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The specific receptor site for aliphatic carboxylate anion in the labellar sugar receptor of the fleshfly
J. insecr Physiol., Vui. 24, pp. 807 to 811. ..< Pergamon Press Ltd. 1978 FrPttedin Great Britain
0022-1910/78/1201-0807 SO2.00/0
THE SPECIFIC. RECEPTOR SITE FOR ALIPHATIC CARBOXYLATE ANION IN THE LABELLAR SUGAR RECEPTOR OF THE FLESHFLY Icmw SHIMADA and K~IO ISOXO Department of Biological Science, Tohoku University, Kawauchi, Sendai 980, Japan jReceived
27 February 1978; revised 2 June 1978)
Abstract-A two minute treatment of a single sugar receptor cell with 10 mg/ml pronase did not affect its response to D-fructose, but depressed markedly its response to L-valine. This is the first direct evidence for a specific site for certain aliphatic amino acids. All. six amino acids that can stimulate the sugar receptor were examined and classified into two groups according to the presence or absence of the inhibitory effects of pronase treatment. Responses to certain alipbatic amino acids and a corresponding fatty acid were depressed whereas responses to pbenylalanine and trytophan were not. Furtherevidence for the existence of two classes of amino acids comes from the fact that the r amino group of valine is not essential whereas that of phenylalanine is. It was concluded that the first class of amino acids react with a specific receptive site for carboxylate anions whereas the second react with the furanose site,
~RODWC~ON MuLTrPLEchemoreceptor sites in a single cell are now well documented in lower organisms.. About 20 receptor molecules have been identified in E. co& and an equivalent number have been identified in o&er species (KOSHLAND,1977). In higher organisms, however, there has been little information about the multiple taste receptor sites except for the different sites in the single sugar receptor cell of the fly (DETHIER, 1955; EVANS, 1963; MORITA and SHIRAISHI, 1968; OMAND and DETIXIER,1969; JAK~NOVICWet al., 1971). SHIAXADAet al. (1974) provided the first direct evidence for at least two different sites (pyranose and furanose sites) in the sugar receptor cell by studying the effects of ~eatment with p-chloromercuribenzoate (PCMB). They found that the response of the sugar receptor cell to glucose was depressed after PCMB treatment, but its response to fructose was not affected. PCMB treatment likewise had no noticeable effect on the response to any of the six amino acids that can stimulate this cell (cf. SIXIFUISHIand KUWABARA,1970; GOLDRICH, 1973), while 2,4,6+rinitrobenzene sulphonic acid (TNBS) markedly decreased the response to both fructose and amino acids (SHIMADA,1975). Thus the amino acid sensitivity of the receptor appears to change always in parallel with fructose responsiveness and contrary to that to glucose. SHIMADA(1975) concluded that the amino acids as well as fructose react with the furanose site and suggested that essential factors for stimulation were a carbon chain with adequate length, a carboxyl group and uamino group in the amino acids, comparing the basic structure of the six amino acids with that of monosa~~ha~des in the furanose fom essential for stimulation. In the course of studying the stereospecificity for these amino acids, however, SHIMADA (in press)+’proved that the cc-amino group was not essential for stimulation by certain amino acids and further showed that certain fatty acids could stimulate
the sugar receptor dell once the solutions were buffered at neutrai pH. Ion substitution experiments indicated that some aliphatic carboxylate anions were intrinsically as stimulative as the corresponding amino acids. This raises a problem, for there are considerable differences between the structure of the aliphatic carboxylate anion and monosaccharides in the furanose form, yet it appeared from work hitherto that both reacted with the same receptor site. We report here the effects of treatment with pronase, a powerful protease, on the responses to fructose and certain amino acids including a fatty acid.
~TERIA~S
AND ~E~ODS
The fleshflies, Boettcherisca peregrina, 4-6 days old were used. The adult flies were usually kept on a 3% sucrose dietudlibitum at room temperature just before the experiment. The chemosensory setae used were of the largest type. The side wall recording technique was employed (MORITA and YAMASHITA, 1959). The methods have been described in detail previously (SHIMADA,in press). Records of the response usually show three types of spikes. The largest spikes come from the sugar receptor cell. The smallest and medium spikes are from the water and salt receptor cells, respectively (SHIRAISHIand KUWABARA, 1970). The magnitude of the response was defined as the number of the largest impulses during a period from 0: 15-0.35s from the beginning of the stimulus. The intervals betwe& stimuli were usually more than three minutes. Sugars were dissolved in redistilled water and the solutions of amino acids and fatty acids were made up in l/l 5 M phosphate buffer @H 7.2). Pronase was also dissolved in the phosphate buffer. It was used within half a day after being dissolved since its effect decreased over half a day at room temperature, possibly because of autolysis. The treatment of the receptoiwith pronase was performed in the same way as stimulation, i.e. using a glass capillary filled with the
807
SO8
ICHIR~
SHIM.~DA
solution. The ambient temperature in the course of the experiments was 22 + 1°C. Relative humidity was maintained at 62-80% throughout this work. Pronase E was purchased from Kakenkagaku, Tokyo, and all the amino acids were obtained from Takara Kosan Co., Tokyo. D-glucose and D-fructose were the products of Nakarai Chemicals Ltd., Kyoto. Isovaleric acid and 3-phenyl propionic acid were from Wako Pure Chemicals, Osaka.
RESULTS AND DISCUSSION In order to find some clue to the multiple receptor sites it seemed worthwhile to ‘dissect’ receptor proteins by using purified proteases of established substrate s~ifi~ty.TANiMu~etal. (inpreparation)haverecently proved the presence of fructose receptor protein in Droso~~~~~by papain treatment. We used here pronase E purified from Streptonzyces griseus. It has a strong proteolytic action that digests substrate protein to each amino acid by breaking peptide bonds at random. We examined iirst the effects of pronase treatment on the response of the sugar receptor cell to glucose, fructose and valine. Then we examined the effects on the response to several other amino acids including a fatty acid. darken ~~~fere~~~sbetween the effects ofpronase on the response to fructose and that io valine The response of the labellar sugar receptor cell of the fleshfly to 0.2 M D-glucose and 0.01 M L-valine was
After
AND
KUNI~ISO~~O
clearly depressed, while that to 0.1 M D-fructose was
unaffected after treatment with 10 mg/ml pronase in l/l5 M phosphate buffer (pH 7.2) for 2 min. The records in Fig. 1 show the responses of the sugar receptor to glucose (A), fructose (3) and valine (C) before and after pronase treatment. In all cases pronase treatment was followed by washing for one minute in Waterhouse’s saline to remove traces of the enzyme. No change was seen in the shapes of impulses after treatment, but the peak impulse frequency of the response to glucose and valine was markedly decreased compared with that before treatment. The frequency of the smallest spikes (from the water receptor) was also substantially depressed in these records. Pronase treatment for more than five minutes often produced changes in the shape of spikes, and in particular decreased the amplitudes of all impulse classes. The duration of pronase treatment was therefore limited to two minutes except for the experiments shown in Fig. 3. The concentration of pronase was also limited to 10 mgjml in al1 experiments--enough to cause a marked difference between the responses. Phosphate buffer (l/l.5 M) alone had no effect on the sugar receptor even if applied for more than ten minutes. Marked differences were further seen in the time course of depression by pronase treatment, Figure 2 shows a typical example, Immediately after pronad treatment for two minutes the response of the sugar receptor cell to 0.01 M L-valine and 0.2 M D-glucose was completely depressed, while that to 0.1 M Dfructose was almost unaffected. Excitability of the
treatment
Fig. 1. Records of control and depression experiments. Responses to 0.2 ti D-giucuse (A), 0.1 M D-fructos (B), and 0.01 M r_-valine (C) before and after treatment with 10 mg/ml pronase for 2 min.
Receptor site for carboxylate in the fleshfly A
O.lM
FrUCtOSe
0
02M
Glucose
l
O.OlM
Valine
809 0 0.1 M
I
d 0.2b.4
T
O.OlM
1.0
q n,o
Fructose Gi ucose L-Valine
P: Pro~ase~l~mglmi~ R:Waterhouse*s
saline
0 0
2.0
1.0
05
3.0
5.0
min
Fig. 2. Time course of response to fructose, glucose, valine, H,O and NaCl after pronase treatment obtained from a single chemosensory seta. P, treatment with IO mg/ml pronase for 2 min; R. treatment with Waterhouse’s saline for 1 min.
water receptor was also con4etely depressed, that of the salt receptor usually less so. A detectable recovery of the response to glucose occurred spontaneously within 15 min, but the depression of valine responses was complete up to 30 min after the treatment, when it began to recover. The response to fructose, on the other hand, remained almost unchanged during the recovery time of glucose and valine responses. The recovery phenomena may raise the question of whether the depression results from any real action of the enzyme. It might have resulted from an inhibitory contamination such as calcium ions in the enzyme solution or might have been caused as a result of the enzyme protein simply being stuck to the surface of the receptor cell and it could reverse spontaneously as they are washed off by the fluids or the applied stimulus solutions. In order to examine these ~ssibilities, we tested the effects of treatment with heat-inactivated pronase. Inactivated pronase was obtained by heating 10 mg/ml active pronase in l/l5 &I phosphate buffer (pH 7.2) at 100°C for 5 min. Treatment with inactivated pronase for two minutes reduced neither the subsequent response of the sugar receptor to glucose and valine nor those of the water and salt receptors. The depression, therefore, may be concluded to result from a real proteolytic action of pronase.
Figure 3 shows the dependency of the response on the time of pronase treatment up to five minutes in terms of relative response, i.e. the ratio of the magnitude of the response to each stimulant after treatment with 10 mg/ml pronase to that before the treatment. Each point indicates the mean value of relative response within 15 min after the treatment, i.e. before the time when any recovery might be expected. Over a five minute treatment, the response to valine as well as that to glucose decreased profoundly, while that to fructose declined only slightly. The shortest duration of pronase treatment required to differentiate maximally between the response to fructose on the one hand and those to valine or glucose on the other was two minutes. A slight difference could be observed between glucose and valine. The response
Fig. 3. Timedependency ofpronase treatment. Responses fructose, glucose and valine after pronase treatment various durations. Ordinate is value of response relative that before the treatment. The range of standard error shown by bars associated with each circle. concentration pronase was always kept at IO mgjml.
to of to is of
to glucose was already depressed by treatment for as little as 0.5 min, but the response to valine was only depressed after a 1 min treatment. The consistent differences, shown in Figs. 1,2 and 3, prove for the first time that valine does not react with the furanose site of the sugar cell, but that it reacts with a third specific site other than the furanose and pyranose sites since stimulative amino acids have already been shown not to interact with the pyranose site (SHIMADA, 1975). This seems to contradict the previous suggestion that stimulative amino acids react with the furanose site, which was based on the several parallels between the amino acids and fructose, a typical sugar that can interact with the furanose site (SHIMADA, 1975). Two groups depression
of amino aci& with respecf to pronase
We next examined the effect of pronase treatment of the sugar receptor cell on the stimulating effectiveness of various amino acids and also a fatty acid, isovaleric acid, in order to determine which site they reacted with, the furanose site or the third site. When the treatment decreased the response to an amino acid, in the same way as that to valine, it was concluded that it reacted with the third site. On the other hand, it was believed to react with the furanose site when the treatment has no effect on the response, as was the case with the fructose. The results are shown in Table 1. Relative response indicates the mean value for the ratio: (magnitude of response to each amino acid after Table 1. Relative response after pronase treatment Chemicals
D-c&ICOSe
D-Fructose
L-Phenylalanine L-Tryptophane L-Valine L-Valine Isovaleric,acid L-Leucine
Concentration fM X 103) 200 100 10 10 10 50 10 10
Relative No. response f S.D. tests 0.24 0.91 0.88 0.98 0.17 0.27 0.17 0.19
f 0.18 2 0.15 * 0.16 +0.18 + 0.17 + 0.10 + 0.24 + 0.25
18 18 17 8 18 6 4 14
810
ICHIRO SHIMADA
pronase treatment)/(magnitude before treatment), as in Fig. 3. All the data used were obtained within 15 min after the treatment so as to avoid the effect of recovery. The amino acids tested can be classified into two groups. One group consisted of the aromatic amino acids phenylalanine and tryptophan. These behaved like fructose, and exhibited a relative response >0.85. The other group, including valine, leucine and isovaleric acid, yielded values of relative response < 0.3. Isoleucine and methionine were also examined and classified with the latter group (data not shown). One notable feature of the results is that the latter group includes valine and a corresponding fatty acid, isovaleric acid. Thus, proteolytic ‘dissection’ provides further direct evidence supporting the conclusion proposed by SHIMADA(in press) that some aliphatic carboxylate anions are intrinsically as stimulative as the corresponding amino acids. The response to 0.05 M valine was also depressed. At this concentration, in the absence of pronase treatment, the magnitude of the response reached maximum and went to excess. The depression suggests that the main effect of treatment on the response to valine is rather a decrease of the maximum response. On the whole, the former group of aromatic amino acids is clearly different from the latter aliphatic amino acids in the character of depression and can be inferred to react with the .furanose site. It may be concluded that the latter group reacts with the third specific site for aliphatic carboxylate anion. The effect of pronase treatment concentration relationship
on the response-
An unchanged response by the sugar cell to and tryptophan at one fixed phenylalanine concentration, 0.01 M, may, however, be insufficient to prove the presence of intact specific sites for these amino acids after pronase treatment. In order to get quantitative evidence for the presence of intact furanose sites after the treatment, we examined the effect of pronase treatment on the responserelationship with phenylalanine concentration stimulation. Figure 4 shows a typical example of the effects of 0
Before
e
After
Pronase
Pronase
treatment
treat
meni
a 1.0 YI I
AND KUNIO ISONO 0
O.OlM
Phenylaianine
A
0.OlM
Vaiine
min
Fig. 4(b). Time course of response to phenylalanine and valine after pronase treatment. ~0.01 M phenylalanine: A, 0.01 M valine. The ordinate is the same as in Fig. 4(a). These experiments were performed simultaneously with those in Fig. 4(a). The stimulations by a second series of concentrations of phenylalanine in Fig. 4(a) were performed between the two responses to 0.01 M phenylalanine after pronase treatment in this figure.
pronase treatment, where the magnitude of all responses is normalized so that the response to 0.01 M phenylalanine before the treatment is unity. The seta was stimulated first with an ascending series of concentrations of phenylalanine, then treated with 10 mg/ml pronase for two minutes, washed with Waterhouse’s saline (BUCK, 1953) for one minute, and finally stimulated with a second identical series (Fig. 4(a)). Immediately after washing with Waterhouse’s saline, the response to 0.01 M valine decreased to zero, while that to 0.01 M phenylalanine was almost unchanged (Fig. 4(b)). The pronase treatment seemed to produce no distinct effect on the response to phenylalanine over a wide range of concentrations although a slight divergence in the lower concentrations was often detectable. More or less recovery of the response to 0.01 M valine was sometimes observed after a second series of stimulations. It may be concluded that most of the sites for phenylalanine remain intact, while those for valine are drastically modified after treatment with 10 mg/ml pronase for two minutes. Pronase, therefore, may selectively attack the specific site for valine under this condition. Further support for intrinsic differences between the receptive sites for aliphatic and aromatic amino acids comes from a study of the stimulating effectiveness of amino acid derivatives. The essential role of the a-amino group ofphenylalanine
I 001
0.1 Phenylalanine
1
10
(MxlOq)
Fig. 4(a). The response to phenylalanine of various concentrations before andafter pronase treatment. 0, Before treatment; l, after treatment. The ordinate shows the level of excitation expressed relative to the response of the untreated receptor to 0.01 M phenylalanine.
SHIMADA(in press) showed that some aliphatic carboxylate anions were as stimulative as the corresponding amino acids and concluded that the aamino group was not always essential for stimulation. The similarity in stimulating effectiveness between 0.01 M isovaleric acid (IVA) and 0.01 M valine (VAL) is shown in Fig. 5. ‘Relative response’ here is the ratio: (response to each chemical)/(response to 0.01 valine). All the chemicals were dissolved in 1/15 M phosphate buffer (pH 7.2). The complete effectiveness of 0.01 M 3-phenyl propionic acid (PPA) contrasted with the
Receptor
site for carboxylate
in the fleshfly
811
digest the specific receptor proteins for the aliphatic carboxylate anion in the labellar sugar recentor cell of the fly. 5.
Acknowledgemenrs-This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan.
REFERENCES
0,
5
I-
-
: VAL
1 IVA
PHE
L
PPA
Fig. 5. Comparison of relative response to valine, phenvlalanine and the corresponding derivatives. The ordinate represents the ratio of the magnitude of the response to each chemical to the response to 0.01 M valine. The range of standard deviation is shown by bars associated with each rectangle. VAL, 0.01 M valine (n = 11); IVA, 0.01 M isovaleric acid (n = 5); PHE, 0.01 M phenylalanine (N = 6);
PPA, 0.01 M 3-phenylpropionic acid (n = 6). effectiveness of 0.01 M marked stimulating phenylalanine. The replacement of the a-amino group of phenyialanine with a hydrogen atom decreased its effectiveness dramatically, which means that the namino group is essential for stimulation by phenylalanine, and agrees well with the proposed stereospecificity of the furanose site for amino acids; it requires an cc-amino group as well as a carboxyl group and a carbon chain having an adequate length. Based on these concentrations, together with the similarities to the fructose responses reported above, aromatic amino acids, therefore, must react with the furanose site. Differences between valine and phenylalanine in the importance of the amino group on the other hand, constitute a significant difference between aliphatic and aromatic amino acid receptive sites, and provide additional support for the existence of a specific site for the aliphatic carboxylate anion in addition to the furanose and pyranose sites. Pronase can selectively
BUCK
J. B. (1953) Physiological properties and chemical composition of insect blood. In Insect Physiology. (Ed. by ROEDER K. D.). John Wiley. New York. DETHIER V. G. (1955) The physiology and histology of the contact chemoreceptors of the blowfly. Q. Rev. Riol. 30, 348-371. EVANS D. R. (1963) Chemical structure and stimulation by carbohydrates. In Olfaction and Tasfe-I. (Ed. by ZOTTERMAN Y.) pp. 165-192. Pergamon Press, Oxford. GOLDRICH N. R. (1973) Behavioral responses of Phormia regina (Meigen) to labellar stimulation with amino acids. J. Ren. Physiol. 61, 74-88. JA~INOVI~H W., Jr, GOLDSTEIN I.J., VON BAUMGARTENR. J. and AORANOFF B. W. f 1971) Sugar receptor snecificitv in the fleshfly, Sarcophagi buliata.Brain ies. 35, 369-378. KOSHLAND D. E., Jr. (1977) A response regulator model in a simple sensory system. Science 196, 1055-1063. MORITA H. and -SHIRAJSHJ A. (1968) Stimulation of the labellar sugar receptor of the fleshfly by mono- and disaccharides. J. gen. Physiol. 52, 559-583. MORITA H. and YAMASHITAS. (1959) Generator potential of insect chemoreceptor. Science < Wash. j 130,922. OMAND E. and DETHIER V. G. (1969)An electrophysiological analysis of the action of carbohydrates on the sugar receptor of the blowfly. Proc. Nat. Acad. Sci. U.S.A. 62, 136143. SHIMADA I. (1975) Two receptor sites and their relation to amino acid stimulation in the labellar sugar receptor of the fleshfly. J. Insect Physiol. 21, 1675-168%. SHIMADA I. (1978) The stimulating effect of fattv acids and amino acid derivatives on the label& sugar receptor &the fleshfly. J. gen. Physiol. (in press). SHIMADA I., SHIRAISHI A., KIJIMA H. and MORITA H. (1974) Separation of two receptor sites in a single labellar sugar receptor of the fleshfly by treatment with pchloromercuribenzoate.’ J. Insect Physiol. 30, 605-621. SHIRAISHIA. and KUWABARA M. (1970) The effects of amino acids on the labellar hair chemosensorv cells of the fly. J. gen. Physiol. 56, 768-782. TANIMURA T., ~SONO K. and KIKUCHI T. Multiple receptor proteins for sweet taste discriminated by papain treatment in Drosophila. (in preparation).
0022-1910/78/1201-0807 SO2.00/0
THE SPECIFIC. RECEPTOR SITE FOR ALIPHATIC CARBOXYLATE ANION IN THE LABELLAR SUGAR RECEPTOR OF THE FLESHFLY Icmw SHIMADA and K~IO ISOXO Department of Biological Science, Tohoku University, Kawauchi, Sendai 980, Japan jReceived
27 February 1978; revised 2 June 1978)
Abstract-A two minute treatment of a single sugar receptor cell with 10 mg/ml pronase did not affect its response to D-fructose, but depressed markedly its response to L-valine. This is the first direct evidence for a specific site for certain aliphatic amino acids. All. six amino acids that can stimulate the sugar receptor were examined and classified into two groups according to the presence or absence of the inhibitory effects of pronase treatment. Responses to certain alipbatic amino acids and a corresponding fatty acid were depressed whereas responses to pbenylalanine and trytophan were not. Furtherevidence for the existence of two classes of amino acids comes from the fact that the r amino group of valine is not essential whereas that of phenylalanine is. It was concluded that the first class of amino acids react with a specific receptive site for carboxylate anions whereas the second react with the furanose site,
~RODWC~ON MuLTrPLEchemoreceptor sites in a single cell are now well documented in lower organisms.. About 20 receptor molecules have been identified in E. co& and an equivalent number have been identified in o&er species (KOSHLAND,1977). In higher organisms, however, there has been little information about the multiple taste receptor sites except for the different sites in the single sugar receptor cell of the fly (DETHIER, 1955; EVANS, 1963; MORITA and SHIRAISHI, 1968; OMAND and DETIXIER,1969; JAK~NOVICWet al., 1971). SHIAXADAet al. (1974) provided the first direct evidence for at least two different sites (pyranose and furanose sites) in the sugar receptor cell by studying the effects of ~eatment with p-chloromercuribenzoate (PCMB). They found that the response of the sugar receptor cell to glucose was depressed after PCMB treatment, but its response to fructose was not affected. PCMB treatment likewise had no noticeable effect on the response to any of the six amino acids that can stimulate this cell (cf. SIXIFUISHIand KUWABARA,1970; GOLDRICH, 1973), while 2,4,6+rinitrobenzene sulphonic acid (TNBS) markedly decreased the response to both fructose and amino acids (SHIMADA,1975). Thus the amino acid sensitivity of the receptor appears to change always in parallel with fructose responsiveness and contrary to that to glucose. SHIMADA(1975) concluded that the amino acids as well as fructose react with the furanose site and suggested that essential factors for stimulation were a carbon chain with adequate length, a carboxyl group and uamino group in the amino acids, comparing the basic structure of the six amino acids with that of monosa~~ha~des in the furanose fom essential for stimulation. In the course of studying the stereospecificity for these amino acids, however, SHIMADA (in press)+’proved that the cc-amino group was not essential for stimulation by certain amino acids and further showed that certain fatty acids could stimulate
the sugar receptor dell once the solutions were buffered at neutrai pH. Ion substitution experiments indicated that some aliphatic carboxylate anions were intrinsically as stimulative as the corresponding amino acids. This raises a problem, for there are considerable differences between the structure of the aliphatic carboxylate anion and monosaccharides in the furanose form, yet it appeared from work hitherto that both reacted with the same receptor site. We report here the effects of treatment with pronase, a powerful protease, on the responses to fructose and certain amino acids including a fatty acid.
~TERIA~S
AND ~E~ODS
The fleshflies, Boettcherisca peregrina, 4-6 days old were used. The adult flies were usually kept on a 3% sucrose dietudlibitum at room temperature just before the experiment. The chemosensory setae used were of the largest type. The side wall recording technique was employed (MORITA and YAMASHITA, 1959). The methods have been described in detail previously (SHIMADA,in press). Records of the response usually show three types of spikes. The largest spikes come from the sugar receptor cell. The smallest and medium spikes are from the water and salt receptor cells, respectively (SHIRAISHIand KUWABARA, 1970). The magnitude of the response was defined as the number of the largest impulses during a period from 0: 15-0.35s from the beginning of the stimulus. The intervals betwe& stimuli were usually more than three minutes. Sugars were dissolved in redistilled water and the solutions of amino acids and fatty acids were made up in l/l 5 M phosphate buffer @H 7.2). Pronase was also dissolved in the phosphate buffer. It was used within half a day after being dissolved since its effect decreased over half a day at room temperature, possibly because of autolysis. The treatment of the receptoiwith pronase was performed in the same way as stimulation, i.e. using a glass capillary filled with the
807
SO8
ICHIR~
SHIM.~DA
solution. The ambient temperature in the course of the experiments was 22 + 1°C. Relative humidity was maintained at 62-80% throughout this work. Pronase E was purchased from Kakenkagaku, Tokyo, and all the amino acids were obtained from Takara Kosan Co., Tokyo. D-glucose and D-fructose were the products of Nakarai Chemicals Ltd., Kyoto. Isovaleric acid and 3-phenyl propionic acid were from Wako Pure Chemicals, Osaka.
RESULTS AND DISCUSSION In order to find some clue to the multiple receptor sites it seemed worthwhile to ‘dissect’ receptor proteins by using purified proteases of established substrate s~ifi~ty.TANiMu~etal. (inpreparation)haverecently proved the presence of fructose receptor protein in Droso~~~~~by papain treatment. We used here pronase E purified from Streptonzyces griseus. It has a strong proteolytic action that digests substrate protein to each amino acid by breaking peptide bonds at random. We examined iirst the effects of pronase treatment on the response of the sugar receptor cell to glucose, fructose and valine. Then we examined the effects on the response to several other amino acids including a fatty acid. darken ~~~fere~~~sbetween the effects ofpronase on the response to fructose and that io valine The response of the labellar sugar receptor cell of the fleshfly to 0.2 M D-glucose and 0.01 M L-valine was
After
AND
KUNI~ISO~~O
clearly depressed, while that to 0.1 M D-fructose was
unaffected after treatment with 10 mg/ml pronase in l/l5 M phosphate buffer (pH 7.2) for 2 min. The records in Fig. 1 show the responses of the sugar receptor to glucose (A), fructose (3) and valine (C) before and after pronase treatment. In all cases pronase treatment was followed by washing for one minute in Waterhouse’s saline to remove traces of the enzyme. No change was seen in the shapes of impulses after treatment, but the peak impulse frequency of the response to glucose and valine was markedly decreased compared with that before treatment. The frequency of the smallest spikes (from the water receptor) was also substantially depressed in these records. Pronase treatment for more than five minutes often produced changes in the shape of spikes, and in particular decreased the amplitudes of all impulse classes. The duration of pronase treatment was therefore limited to two minutes except for the experiments shown in Fig. 3. The concentration of pronase was also limited to 10 mgjml in al1 experiments--enough to cause a marked difference between the responses. Phosphate buffer (l/l.5 M) alone had no effect on the sugar receptor even if applied for more than ten minutes. Marked differences were further seen in the time course of depression by pronase treatment, Figure 2 shows a typical example, Immediately after pronad treatment for two minutes the response of the sugar receptor cell to 0.01 M L-valine and 0.2 M D-glucose was completely depressed, while that to 0.1 M Dfructose was almost unaffected. Excitability of the
treatment
Fig. 1. Records of control and depression experiments. Responses to 0.2 ti D-giucuse (A), 0.1 M D-fructos (B), and 0.01 M r_-valine (C) before and after treatment with 10 mg/ml pronase for 2 min.
Receptor site for carboxylate in the fleshfly A
O.lM
FrUCtOSe
0
02M
Glucose
l
O.OlM
Valine
809 0 0.1 M
I
d 0.2b.4
T
O.OlM
1.0
q n,o
Fructose Gi ucose L-Valine
P: Pro~ase~l~mglmi~ R:Waterhouse*s
saline
0 0
2.0
1.0
05
3.0
5.0
min
Fig. 2. Time course of response to fructose, glucose, valine, H,O and NaCl after pronase treatment obtained from a single chemosensory seta. P, treatment with IO mg/ml pronase for 2 min; R. treatment with Waterhouse’s saline for 1 min.
water receptor was also con4etely depressed, that of the salt receptor usually less so. A detectable recovery of the response to glucose occurred spontaneously within 15 min, but the depression of valine responses was complete up to 30 min after the treatment, when it began to recover. The response to fructose, on the other hand, remained almost unchanged during the recovery time of glucose and valine responses. The recovery phenomena may raise the question of whether the depression results from any real action of the enzyme. It might have resulted from an inhibitory contamination such as calcium ions in the enzyme solution or might have been caused as a result of the enzyme protein simply being stuck to the surface of the receptor cell and it could reverse spontaneously as they are washed off by the fluids or the applied stimulus solutions. In order to examine these ~ssibilities, we tested the effects of treatment with heat-inactivated pronase. Inactivated pronase was obtained by heating 10 mg/ml active pronase in l/l5 &I phosphate buffer (pH 7.2) at 100°C for 5 min. Treatment with inactivated pronase for two minutes reduced neither the subsequent response of the sugar receptor to glucose and valine nor those of the water and salt receptors. The depression, therefore, may be concluded to result from a real proteolytic action of pronase.
Figure 3 shows the dependency of the response on the time of pronase treatment up to five minutes in terms of relative response, i.e. the ratio of the magnitude of the response to each stimulant after treatment with 10 mg/ml pronase to that before the treatment. Each point indicates the mean value of relative response within 15 min after the treatment, i.e. before the time when any recovery might be expected. Over a five minute treatment, the response to valine as well as that to glucose decreased profoundly, while that to fructose declined only slightly. The shortest duration of pronase treatment required to differentiate maximally between the response to fructose on the one hand and those to valine or glucose on the other was two minutes. A slight difference could be observed between glucose and valine. The response
Fig. 3. Timedependency ofpronase treatment. Responses fructose, glucose and valine after pronase treatment various durations. Ordinate is value of response relative that before the treatment. The range of standard error shown by bars associated with each circle. concentration pronase was always kept at IO mgjml.
to of to is of
to glucose was already depressed by treatment for as little as 0.5 min, but the response to valine was only depressed after a 1 min treatment. The consistent differences, shown in Figs. 1,2 and 3, prove for the first time that valine does not react with the furanose site of the sugar cell, but that it reacts with a third specific site other than the furanose and pyranose sites since stimulative amino acids have already been shown not to interact with the pyranose site (SHIMADA, 1975). This seems to contradict the previous suggestion that stimulative amino acids react with the furanose site, which was based on the several parallels between the amino acids and fructose, a typical sugar that can interact with the furanose site (SHIMADA, 1975). Two groups depression
of amino aci& with respecf to pronase
We next examined the effect of pronase treatment of the sugar receptor cell on the stimulating effectiveness of various amino acids and also a fatty acid, isovaleric acid, in order to determine which site they reacted with, the furanose site or the third site. When the treatment decreased the response to an amino acid, in the same way as that to valine, it was concluded that it reacted with the third site. On the other hand, it was believed to react with the furanose site when the treatment has no effect on the response, as was the case with the fructose. The results are shown in Table 1. Relative response indicates the mean value for the ratio: (magnitude of response to each amino acid after Table 1. Relative response after pronase treatment Chemicals
D-c&ICOSe
D-Fructose
L-Phenylalanine L-Tryptophane L-Valine L-Valine Isovaleric,acid L-Leucine
Concentration fM X 103) 200 100 10 10 10 50 10 10
Relative No. response f S.D. tests 0.24 0.91 0.88 0.98 0.17 0.27 0.17 0.19
f 0.18 2 0.15 * 0.16 +0.18 + 0.17 + 0.10 + 0.24 + 0.25
18 18 17 8 18 6 4 14
810
ICHIRO SHIMADA
pronase treatment)/(magnitude before treatment), as in Fig. 3. All the data used were obtained within 15 min after the treatment so as to avoid the effect of recovery. The amino acids tested can be classified into two groups. One group consisted of the aromatic amino acids phenylalanine and tryptophan. These behaved like fructose, and exhibited a relative response >0.85. The other group, including valine, leucine and isovaleric acid, yielded values of relative response < 0.3. Isoleucine and methionine were also examined and classified with the latter group (data not shown). One notable feature of the results is that the latter group includes valine and a corresponding fatty acid, isovaleric acid. Thus, proteolytic ‘dissection’ provides further direct evidence supporting the conclusion proposed by SHIMADA(in press) that some aliphatic carboxylate anions are intrinsically as stimulative as the corresponding amino acids. The response to 0.05 M valine was also depressed. At this concentration, in the absence of pronase treatment, the magnitude of the response reached maximum and went to excess. The depression suggests that the main effect of treatment on the response to valine is rather a decrease of the maximum response. On the whole, the former group of aromatic amino acids is clearly different from the latter aliphatic amino acids in the character of depression and can be inferred to react with the .furanose site. It may be concluded that the latter group reacts with the third specific site for aliphatic carboxylate anion. The effect of pronase treatment concentration relationship
on the response-
An unchanged response by the sugar cell to and tryptophan at one fixed phenylalanine concentration, 0.01 M, may, however, be insufficient to prove the presence of intact specific sites for these amino acids after pronase treatment. In order to get quantitative evidence for the presence of intact furanose sites after the treatment, we examined the effect of pronase treatment on the responserelationship with phenylalanine concentration stimulation. Figure 4 shows a typical example of the effects of 0
Before
e
After
Pronase
Pronase
treatment
treat
meni
a 1.0 YI I
AND KUNIO ISONO 0
O.OlM
Phenylaianine
A
0.OlM
Vaiine
min
Fig. 4(b). Time course of response to phenylalanine and valine after pronase treatment. ~0.01 M phenylalanine: A, 0.01 M valine. The ordinate is the same as in Fig. 4(a). These experiments were performed simultaneously with those in Fig. 4(a). The stimulations by a second series of concentrations of phenylalanine in Fig. 4(a) were performed between the two responses to 0.01 M phenylalanine after pronase treatment in this figure.
pronase treatment, where the magnitude of all responses is normalized so that the response to 0.01 M phenylalanine before the treatment is unity. The seta was stimulated first with an ascending series of concentrations of phenylalanine, then treated with 10 mg/ml pronase for two minutes, washed with Waterhouse’s saline (BUCK, 1953) for one minute, and finally stimulated with a second identical series (Fig. 4(a)). Immediately after washing with Waterhouse’s saline, the response to 0.01 M valine decreased to zero, while that to 0.01 M phenylalanine was almost unchanged (Fig. 4(b)). The pronase treatment seemed to produce no distinct effect on the response to phenylalanine over a wide range of concentrations although a slight divergence in the lower concentrations was often detectable. More or less recovery of the response to 0.01 M valine was sometimes observed after a second series of stimulations. It may be concluded that most of the sites for phenylalanine remain intact, while those for valine are drastically modified after treatment with 10 mg/ml pronase for two minutes. Pronase, therefore, may selectively attack the specific site for valine under this condition. Further support for intrinsic differences between the receptive sites for aliphatic and aromatic amino acids comes from a study of the stimulating effectiveness of amino acid derivatives. The essential role of the a-amino group ofphenylalanine
I 001
0.1 Phenylalanine
1
10
(MxlOq)
Fig. 4(a). The response to phenylalanine of various concentrations before andafter pronase treatment. 0, Before treatment; l, after treatment. The ordinate shows the level of excitation expressed relative to the response of the untreated receptor to 0.01 M phenylalanine.
SHIMADA(in press) showed that some aliphatic carboxylate anions were as stimulative as the corresponding amino acids and concluded that the aamino group was not always essential for stimulation. The similarity in stimulating effectiveness between 0.01 M isovaleric acid (IVA) and 0.01 M valine (VAL) is shown in Fig. 5. ‘Relative response’ here is the ratio: (response to each chemical)/(response to 0.01 valine). All the chemicals were dissolved in 1/15 M phosphate buffer (pH 7.2). The complete effectiveness of 0.01 M 3-phenyl propionic acid (PPA) contrasted with the
Receptor
site for carboxylate
in the fleshfly
811
digest the specific receptor proteins for the aliphatic carboxylate anion in the labellar sugar recentor cell of the fly. 5.
Acknowledgemenrs-This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan.
REFERENCES
0,
5
I-
-
: VAL
1 IVA
PHE
L
PPA
Fig. 5. Comparison of relative response to valine, phenvlalanine and the corresponding derivatives. The ordinate represents the ratio of the magnitude of the response to each chemical to the response to 0.01 M valine. The range of standard deviation is shown by bars associated with each rectangle. VAL, 0.01 M valine (n = 11); IVA, 0.01 M isovaleric acid (n = 5); PHE, 0.01 M phenylalanine (N = 6);
PPA, 0.01 M 3-phenylpropionic acid (n = 6). effectiveness of 0.01 M marked stimulating phenylalanine. The replacement of the a-amino group of phenyialanine with a hydrogen atom decreased its effectiveness dramatically, which means that the namino group is essential for stimulation by phenylalanine, and agrees well with the proposed stereospecificity of the furanose site for amino acids; it requires an cc-amino group as well as a carboxyl group and a carbon chain having an adequate length. Based on these concentrations, together with the similarities to the fructose responses reported above, aromatic amino acids, therefore, must react with the furanose site. Differences between valine and phenylalanine in the importance of the amino group on the other hand, constitute a significant difference between aliphatic and aromatic amino acid receptive sites, and provide additional support for the existence of a specific site for the aliphatic carboxylate anion in addition to the furanose and pyranose sites. Pronase can selectively
BUCK
J. B. (1953) Physiological properties and chemical composition of insect blood. In Insect Physiology. (Ed. by ROEDER K. D.). John Wiley. New York. DETHIER V. G. (1955) The physiology and histology of the contact chemoreceptors of the blowfly. Q. Rev. Riol. 30, 348-371. EVANS D. R. (1963) Chemical structure and stimulation by carbohydrates. In Olfaction and Tasfe-I. (Ed. by ZOTTERMAN Y.) pp. 165-192. Pergamon Press, Oxford. GOLDRICH N. R. (1973) Behavioral responses of Phormia regina (Meigen) to labellar stimulation with amino acids. J. Ren. Physiol. 61, 74-88. JA~INOVI~H W., Jr, GOLDSTEIN I.J., VON BAUMGARTENR. J. and AORANOFF B. W. f 1971) Sugar receptor snecificitv in the fleshfly, Sarcophagi buliata.Brain ies. 35, 369-378. KOSHLAND D. E., Jr. (1977) A response regulator model in a simple sensory system. Science 196, 1055-1063. MORITA H. and -SHIRAJSHJ A. (1968) Stimulation of the labellar sugar receptor of the fleshfly by mono- and disaccharides. J. gen. Physiol. 52, 559-583. MORITA H. and YAMASHITAS. (1959) Generator potential of insect chemoreceptor. Science < Wash. j 130,922. OMAND E. and DETHIER V. G. (1969)An electrophysiological analysis of the action of carbohydrates on the sugar receptor of the blowfly. Proc. Nat. Acad. Sci. U.S.A. 62, 136143. SHIMADA I. (1975) Two receptor sites and their relation to amino acid stimulation in the labellar sugar receptor of the fleshfly. J. Insect Physiol. 21, 1675-168%. SHIMADA I. (1978) The stimulating effect of fattv acids and amino acid derivatives on the label& sugar receptor &the fleshfly. J. gen. Physiol. (in press). SHIMADA I., SHIRAISHI A., KIJIMA H. and MORITA H. (1974) Separation of two receptor sites in a single labellar sugar receptor of the fleshfly by treatment with pchloromercuribenzoate.’ J. Insect Physiol. 30, 605-621. SHIRAISHIA. and KUWABARA M. (1970) The effects of amino acids on the labellar hair chemosensorv cells of the fly. J. gen. Physiol. 56, 768-782. TANIMURA T., ~SONO K. and KIKUCHI T. Multiple receptor proteins for sweet taste discriminated by papain treatment in Drosophila. (in preparation).