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Detoxication of benzoate by glycine conjugation in the silkworm, Bombyx mori L.
J. Ins. Physiol., 1964, Vol. 10, pp. 385 to 391. Pergamon Press Ltd.
Printed in G+eaz B&air
DETOXICATION OF BENZOATE CONJUGATION IN THE SILKWORM,
BY GLYCINE BOMBYX MORI L.
M. B. SHYAMALA Fermentation
Technology
Laboratory,
Indian
Institute
of Science,
Bangalore
12
(Received 13 August 1963)
Abstract-The role of hippuric acid formation as a mechanism for detoxication of benzoic acid in the silkworm has been investigated. Benzoate is inhibitory to the growth of the silkworm and excreted as hippuric acid, which is not toxic. Hippuric acid is not a normal constituent of excreta. Synthesis of hippuric acid has been shown to occur in the intestines of the silkworm. Hippuricase activity is present in the fat body and silk-gland tissue.
INTRODUCTION
CONJUGATION of aromatic acids with glycine is a well-known mode of detoxication in higher animals. That the detoxication mechanism functions in the same manner in insects was demonstrated by FRIEDLER and SMITH (1954) in Locusta migratoriu and by CASSIDA (1955) in the mosquito A&es aegypti. CASSIDA (1955) was also able to demonstrate that when whole larvae were incubated with benzoate and glycine, hippurate was found in both the larvae as well as in the supernatant. In the silkworm itself, conjugation of anthranilic acid was found to precede excretion (KIKKAWA, 1953). The occurrence of glycine conjugation as a detoxicating mechanism was also suggested by the observations of MURTHY (1954). He found that the toxicity produced by feeding excess nicotinic acid was counteracted by an additional These studies did not, however, contain much information supplement of glycine. on the in vitro conjugation of aromatic acids (SMITH, 1955, 1962; GILMOUR, 1961) or on the sites of formation of such conjugates. Thus the nature of the enzymes involved in the process has not been understood in insects in general and in the The purpose of the present communication is to give silkworm in particular. details of experiments conducted to show (1) the relative non-toxicity of hippurate as compared to benzoate, (2) the excretion of hippurate in benzoate-fed larvae, (3) hippurate synthesis in silkworm tissues in vitro, and (4) the presence of hippuricase activity in the tissues. MATERIALS
AND
METHODS
(i) Growth studies These were conducted with the Mysore silkworm during the fourth instar. For each treatment eight replicates of five worms each were used. Benzoic and hippuric acids were fed as aqueous solutions of sodium salts (pH adjusted to 7-O). Aliquots of 0.1-0.2 ml were spread uniformly on mulberry leaves. In studying the relative toxicities of benzoate and 25
385
M. B. SHYAMALA
386
were used. For studying the effect of benzoate and glycine hippurate, l-5 y& concentrations individually and in combination a 0.05 M concentration of each of the substances was used. Weights were recorded daily till the fourth ecdysis. (ii) Analysis
of excretu
Excreta samples obtained in the growth experiments were collected daily in the morning and dried. The dry samples collected on the different days from the same treatments were pooled together. About 1.5 g portions of pooled samples were extracted with 30 ml of boiling water. The clear aqueous extracts obtained by centrifuging were evaporated to a small volume and taken in about 50 ml of 80% alcohol. The alcoholic extracts were filtered and the filtrates concentrated to a small volume (1.5-2.0 ml). Hippuric acid in the extract was detected by chromatography of 40 ~1 aliquots using the solvent n-butanol : acetic acid : water (4 : 1 : 1). Hippuric acid on the chromatograms was detected by its characteristic property of azlactone formation with p-dimethylaminobenzaldehyde-acetic anhydride reagent (GAFFNEY et al., 1954). This method also proved useful for estimating the hippuric acid formation and hippuricase activity of tissue since recovery of added hippuric acid to tissue homogenates was complete in the range studied S-50 pg. Hippuric acid formation. Suitable aliquots of crude homogenates prepared from the tissues of silkworms were incubated with benzoate and glycine in the presence of cysteine, MgSO,, and ATP (details of incubations are specified in the Tables and Figs.). The reaction was stopped by adding alcohol to SO”,/0 concentration. The precipitated protein was centrifuged off, and suitable aliquots were concentrated ten times by evaporating to dryness on a water bath and taking up the residue in 10% isopropanol. Aliquots of 30 ~1 were used for spotting. The orange-coloured azlactone obtained by spraying with p-dimethylaminobenzaldehyde reagent (4% in acetic anhydride containing a few crystals of sodium acetate) was extracted into 6.0 ml of redistilled methanol and the colour measured in a Bausch and Lomb Spectronic 20 Spectrophotometer at 460 mp (GAFFNEY et al., 1954). All reactions were carried out in duplicate. Hippuricase actiwity. Aliquots of the crude tissue homogenates were incubated with buffer and hippurate. After incubation, the enzyme was inactivated by adding alcohol to 50% concentration. The precipitated protein was centrifuged off and the residual hippuric Hippuricase activity could also acid in the supernatant was estimated as described above. This method proved satisfactory when be estimated by measuring the glycine released. dialysed extracts were used. In the present experiments, however, undialysed extracts were used and hence the former method of estimating the residual hippuric acid was used. RESULTS
Fig. 1 shows the response of silkworms to different concentrations of benzoate and hippurate at the end of 4 days, and it is evident from this that benzoate is toxic to the silkworm when solutions above 2% in concentration are applied externally on mulberry leaves (P = O-02). But hippurate at similar concentrations is not toxic. On the other hand, there is a significant stimulation in growth (P = O*OZ). The role of glycine in the metabolism of benzoate was also investigated and the results recorded are presented in Table 1. These are the consolidated results of two experiments. Since each experiment had eight replicates the averages presented are for sixteen replicates. It is seen that at the low level of benzoic acid there is no significant effect on growth. Glycine, on the other hand, stimulates growth (P = 0.01). Feeding of benzoate and glycine together decreases the stimulatory influence due to glycine, probably because part of it is being utilized for the synthesis of hippuric acid.
DETOXICATION OF BENZOATE BY GLYCINECONJUCATION IN THE SILKWORM
387
TABLE ~-EFFECT OF BENZOATE,GLYCINE,AND HIPPURATEON GROWTHOF THE SILKWORM (expressed as mg/S larvae on the fourth day of fourth instar)
Av. weight and standard error P (w.r.t. water)
Water
Benzoate 0.05 M
Glycine 0.05 M
Benzoate (0.05 M) plus glycine (0.05 M)
Hippurate (0.05 M)
965 f 5.6 -
984 +_33.6 ns.
1047 f 20.9 0.01
1004 f 23.3 n.s.
1021 f 34.2 n.s.
ns. = not significant.
Analysis of the excreta showed that normal worms do not excrete hippuric acid. Since the present mode of detection is both specific and sensitive (quantities as low as 2 pg can be detected visually), it is reasonable to conclude that the
-
Hippurole
-
Benmate
* 0
670
I
0
1 -
I 3 2 CoNcN OF SUPPLE~~ENT I
1
4 IN 7.
I 5
FIG. 1. Effect of benzoate and hippurate on growth of the silkworm (weights on fifth day of fourth instar).
silkworm does not normally excrete hippuric acid. FRIEDLER and SMITH (1954) had made similar observations on the locust. It would appear from these data that hippuric acid is not a normal constituent of the excreta of phytophagous insects. It must, however, be mentioned here that hippuric acid was present in traces in the intestines and the fat body of the silkworm inasmuch as homogenate blanks used in enzyme studies revealed its presence. Feeding of benzoate alone or in combination with glycine led to the excretion of hippuric acid in appreciable quantities.
388
M. IS. SHYAMALA
Enzyme activity studies on both hippuric acid formation and its breakdown were made on intestinal tissue, fat body, and posterior silk gland. Synthetic activity was higher in the intestines than in the fat body. It is possible that the synthesis of hippuric acid (in the fat body) is masked as a result of its hippuricase activity. The silk gland did not show any hippuric acid synthetase activity (Table 2). The results of detailed experiments conducted with intestinal homogenates on the effect TABLE
2-HIPPURIC
ACID SYNTHESIS,
HIPPURICASE
SILKWORM
Enzyme
activity
AND
Intestine
Hippuric acid synthesis* Hippuricaset Amino acylasez
ACYLASE ACTIVITY IN
AMINO
TISSUE
Fat
43.3 nil 110
body
Silk gland
18.6 168 113
nil 134.6 3200
* Expressed as pg of hippuric acid formed/mg of nitrogen. Incubation mixture: benzoate (0.1 M), 0.1 ml; glycine (0.1 M), 0.1 ml; ATP (0.05 M), 0.1 ml; MgS04 (0.05 M) 0.1 ml; cysteine (0.05 M), 0.1 ml; M/l5 phosphate buffer (pH 8.0), 0.5 ml; homogenate I.0 ml; time 2 hr; temp. 30°C. 7 Expressed as pg of hippuric acid cleaved/mg of nitrogen. Hippurate (0.1 M), 0.1 ml; M/l5 phosphate buffer (pH 8.0), 0.4 ml; homogenate, I.0 ml; time 2 hr; temp. 30°C. 1 Taken from BHEEMESWAR (1954). Activity expressed as pg of leucine formed/mg of nitrogen. buffer
(pH
Chloroacetyl-leucine 7.4), 0.05 ml; time
(l.O%),
0.1 ml;
1 hr; temp.
homogenate,
0.05
ml
;
M/15
phosphate
37°C.
of substrate concentrations using equimolar concentrations of glycine and benzoate are presented in Table 3. It is evident from Table 3 that much larger concentrations of benzoate were required for activity than those used in the rat enzyme studies (BORSOOK and DUBNOFF, 1940; CHANTRENNE, 1955). TABLE
j--EFFECT
OF SUBSTRATE CONCENTRATION
Final concentration of benzoate and glycine Total hippuric acid produced in _wg
0.01 M 25.0
0.02 M 50.0
0.025 65.0
ON HIPPURIC
M
0.03 M 120.0
ACID SYNTHESIS
0.035 95.0
M
0.04 M 85.0
Incubation mixture: benzoate, 0.1 ml; glycine, 0.1 ml; ATP (0.05 M), 0.1 ml; MgSO, (0.05 M), 0.1 ml; cysteine (0.05 M), 0.1 ml; homogenate prepared in M/l5 phosphate buffer (pH 8.0), 0.5 ml; time 2 hr; temp. 30°C.
The enzyme activity was found to increase gradually from pH 8.0 to 10.5, the maximum pH at which the activity was studied in the present series of experiments (Fig. 2). This can be readily explained on the basis of the high alkaline pH of the silkworm gut. The enzyme is well suited to act in this range, whereas for rat liver From the progress of the reaction enzyme a pH of 7.5 is used (CHANTRENNE, 1955). with time it is evident that the reaction is complete in 2 hr (Fig. 3).
DETOXICATION
OF BENZOATE
BY
GLYCINE
CONJUGATION
IN
THE
SILKWORM
389
Acetone powder preparations of silkworm intestines were found to require coenzyme A for activity unlike the rat liver acetone powder (CHANTRENNE, 1955).
201 %O
I
I
70
%O
-c
1 9.0
I
100
I ll.0
P”
FIG. 2. Effect of pH on hippuric acid synthesis. Incubation mixture: benzoate (0.7 M), 0.1 ml; glycine (O-7 M), 0.1 ml; ATP (0.05 M), 0.1 ml; MgSO, (0.05 M), 0.1 ml; cysteine (0.05 M), O-1 ml; homogenate, O-5 ml; buffer, 0.5 ml. From pH 5.0 to 8.0, phosphate buffer (M/15) used; for pH 8.6 and 9.0 tris buffer (O-2 M) used. From pH 9.5 to 10.5 bicarbonate buffer (0.2 M) used; time, 2 hr; temp. 30°C.
Hippuricase activity was found in fat body and silk-gland tissue. Intestinal tissue, however, did not possess this activity (Table 2). In the past, hippuricase activity has been attributed to the amino acylases (LEUTHARDT, 1951). BHEEMESWAR
FIG. 3. Time (O-7 M), 1-O ml; 1.0 ml; cysteine buffer, pH 10.0);
course of hippuric acid synthesis. Incubation mixture: benzoate glycine (0.7 M), 1.0 ml; ATP (O-05 M), 1.0 ml; MgSO, (0.05 M), (0.05 M), 1.0 ml; homogenate, 5.0 ml (prepared in bicarbonate temp. 30°C. Aliquots of 1.0 ml each inactivated at different intervals.
M. B. SHYAMALA
390
(1954) made a study of amino acylases in the silkworm. Using chloracetyl-L-leucine as the substrate, he reported amino acylase activity in intestines, fat body, body fluid, and silk-gland extracts, especially high activity being recorded with silk-gland extracts. For comparison, the amino acylase activity in the different tissues is also included in Table 2. Acetone powder preparations of fat body showed hippuricase activity, the activity tending to be maximum at neutral pH (Table 4). TABLE 4-EFFECX
OF pH ON HIPPURICASEACTIVITY OF ACETONEPOWDEREXTRACTOF FAT BODY
PH Hipp. acid cleaved*
5.0 nil
OF THE
SILKWORM
6.0 nil
7.0 60.8
8.0 56.1
9.0 19.4
* Expressed as pg/lO mg of acetone powder. DISCUSSION
From the growth studies it is brought to light that hippurate is less inhibitory to the silkworm than benzoate. In fact, hippurate at low concentration stimulates growth. When benzoate is fed to the silkworm, part of it is converted to hippurate and is eliminated in this form. In silkworms fed normally on mulberry leaves, however, hippuric acid is not excreted, although hippuric acid can be detected in the intestines and fat body. It would therefore appear that at normal physiological levels of hippuric acid in the silkworm, the benzoate released on hydrolysis, not being high enough to inhibit growth, is eliminated as such. In fact, this would enable the organism to conserve glycine which is valuable for the silkworm both for growth as well as silk synthesis. The growth studies also reveal the significant stimulatory etiect of glycine. Hippuric acid would then act as an endogenous source of glycine. The presence of hippuricase activity in the fat body and silk gland ensures such an endogenous, hydrolytic release of glycine as occasion demands. The presence of both the synthetic and hydrolytic activity bestows on the organism not only a protective mechanism aimed at detoxication but also a means to conserve the glycine moiety of hippuric acid under normal conditions when such protection is not needed. It is also possible that the synthetic activity is not restricted to the substrates benzoate and glycine alone, in which event synthesis of other amino acid conjugates of physiological significance might also be expected to be mediated by this. This point, however, cannot be answered at this stage since very little is known about the specificity of the enzymes involved. The conditions required for optimum activity are different from those observed in rat liver (BORSOOK and DUBNOFF, 1940). The silkworm enzyme extracts required much higher concentrations of benzoate (0.03 M) as compared to rat liver (0.0025 M). The time taken for complete reaction was about 2 hr with the former and 4 hr with the latter. The silkworm enzyme acts at higher pH than the rat liver enzyme.
DETOXICATION OFBENZOATE BY GLYCINECONJUGATION IN THESILKWORM
391
Doubts regarding the existence of a specific hippuric acid cleaving enzyme as distinct from amino acylases have long been expressed (LEUTHARDT, 1951). The same doubt also arises with the silkworm, since high amino acylase activity is also encountered in the organs. However, it is apparent from Table 3 that even if the two activities could be attributed to one and the same enzyme, the specificity of the amino acylases of the different organs is not the same. Although a strict, quantitative comparison cannot be made, the extreme case of intestinal tissue which shows moderate amino acylase but not hippuricase activity amply justifies the conclusion. If amino acylase is also responsible for hippuricase activity this would lead to the question of whether multiple forms of enzymes (isoenzymes) are present in the silkworm (cf. RAMAKRISHNAN, 1962). The presence of multiple forms of esterase and lactic dehydrogenase have, in fact, been demonstrated in Hylaphora, Cecropia, and Cynthia silkworms (LAUFLER, 1961). Purification of the enzymes from the different tissues, and a study of the two activities at different stages of purification and with different inhibitors, might provide fruitful lines of investigations in elucidating this problem. Acknowledgements-The author wishes to thank Dr. J. V. BHAT, Assistant Professor, Fermentation Technology Laboratory, for his advice and keen interest in this work and the Director, Indian Institute of Science, for his keen interest. REFERENCES BHEEMESWAR B. (1954) Biochemical studies on the silkworm Bombyx mori, L. Ph.D thesis, Univ. Bombay, India. BORSOOKH. and DUBNOFFJ. W. (1940) The biological synthesis of hippuric acid in oitro. J. Biol. Chem. 132, 307-324. CASSIDAJ. E. (195.5) Toxicity of aromatic acids to the larvae of the mosquito Aedes aegypti L. and the counteracting influence of amino acids. Biochem. J. 59, 216-221. CHANTRENNEH. (1955) Hippuric acid synthesis. In Methods in Enzymology (Ed. by COLOWICKS. P. and KAPLANN. O.), Vol. II, pp. 346-350. FRIEDLERL. and SMITH J. N. (1954) Comparative detoxication: 3 Hippuric acid formation in adult locusts. Bi0chem.J. 57, 396400. GAFFNEYG. W., SCHREIERK., DIFERRANTE N., and ALTMANNK. (1954) The quantitative determination of hippuric acid. J. biol. Chem. 206, 695-698. GILMOURD. (1961) The Biochemistry ofInsects, p. 227. Acad. Press, New York. KIKKAWAH. (1953) Biochemical genetics of Bombyx mori, L., Silkworm. Advanc. Genet. 5, 107-140. LAUFLERH. (1961) Forms of enzymes in insect development. Ann. N.Y. Acud. Sci. 94, 825-835. LEUTHARDT F. (1951) Hippuricase. In The Enzymes (Ed. by SUMNERJ. B. and MYRBACKK.), Vol. I, Part 2, pp. 9.5-955. MURTHY M. R. V. (1954) Studies on the nutrition of the silkworm, Bombyxmori, L. Ph.D. thesis Univ. Mysore, India. RAMAKRISHNAN T. (1962) Isoenzymes. J. Sci. Industr. Res. 21A, 177-178. SMITH J. N. (19.55) Detoxication mechanisms in insects. Biol. Rev. 30,455475. SMITH J. N. (1962) Detoxication mechanisms. Annu. Rev. Ent. 7, 46.5480.
Printed in G+eaz B&air
DETOXICATION OF BENZOATE CONJUGATION IN THE SILKWORM,
BY GLYCINE BOMBYX MORI L.
M. B. SHYAMALA Fermentation
Technology
Laboratory,
Indian
Institute
of Science,
Bangalore
12
(Received 13 August 1963)
Abstract-The role of hippuric acid formation as a mechanism for detoxication of benzoic acid in the silkworm has been investigated. Benzoate is inhibitory to the growth of the silkworm and excreted as hippuric acid, which is not toxic. Hippuric acid is not a normal constituent of excreta. Synthesis of hippuric acid has been shown to occur in the intestines of the silkworm. Hippuricase activity is present in the fat body and silk-gland tissue.
INTRODUCTION
CONJUGATION of aromatic acids with glycine is a well-known mode of detoxication in higher animals. That the detoxication mechanism functions in the same manner in insects was demonstrated by FRIEDLER and SMITH (1954) in Locusta migratoriu and by CASSIDA (1955) in the mosquito A&es aegypti. CASSIDA (1955) was also able to demonstrate that when whole larvae were incubated with benzoate and glycine, hippurate was found in both the larvae as well as in the supernatant. In the silkworm itself, conjugation of anthranilic acid was found to precede excretion (KIKKAWA, 1953). The occurrence of glycine conjugation as a detoxicating mechanism was also suggested by the observations of MURTHY (1954). He found that the toxicity produced by feeding excess nicotinic acid was counteracted by an additional These studies did not, however, contain much information supplement of glycine. on the in vitro conjugation of aromatic acids (SMITH, 1955, 1962; GILMOUR, 1961) or on the sites of formation of such conjugates. Thus the nature of the enzymes involved in the process has not been understood in insects in general and in the The purpose of the present communication is to give silkworm in particular. details of experiments conducted to show (1) the relative non-toxicity of hippurate as compared to benzoate, (2) the excretion of hippurate in benzoate-fed larvae, (3) hippurate synthesis in silkworm tissues in vitro, and (4) the presence of hippuricase activity in the tissues. MATERIALS
AND
METHODS
(i) Growth studies These were conducted with the Mysore silkworm during the fourth instar. For each treatment eight replicates of five worms each were used. Benzoic and hippuric acids were fed as aqueous solutions of sodium salts (pH adjusted to 7-O). Aliquots of 0.1-0.2 ml were spread uniformly on mulberry leaves. In studying the relative toxicities of benzoate and 25
385
M. B. SHYAMALA
386
were used. For studying the effect of benzoate and glycine hippurate, l-5 y& concentrations individually and in combination a 0.05 M concentration of each of the substances was used. Weights were recorded daily till the fourth ecdysis. (ii) Analysis
of excretu
Excreta samples obtained in the growth experiments were collected daily in the morning and dried. The dry samples collected on the different days from the same treatments were pooled together. About 1.5 g portions of pooled samples were extracted with 30 ml of boiling water. The clear aqueous extracts obtained by centrifuging were evaporated to a small volume and taken in about 50 ml of 80% alcohol. The alcoholic extracts were filtered and the filtrates concentrated to a small volume (1.5-2.0 ml). Hippuric acid in the extract was detected by chromatography of 40 ~1 aliquots using the solvent n-butanol : acetic acid : water (4 : 1 : 1). Hippuric acid on the chromatograms was detected by its characteristic property of azlactone formation with p-dimethylaminobenzaldehyde-acetic anhydride reagent (GAFFNEY et al., 1954). This method also proved useful for estimating the hippuric acid formation and hippuricase activity of tissue since recovery of added hippuric acid to tissue homogenates was complete in the range studied S-50 pg. Hippuric acid formation. Suitable aliquots of crude homogenates prepared from the tissues of silkworms were incubated with benzoate and glycine in the presence of cysteine, MgSO,, and ATP (details of incubations are specified in the Tables and Figs.). The reaction was stopped by adding alcohol to SO”,/0 concentration. The precipitated protein was centrifuged off, and suitable aliquots were concentrated ten times by evaporating to dryness on a water bath and taking up the residue in 10% isopropanol. Aliquots of 30 ~1 were used for spotting. The orange-coloured azlactone obtained by spraying with p-dimethylaminobenzaldehyde reagent (4% in acetic anhydride containing a few crystals of sodium acetate) was extracted into 6.0 ml of redistilled methanol and the colour measured in a Bausch and Lomb Spectronic 20 Spectrophotometer at 460 mp (GAFFNEY et al., 1954). All reactions were carried out in duplicate. Hippuricase actiwity. Aliquots of the crude tissue homogenates were incubated with buffer and hippurate. After incubation, the enzyme was inactivated by adding alcohol to 50% concentration. The precipitated protein was centrifuged off and the residual hippuric Hippuricase activity could also acid in the supernatant was estimated as described above. This method proved satisfactory when be estimated by measuring the glycine released. dialysed extracts were used. In the present experiments, however, undialysed extracts were used and hence the former method of estimating the residual hippuric acid was used. RESULTS
Fig. 1 shows the response of silkworms to different concentrations of benzoate and hippurate at the end of 4 days, and it is evident from this that benzoate is toxic to the silkworm when solutions above 2% in concentration are applied externally on mulberry leaves (P = O-02). But hippurate at similar concentrations is not toxic. On the other hand, there is a significant stimulation in growth (P = O*OZ). The role of glycine in the metabolism of benzoate was also investigated and the results recorded are presented in Table 1. These are the consolidated results of two experiments. Since each experiment had eight replicates the averages presented are for sixteen replicates. It is seen that at the low level of benzoic acid there is no significant effect on growth. Glycine, on the other hand, stimulates growth (P = 0.01). Feeding of benzoate and glycine together decreases the stimulatory influence due to glycine, probably because part of it is being utilized for the synthesis of hippuric acid.
DETOXICATION OF BENZOATE BY GLYCINECONJUCATION IN THE SILKWORM
387
TABLE ~-EFFECT OF BENZOATE,GLYCINE,AND HIPPURATEON GROWTHOF THE SILKWORM (expressed as mg/S larvae on the fourth day of fourth instar)
Av. weight and standard error P (w.r.t. water)
Water
Benzoate 0.05 M
Glycine 0.05 M
Benzoate (0.05 M) plus glycine (0.05 M)
Hippurate (0.05 M)
965 f 5.6 -
984 +_33.6 ns.
1047 f 20.9 0.01
1004 f 23.3 n.s.
1021 f 34.2 n.s.
ns. = not significant.
Analysis of the excreta showed that normal worms do not excrete hippuric acid. Since the present mode of detection is both specific and sensitive (quantities as low as 2 pg can be detected visually), it is reasonable to conclude that the
-
Hippurole
-
Benmate
* 0
670
I
0
1 -
I 3 2 CoNcN OF SUPPLE~~ENT I
1
4 IN 7.
I 5
FIG. 1. Effect of benzoate and hippurate on growth of the silkworm (weights on fifth day of fourth instar).
silkworm does not normally excrete hippuric acid. FRIEDLER and SMITH (1954) had made similar observations on the locust. It would appear from these data that hippuric acid is not a normal constituent of the excreta of phytophagous insects. It must, however, be mentioned here that hippuric acid was present in traces in the intestines and the fat body of the silkworm inasmuch as homogenate blanks used in enzyme studies revealed its presence. Feeding of benzoate alone or in combination with glycine led to the excretion of hippuric acid in appreciable quantities.
388
M. IS. SHYAMALA
Enzyme activity studies on both hippuric acid formation and its breakdown were made on intestinal tissue, fat body, and posterior silk gland. Synthetic activity was higher in the intestines than in the fat body. It is possible that the synthesis of hippuric acid (in the fat body) is masked as a result of its hippuricase activity. The silk gland did not show any hippuric acid synthetase activity (Table 2). The results of detailed experiments conducted with intestinal homogenates on the effect TABLE
2-HIPPURIC
ACID SYNTHESIS,
HIPPURICASE
SILKWORM
Enzyme
activity
AND
Intestine
Hippuric acid synthesis* Hippuricaset Amino acylasez
ACYLASE ACTIVITY IN
AMINO
TISSUE
Fat
43.3 nil 110
body
Silk gland
18.6 168 113
nil 134.6 3200
* Expressed as pg of hippuric acid formed/mg of nitrogen. Incubation mixture: benzoate (0.1 M), 0.1 ml; glycine (0.1 M), 0.1 ml; ATP (0.05 M), 0.1 ml; MgS04 (0.05 M) 0.1 ml; cysteine (0.05 M), 0.1 ml; M/l5 phosphate buffer (pH 8.0), 0.5 ml; homogenate I.0 ml; time 2 hr; temp. 30°C. 7 Expressed as pg of hippuric acid cleaved/mg of nitrogen. Hippurate (0.1 M), 0.1 ml; M/l5 phosphate buffer (pH 8.0), 0.4 ml; homogenate, I.0 ml; time 2 hr; temp. 30°C. 1 Taken from BHEEMESWAR (1954). Activity expressed as pg of leucine formed/mg of nitrogen. buffer
(pH
Chloroacetyl-leucine 7.4), 0.05 ml; time
(l.O%),
0.1 ml;
1 hr; temp.
homogenate,
0.05
ml
;
M/15
phosphate
37°C.
of substrate concentrations using equimolar concentrations of glycine and benzoate are presented in Table 3. It is evident from Table 3 that much larger concentrations of benzoate were required for activity than those used in the rat enzyme studies (BORSOOK and DUBNOFF, 1940; CHANTRENNE, 1955). TABLE
j--EFFECT
OF SUBSTRATE CONCENTRATION
Final concentration of benzoate and glycine Total hippuric acid produced in _wg
0.01 M 25.0
0.02 M 50.0
0.025 65.0
ON HIPPURIC
M
0.03 M 120.0
ACID SYNTHESIS
0.035 95.0
M
0.04 M 85.0
Incubation mixture: benzoate, 0.1 ml; glycine, 0.1 ml; ATP (0.05 M), 0.1 ml; MgSO, (0.05 M), 0.1 ml; cysteine (0.05 M), 0.1 ml; homogenate prepared in M/l5 phosphate buffer (pH 8.0), 0.5 ml; time 2 hr; temp. 30°C.
The enzyme activity was found to increase gradually from pH 8.0 to 10.5, the maximum pH at which the activity was studied in the present series of experiments (Fig. 2). This can be readily explained on the basis of the high alkaline pH of the silkworm gut. The enzyme is well suited to act in this range, whereas for rat liver From the progress of the reaction enzyme a pH of 7.5 is used (CHANTRENNE, 1955). with time it is evident that the reaction is complete in 2 hr (Fig. 3).
DETOXICATION
OF BENZOATE
BY
GLYCINE
CONJUGATION
IN
THE
SILKWORM
389
Acetone powder preparations of silkworm intestines were found to require coenzyme A for activity unlike the rat liver acetone powder (CHANTRENNE, 1955).
201 %O
I
I
70
%O
-c
1 9.0
I
100
I ll.0
P”
FIG. 2. Effect of pH on hippuric acid synthesis. Incubation mixture: benzoate (0.7 M), 0.1 ml; glycine (O-7 M), 0.1 ml; ATP (0.05 M), 0.1 ml; MgSO, (0.05 M), 0.1 ml; cysteine (0.05 M), O-1 ml; homogenate, O-5 ml; buffer, 0.5 ml. From pH 5.0 to 8.0, phosphate buffer (M/15) used; for pH 8.6 and 9.0 tris buffer (O-2 M) used. From pH 9.5 to 10.5 bicarbonate buffer (0.2 M) used; time, 2 hr; temp. 30°C.
Hippuricase activity was found in fat body and silk-gland tissue. Intestinal tissue, however, did not possess this activity (Table 2). In the past, hippuricase activity has been attributed to the amino acylases (LEUTHARDT, 1951). BHEEMESWAR
FIG. 3. Time (O-7 M), 1-O ml; 1.0 ml; cysteine buffer, pH 10.0);
course of hippuric acid synthesis. Incubation mixture: benzoate glycine (0.7 M), 1.0 ml; ATP (O-05 M), 1.0 ml; MgSO, (0.05 M), (0.05 M), 1.0 ml; homogenate, 5.0 ml (prepared in bicarbonate temp. 30°C. Aliquots of 1.0 ml each inactivated at different intervals.
M. B. SHYAMALA
390
(1954) made a study of amino acylases in the silkworm. Using chloracetyl-L-leucine as the substrate, he reported amino acylase activity in intestines, fat body, body fluid, and silk-gland extracts, especially high activity being recorded with silk-gland extracts. For comparison, the amino acylase activity in the different tissues is also included in Table 2. Acetone powder preparations of fat body showed hippuricase activity, the activity tending to be maximum at neutral pH (Table 4). TABLE 4-EFFECX
OF pH ON HIPPURICASEACTIVITY OF ACETONEPOWDEREXTRACTOF FAT BODY
PH Hipp. acid cleaved*
5.0 nil
OF THE
SILKWORM
6.0 nil
7.0 60.8
8.0 56.1
9.0 19.4
* Expressed as pg/lO mg of acetone powder. DISCUSSION
From the growth studies it is brought to light that hippurate is less inhibitory to the silkworm than benzoate. In fact, hippurate at low concentration stimulates growth. When benzoate is fed to the silkworm, part of it is converted to hippurate and is eliminated in this form. In silkworms fed normally on mulberry leaves, however, hippuric acid is not excreted, although hippuric acid can be detected in the intestines and fat body. It would therefore appear that at normal physiological levels of hippuric acid in the silkworm, the benzoate released on hydrolysis, not being high enough to inhibit growth, is eliminated as such. In fact, this would enable the organism to conserve glycine which is valuable for the silkworm both for growth as well as silk synthesis. The growth studies also reveal the significant stimulatory etiect of glycine. Hippuric acid would then act as an endogenous source of glycine. The presence of hippuricase activity in the fat body and silk gland ensures such an endogenous, hydrolytic release of glycine as occasion demands. The presence of both the synthetic and hydrolytic activity bestows on the organism not only a protective mechanism aimed at detoxication but also a means to conserve the glycine moiety of hippuric acid under normal conditions when such protection is not needed. It is also possible that the synthetic activity is not restricted to the substrates benzoate and glycine alone, in which event synthesis of other amino acid conjugates of physiological significance might also be expected to be mediated by this. This point, however, cannot be answered at this stage since very little is known about the specificity of the enzymes involved. The conditions required for optimum activity are different from those observed in rat liver (BORSOOK and DUBNOFF, 1940). The silkworm enzyme extracts required much higher concentrations of benzoate (0.03 M) as compared to rat liver (0.0025 M). The time taken for complete reaction was about 2 hr with the former and 4 hr with the latter. The silkworm enzyme acts at higher pH than the rat liver enzyme.
DETOXICATION OFBENZOATE BY GLYCINECONJUGATION IN THESILKWORM
391
Doubts regarding the existence of a specific hippuric acid cleaving enzyme as distinct from amino acylases have long been expressed (LEUTHARDT, 1951). The same doubt also arises with the silkworm, since high amino acylase activity is also encountered in the organs. However, it is apparent from Table 3 that even if the two activities could be attributed to one and the same enzyme, the specificity of the amino acylases of the different organs is not the same. Although a strict, quantitative comparison cannot be made, the extreme case of intestinal tissue which shows moderate amino acylase but not hippuricase activity amply justifies the conclusion. If amino acylase is also responsible for hippuricase activity this would lead to the question of whether multiple forms of enzymes (isoenzymes) are present in the silkworm (cf. RAMAKRISHNAN, 1962). The presence of multiple forms of esterase and lactic dehydrogenase have, in fact, been demonstrated in Hylaphora, Cecropia, and Cynthia silkworms (LAUFLER, 1961). Purification of the enzymes from the different tissues, and a study of the two activities at different stages of purification and with different inhibitors, might provide fruitful lines of investigations in elucidating this problem. Acknowledgements-The author wishes to thank Dr. J. V. BHAT, Assistant Professor, Fermentation Technology Laboratory, for his advice and keen interest in this work and the Director, Indian Institute of Science, for his keen interest. REFERENCES BHEEMESWAR B. (1954) Biochemical studies on the silkworm Bombyx mori, L. Ph.D thesis, Univ. Bombay, India. BORSOOKH. and DUBNOFFJ. W. (1940) The biological synthesis of hippuric acid in oitro. J. Biol. Chem. 132, 307-324. CASSIDAJ. E. (195.5) Toxicity of aromatic acids to the larvae of the mosquito Aedes aegypti L. and the counteracting influence of amino acids. Biochem. J. 59, 216-221. CHANTRENNEH. (1955) Hippuric acid synthesis. In Methods in Enzymology (Ed. by COLOWICKS. P. and KAPLANN. O.), Vol. II, pp. 346-350. FRIEDLERL. and SMITH J. N. (1954) Comparative detoxication: 3 Hippuric acid formation in adult locusts. Bi0chem.J. 57, 396400. GAFFNEYG. W., SCHREIERK., DIFERRANTE N., and ALTMANNK. (1954) The quantitative determination of hippuric acid. J. biol. Chem. 206, 695-698. GILMOURD. (1961) The Biochemistry ofInsects, p. 227. Acad. Press, New York. KIKKAWAH. (1953) Biochemical genetics of Bombyx mori, L., Silkworm. Advanc. Genet. 5, 107-140. LAUFLERH. (1961) Forms of enzymes in insect development. Ann. N.Y. Acud. Sci. 94, 825-835. LEUTHARDT F. (1951) Hippuricase. In The Enzymes (Ed. by SUMNERJ. B. and MYRBACKK.), Vol. I, Part 2, pp. 9.5-955. MURTHY M. R. V. (1954) Studies on the nutrition of the silkworm, Bombyxmori, L. Ph.D. thesis Univ. Mysore, India. RAMAKRISHNAN T. (1962) Isoenzymes. J. Sci. Industr. Res. 21A, 177-178. SMITH J. N. (19.55) Detoxication mechanisms in insects. Biol. Rev. 30,455475. SMITH J. N. (1962) Detoxication mechanisms. Annu. Rev. Ent. 7, 46.5480.