sugars should be relatively slight.
T h e above-mentioned assumption predicts that the complex (fructose-
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ill
O~
0,I
Published October 1, 1968
H. MORITA AND A. SHIRAISHI Mono- and Disaeeharides and Sugar Receptor
575
glucose-receptor site) is formed when the sugar receptor is stimulated b y a
mixture of fructose and glucose, and can be detected as a synergism at low
concentrations. Dethier et al. (1956) reported that this was the case, b u t
probably because of the variability of the K1 value with different preparations
and other factors, the prediction was fulfilled b y only one preparation out of
several (Fig. 15). In this preparation, the responses to the mixtures of 0.02
M fructose with glucose (notice the results below 0.04 ~r glucose) are shown to
be higher than those to the pure solution of 0.02 M fructose, though responses
to glucose below 0.04 M were zero.
~
o
O.02M
•
GLUCOSE ONLY
J
FRUCTOSE + GLUCOSE
x o o2M F
./72
~,~/25
/ /
C,=E
~o
I0
d
if)
bJ
01
.j
a.
16
5
>~
d
'
14
.
0
20
27 .
. . .xs. ./. 7 ~ ' _ _ _ _ _ _ J ~_ __ _£"
XZS
s
.L4
-
0.01
.6,
~1
....
0.05
,
0.1
,
,
= I,,HI
0.5
Xz
X31
,
1.0
, I ....
I
5.0
MOLAR CONCENTRATION OF GLUCOSE
F 1 o u ~ 15. Effects of various concentrations of glucose o n stimulation by a fixed concentration of 0.02 M fructose. Average of response to 0.02 M fructose is represented by
b r o k e n line.
DISCUSSION
Multimolecular Complex Model Formation of a multimolecular complex
between stimulating molecules and the receptor site described here is not the
first example to be shown in chemoreceptors. Quite recently, T a t e d a and
H i d a k a (1966) studied the receptor for sweet substances in the rat, and have
suggested that more than four molecules of glycine can combine with the
single receptor site.
In the present work, we need not have assumed any model of a complex
combining more than two molecules of stimulant. In describing the results,
we have assumed only one type of receptor site, which is divided into two
subunits: when the two subunits are simultaneously filled with stimulating
molecules, excitation results. F r o m this picture of the sugar receptor, it also
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0
1/1
N
Published October 1, 1968
576
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
• VOLUME
52
•
1968
follows that the single receptor site could combine with two molecules of
disaccharides. This point of view might assist in interpreting the slight deviation from the theoretical curve shown in the results with sucrose stimulation.
GSo Sv
where
GKo -- [G][So Sv]
[GSo SF] '
( DI-I )
G -t- So Sv ~ So S~, G,
where
rKa -
[G][So SF]
( D1-2 )
G+
Sa Sv o
and
[So &, G] "
Each symbol in equation (D I) corresponds to each one in equations (1) and
(3)
as
G = A,
[G] = a,
SoS~, = S,
and SoSFG = AS,
GSoSy
and
[SoS,] = s -- (nx + n,.),
[GSaSr] + [SoSFG] = n,.
Comparing equation (D 1) with equations (1) and (3), we obtain
1
1
1
aK-~ + ~K~ - K1 "
(for glucose)
(D2)
T h e second step of reaction is assumed to proceed as
G -t- GSo Sp ,-" GSo SF G,
where
FKo = [G][aSo SF]
[GSo S~, G]"
( D3-1 )
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Differentiation of the Receptor Site The results of strong interaction between
glucose and fructose suggest that no definite groups of receptor sites are
differentiated for combining only with two molecules of glucose or only with
two of fructose. T h e same results, however, do not exclude the possibility that
the two subunits of the receptor site differentiate to combine with glucose
and fructose, respectively. From the affinity, which is measured by the reciprocals of/£1 and Ks values listed in Table II, it is also unlikely that there
is any complete differentiation between the subunits, since the Ks value for
fructose is less than the/£1 value for glucose. (If there is any differentiation
for glucose and for fructose, the first step of the reaction in stimulation by
glucose should occur mainly at the subunit specific for glucose, and the second
step mainly at the one specific for fructose.) However, some degree of differentiation is suggested by the difference in the effects of mannose on stimulation
by fructose and by glucose or sucrose.
If we assume temporarily that the subunits are differentiated, and, therefore, denote them by So and Sp, respectively (the receptor site, S, accordingly,
being expressed as SoS~,) and the glucose molecule by G, equation (1) is divided into
Published October 1, 1968
H. MORITAAND A. SHIm~ISHI Mono- and Disaceharides and Sugar Receptor
577
and
G + So Sv G ~ GSa S~, G,
where
oKo = [G][So S~, G]
[Gso s~ G]
( D3-2 )
Then, comparing equation (D3) with equations (2) and (4), we get
oKo "1- FKo = K2 .
(for glucose)
(D4)
T h e value of a ( = K~/K1) for glucose can be obtained from equations (D2)
and (D4) as
oz = K2/K1 = (aKo -b ~,Ko)2/(oKa • ~,Ko).
(D5)
Allosteric Model Such an interaction as the one mentioned above has
been claimed as strong evidence for allosteric transition in proteins (Wyman,
1963). O u r receptor site is considered in m a n y respects to be composed of
allosteric macromolecules. First, specificity for certain sugars m a y be attributed only to macromolecular structure. Second, noncharge molecules
such as those of sugars m a y induce electrical changes in the receptor membrane only through structural changes in the receptor site, and these changes
should be closely related to, or synonymous with, allosteric transitions. Third,
the existence of subunits has been emphasized in the present paper, and an
interaction between the subunits has been suggested. It is, therefore, justifiable
to examine the present results from the viewpoint of allosteric transitions in
the receptor site.
Monad, Wyman, and Changeux (1965) have proposed a model for allosteric transitions. According to them, let us assume two states of the receptor
site as
Ro R~, ~-. To TF ,
where L - [To Tp]
[Ro R~,]
( D6 )
and R o , R1,, T o , and TF represent two different subunits in the R and T
state, respectively. T h e symbol, L, denotes an equilibrium constant for the
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It can easily be shown from equation (D5) that the value of ~ is m i n i m u m and
is 4 when oKo = FKo • Therefore, any value of ~ below 4 indicates that the
assumption m a d e in equation (D3) is wrong, and that a "stabilizing interaction" exists between the subunits.
T h e analyses of the results of glucose and fructose stimulation are summarized in Table II, and the value of ~ = K2/K1 is unity for glucose and 4 for
fructose. As far as we can assume that there are two subunits in a single
receptor site, we have to conclude that there is a stabilizing interaction between the subunits making a complex with molecules of monosaccharide.
Published October 1, 1968
578
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
• VOLUME
5°
"
I968
transition. T h e dissociation constants between the subunits and ligands
(fructose and glucose) are defined as
oKo
_ [G][Ro]
[Ro G] '
,Ko -
,K, - [F][R,]
oK, -
[Re F] '
[G][Rr]
[R, G]
,
[F][Ro]
[Ro F]'
,
oKo -
[G][To]
,
[To GI
,
[GI[T,]
l,Ko -,
[ T , G]
eK'r -
[F][Tr]
oK; -
F]
[T,
'
[el[rol
[To F] "
For convenience of derivation of the folllwing equations, ratios between the
constants are defined as
!
co
=
oKo/oXo,
bo
oKo/tKo,
c,
=
,X,l,X;.
b, = , K , IOK,,
=
?
bo = o K o / t K o ,
b;
!
=
t
,X, loX,.
relative concentrations of glucose and fructose are denoted by
ao (= [G]/oKo)and
a , ( = [ F ] / , K r ) , respectively. T h e n u m b e r s of all forms
of the site in the R state, ZR, and that in the T state, ~ T, are
~R = [Roa,] + [CRoR,] + [RoR,G] + [CRoR,C] + [aR~,F]
+ [FRoR,a] + [R~R,F] + [FR~,] + [FR,RoF]
( D7-1 )
= [RoR~,]{I + (1 + bo)ao + bo~2o nt- (1 + b~,bo)otoap
+ (1 + b,)a, + b,o31
T~T = [ To Tr] + . . . . .
( t h e same types of complex as in ~R)
t
it
2
2
t
!
= L[RoRa,]{ 1 + (1 + bo)coao + oocoao + (1 + b o b , ) e o c , a o a ,
( D7-2 )
?
71 2 2
+ (1 + b,)c,a,
+ o,c,
oo,}.
T h e function of state (fraction of the site in the R state)• R, is
R-
ZR
ZR + l~T"
(D8)
T h e maximal value of the function of s t a t e , / ~ , when the values of av and
a o are infinitely large in an equimolar mixture of glucose and fructose, is
written as
~ =
1 ?
2
!
1 + co L(~,r + bo)(~'rb, + t)
('t + bo)('rb,, + 1)
•
(D9)
where ¢/ = c p / c o and 3' = o o , / a o . In the case of pure glucose or fructose,
respectively, equation (D9) reduces to
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The
Published October 1, 1968
H. Mortrra AND A. SHIRAISHI Mono- and Disaccharides and Sugar Receptor
579
1
~®o = 1 q- c2o L(bo/bo) '
( DI0-1 )
1
(DIO-2,)
1 + c2r L(b'r/b~,)"
I f we assume t h a t the response is p r o p o r t i o n a l t o / ~ , we c a n test this assumption with a p p r o p r i a t e values for the constants.
1.0
t~
i~8 0.5
-~-~M-.
I
, i l,li,l
,d
I
,0 5
,o 4
FIGURE 16. Test of the experimental results in stimulations by fructose used in Fig. 6
(filled circles) with allosteric model. The continuous curve was obtained from equation
(D8) in the text. The values of the constants used:
L ffi I 0 e,
c r ffi
10-4,
br •0.1,
and b~ ffi 10.
Resultant value of R,or is 0.5, and the estimated value of FKI, is 1.5 X 10-s M.
T h e results of the test are shown in Figs. 16 a n d 17 for fructose a n d for glucose, respectively, a n d the e x p e r i m e n t a l values are the ones used in Figs. 6
a n d 7. T h e constants used are
L = 10s,
c a - - 10.-4,
bo = 0.1,
c~ =
br =
b~, =
b~ = 1,
(D:I)
10 -4 ,
0.1,
10,
so t h a t the m a x i m u m responses for glucose ( / ~ o) a n d for fructose (l~®v) are
c a l c u l a t e d as 0.91 a n d 0.5, respectively; the values of aKo a n d r K r c a n also
b e e s t i m a t e d a p p r o x i m a t e l y as 4 × 10 -6 a n d 1.5 × 10 -5 ~, respectively. I n
Fig. 18, the c a l c u l a t e d values of R (R = 1.0 w h e n all sites are in the R state)
with the same values of constants used in Figs. 16 a n d 17 are p l o t t e d against
m o l a r c o n c e n t r a t i o n , so t h a t it is easy to c o m p a r e t h e m with the e x p e r i m e n t s
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0
Published October 1, 1968
580
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
• VOLUME
5°
•
x968
shown b y Fig. 1. T h e theoretical curves w h i c h were c a l c u l a t e d for glucose
(curve A) a n d for fructose (curve B) well represent the e x p e r i m e n t a l d a t a .
R e l a t i v e c o n c e n t r a t i o n s of 1 M glucose a n d fructose are a p p r o x i m a t e d f r o m
the values of oKa a n d pKF (4 × 10 -5 a n d 1.5 X 10 -5 ~a, respectively) as
a o = 2.5 × 104 a n d a p = 7 X 104. T h e r e f o r e , with these values, the response to the m i x t u r e of 1.0 M glucose a n d 1.0 M fructose c a n be c a l c u l a t e d
b y e q u a t i o n (D8), or as a close a p p r o x i m a t i o n by e q u a t i o n (Dg). T h e resultant v a l u e is 0.77, a n d it is shown b y the b r o k e n line at the u p p e r p a r t of
] .0
i--e--ee~oe
m
•
•
•
.,./:
ee
•
•
o •
... •
0.5
X
IQ~:
:
•
It,
o•
•
oe
•
•
•
•
0
io*
,o
FiGum~ 17. The same as in Fig. 16, but for stimulations by glucose. The experimental
values (filled circles) are the same ones used in Fig. 7. The values of the constants used
for calculation in equation (D8):
L = 106,
ca
=
10 --4,
be = 0.1,
and b~ =
1.
Resultant value of/~o0a is 0.91, and the estimated value of oKo is 4 X 10--5 M.
Fig. 18. T h i s d e m o n s t r a t e s t h a t it is possible for the response to the m i x t u r e
not to d e p a r t so m u c h f r o m the response to 1.0 M glucose, b u t to d o so f r o m
the response to 1.0 M fructose. F o r c o m p a r i s o n with the results in Fig. 10, the
values for the m i x t u r e s of a dilute c o n c e n t r a t i o n of glucose a n d various
c o n c e n t r a t i o n s of fructose ( a o = 103, a~ is variable) are plotted as c u r v e C.
C o m p a r e d with the results shown in Fig. l0 the result is s o m e w h a t too high
at low fructose concentrations.
I t will be noticed t h a t the theoretical curves o b t a i n e d f r o m the allosteric
a n d the classical c o m p l e x models are almost the same. T h i s is q u i t e u n d e r s t a n d a b l e since the q u a d r a t i c terms of c o n c e n t r a t i o n ( a ~ , o~ ,F a n d a o a p )
are b y far the largest in size in the n u m e r a t o r of e q u a t i o n (D8). I n o t h e r
words, in the allosteric model also the response is practically p r o p o r t i o n a l to
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I1~
Published October 1, 1968
H. MORITA AND A. SHIRAISHI Mono-and Disaccharides and Sugar Receptor
581
the n u m b e r of sites occupied by two molecules of ligand. Some i m p o r t a n t
differences between the two models, however, exist in the assumptions on
which the two are based. I t has been shown that the maximal responses are
different with different sugars. According to the allosteric model, such differences result mainly from differences in the ratios between the dissociation constants of ligands for the R state and for the T state of the receptor site. T h e
classical complex model, however, interprets the same result as the difference
in the proportionality constant between the response m a g n i t u d e and the
n u m b e r of the 2:1 complex. I t might be supposed, therefore, that the extent
i.o F
~q~(o(,,-=o)
0
0.5
1.0
MOLAR CONCENTRATION
FIOURE 18. Calculations of fractions of the site in the R state, R, for various concentrations of glucose (curve A), of fructose (curve B), both in plain solutions; and of fructose in mixtures with glucose whose relative concentration is 103 (curve C). Calculated
values of/~ for the mixture of 1 M glucose (ao = 2.5 × 104) and 1 M fructose (c~v =
7 X 104) and for plain glucose at relative concentration of 103 are represented by the
upper and lower broken lines, respectively. The values of the constants used are the
same that were used in Figs. 16 and 17.
of permeability change in the receptor m e m b r a n e could be different with
different types of the complex.
If we assume conventionally that the K1 values in T a b l e II are the dissociation constants and compare them with the values of oKo and vKF estimated
from the allosteric model, the free energy change for forming a complex with
the ligands has to be more negative in the R state of the allosteric model than
in the site of the classical complex model by 7-8 kcal/mole. This amounts to
the free energy change of hydrolysis of so-called " h i g h energy" phosphate
compounds. In fact, in the allosteric model, combination of the ligands with
the subunit in the R state causes the release of the free energy for transition
from T to R.
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0
Published October 1, 1968
5 8`,
THE JOURNAL OF GENERAL PHYBIOLOGY • VOLUME 52 • 1968
This work was mpported in part by the Scientific Research Fund from the Ministry of Education
of Japan and under the sponsorship of the United States Army Research and Development Group
(Far East), Department of the Army, DA-CRD-AG-S92-544-67-G61.
The contents of this paper fulfill in part the requirement for the Doctoral Thesis of one of us (A.S.)
at Kynshu University.
Receivedfor publication 15 April 1968.
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The theory presented here, which is based on the allosteric model, is
rather incomplete. It has not been shown that stimulations by disaccharides
can be interpreted by the same model. It may be possible, however, to describe
them with the allosteric model by using the constants in equation (D7), based
on an assumption that one molecule of disaccharides can combine with each
subunit at two different parts of the monosaccharide. We have assumed in
this model (in the classical complex model, also) that there are only identical
receptor sites, SoS~,, but there could be other types of receptor sites, for example, SoSo and SFSp. Such an additional assumption might give better
agreement with the results of experiments on fructose effects on glucose
stimulation, though a discrepancy has been pointed out between the theoretical curve and experimental values at low concentrations of fructose in
Fig. 18 (curve C).
Monod et al. (1965) have based their theory on polymers of identical subunits, in which case they have proved that any intermediate state such as R T
is unstable and can be neglected. Some recent papers (Antonini, Bucci,
Fronticelli, Wyman, and Rossi-Fanelli, 1965; Tyuma, Benesch, and Benesch,
1966) show, however, that artificially synthesized hemoglobin molecules composed of the same four subunits have weaker allosteric activities compared
with those of two a and two/3 chains. Therefore, it may be justifiable to assume that the two subunits introduced here have different structures.
At present, we have insufficient data to decide whether the classical complex model or the allosteric one is really correct, though the latter seems to
give a somewhat better interpretation of the experimental results.
Published October 1, 1968
H. MORITA AND A. SmRAISm Mono- and Disaccharides and Sugar Receptor
583
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