Phenylalanine metabolism in cockroaches, Periplaneta americana: Tyrosine and benzoyl-β-glucoside biosynthesis

Comp. Biochem. Physiol., 1970, Vol. 36, pp. 535 to 545. PergamonPress. Printed in Great Britain

PHENYLALANINE METABOLISM IN COCKROACHES, P E R I P L A N E T A A M E R I C A N A : TYROSINE AND BENZOYL-/%GLUCOSIDE BIOSYNTHESIS* L. L. MURDOCK~f, T. L. H O P K I N S and R. A. W I R T Z Department of Entomology, Kansas State University, Manhattan, Kansas 66502 (Received 31 March 1970) Abstract--1. Teneral adult male cockroaches, Periplaneta americana, produced more 14CO8 from phenylalanine-l-'4C than did the mature, fully tanned males. This would indicate utilization of phenylalanine-derived tyrosine for Nacetyl-dopamine biosynthesis during the time of cuticular sclerotization. 2. Tyrosine biosynthesis from phenylalanine occurred from ecdysis throughout adult life. Less tyrosine accumulated during the teneral period due to increased utilization for cuticular tanning. 3. A second major metabolite of phenylalanine was benzoic acid, most of which was conjugated as benzoyl-fl-glucoside. The glucoside was not produced during the teneral period, suggesting changes in phenylalanine metabolism during ecdysis similar to those of tyrosine. 4. Phenylpyruvic acid-U-t4C was rapidly metabolized to phenylalanine and tyrosine, indicating a transaminase in cockroach tissues with the equilibrium of transamination in the direction of the amino acid. INTRODUCTION

ALTHOUGH phenylalanine is an essential amino acid for most insect species thus far studied (House, 1965), its metabolic fate has been examined in only a few cases. Tyrosine is formed from phenylalanine in larval Chilo suppressalis (Ishii & Hirano, 1958), Bombyx mori (Bricteaux-Gr6goire et al., 1956; Fukuda, 1956; Belzecka et al., 1964), and Calliphora erythrocephala (Belzecka et al., 1964; Price, 1965); and in larvae of Celerio euphorbia, Ephestia kuehniella, and Carausius morosus (Belzecka et al., 1964). Tyrosine biosynthesis is catalyzed by a phenylalanine 4-hydroxylase (E.C. 1.99.1.2) similar in its cofactor requirements to the vertebrate enzyme (Belzecka et al., 1964). Tyrosine may be metabolized by alternate pathways, depending upon the stage of development (Karlson et al., 1962). At the time of cuticular sclerotization it is utilized for biosynthesis of N-acetyldopamine, the tanning quinone precursor in the insect species so far examined. Between ecdyses, tyrosine metabolism apparently takes a degradative pathway leading to a series of * Contribution No. 1021, Department of Entomology, Kansas Agricultural Experiment Station, Manhattan 66502. Research supported in part by National Institutes of Health Grant A1-04643 to T. L. H. and by a National Aeronautics and Space Administration Pre-doctoral Traineeship to L. L. M. t Present address: Fachbereich Biologie, Universit~it Konstanz, 775 Konstanz, Germany. 535

536

L. L. MtmnocK, T. L. HOPKINSAND R. A. Wmrz

hydroxyphenylcarboxylic acids. T h e shift in tyrosine metabolism has been shown to occur in several species: C. erythrocephala (Karlson et aL, 1962; Sekeris & Karlson, 1962); Locusta migratoria (Karlson & Herrlich, 1965); Tenebrio molitor and Drosophila melanogaster (Sekeris & Herrlich, 1966), and Periplaneta americana (Murdock et al., 1970a). Because phenylalanine is a precursor of tyrosine and tyrosine is rapidly utilized at the time of ecdysis, it seemed possible that phenylalanine metabolism might also vary quantitatively and/or qualitatively, paralleling changes in tyrosine utilization. With this working hypothesis, we investigated the metabolism of phenylalanine in the cockroach in relation to ecdysis and maturation. MATERIALS AND METHODS

Insect cultures Periplaneta americana were reared and maintained at 27°C in tubs having a layer of wood shavings; water and laboratory-animal pellets were provided ad lib. The photoperiod regimen was 16 hr of light and 8 hours of dark. Adults of known age were obtained by collecting newly ecdysed insects and isolating them with food and water in pint jars. In some experiments tanned adult males of unknown age were taken directly from the colony; only those that had lost the characteristic softness, which persists several days after the imaginal ecdysis, were selected.

Radiosotope-labeled compounds Solutions of L-phenylalanine-l-laC (52.6 mc/mM), DL-phenylalanine-l-ring-14C (3-48 mc/mM) (New England Nuclear Corp., Boston, Mass.), and L-phenylalanine-U-14C (459 mc/mM) (Amersham/Searle, Des Plaines, ILL), in distilled water were checked regularly for radiochemical purity by several paper chromatographic systems. The 14C-labeled compound was injected into the abdominal haemocoele in 5-10/zl of solution using a Hamilton 50/zl syringe with a stainless steel plunger and needle. Phenylpyruvic acid-U-14C was synthesized as outlined by Meister (1957). To a 1-mlvol. flask, held at 23°C, 0-5 ml of 0"2M Tris-HCl buffer, pH 7"2 was added, followed by 2/~1 catalase (206,500 U/ml, Mann Res. Labs., New York, N.Y.), 50/zl L-phenylalanineU-14C (3"1 /zc), then 2/~1 Crotalus adamentus venom L-amino acid oxidase (0"1 unit, Mann Res. Labs., New York, N.Y.). The mixture was incubated with occasional swirling for one hour and then frozen until used. The yield of phenylpyruvic acid, shown by paper chromatography, was > 98"5 per cent. No further purification of the produce was necessary, because freezing destroys L-amino acid oxidase. Injecting the cockroaches with small volumes of Tris buffer had no observable effect on them. To check buffer effects, parallel experiments were done using 0.2 M, pH 7.2, phosphate buffer in the synthesis ; no differences were found.

Radiorespirometry With radiorespirometry techniques the oxidative metabolism of specific carbon atoms of a labeled precursor can be traced by observing the production of x~CO~. The technique has the particular advantage of permitting metabolism to be traced in vivo with insects in different physiological states. Combined with such techniques as paper or thin-layer chromatography for identification metabolites, the method provides substantial insight about the fate of a given compound. The techniques used were those of Murdock et al. (1970a).

Extraction Cockroaches (4 ml/insect of 80% methanol in water containing 20 mM ascorbic acid) were homogenized at 0°C in a Sorvall Omnimixer for 5 rain. The crude homogenate was

PHENYLALANINE METABOLISM IN COCKROACHES

537

centrifuged at 10,000 g for 10 min at 0-2°C; the supernatant fluid removed and used directly for chromatography. In some cases, portions of the extract were taken to dryness and hydrolyzed with 6N HC1 for 18-20 hr at 100°C. The HC1 was removed in vacuo and the residue taken up in a small volume of methanol. To check for/g-glucoside conjugates of phenylalanine metabolites, fl-glucosidase was incubated with whole extracts or with metabolites isolated by paper chromatography. The method was essentially that of Pau and Acheson (1968): methanol aliquots containing extractives or metabolites were evaporated to dryness in tubes and then, for 4 hr at 37°C, incubated with 1"0 ml 0-065 M acetate buffer, pH 5, containing 0-1 mg/ml of/3-glucosidase (Mann Res. Labs., New York, N.Y.). Controls contained only buffer or enzyme inactivated by boiling. The reaction was stopped by adding 3 ml methanol; the mixture then was evaporated to dryness in vacuo and the residue redissolved in a small volume of methanol for chromatography.

Paper chromatography Ascending paper chromatography on Whatman No. 1 paper strips was used to identify phenylalanine metabolites. Extracts were streaked 3 cm from the lower edge and the mobile phases allowed to ascend 20 cm. Solvent systems used were: (1) n-butanol-glacial acetic acid-H~O (4 : 1 : 5); (2) n-butanol saturated with 1N HC1; (3) aqueous phenol (88%)-con. HC1-KCN (400 ml : 1 ml : 20 mg) saturated with SO2; (4) n-butanol saturated with 2 N NH4OH; (5) tert-amyl alcohol saturated with H~O; (6) n-butanol-95% ethanol-HzO ( 2 : 2 : 1 ) ; (7) n-butanol saturated with 1"5 N NH4OH and (NH,)~COs. Radioactive areas on the strips were located by a windowless, 4zr-chromatogram scanner. In most cases, radioactive metabolites were identified by eluting radioactive areas from the strips and rechromatographing recovered material with standard compounds; basis for identification was correspondence of RI values of the radioactive peaks with the standard chemicals in three or more chromatographic systems. Standards of tyrosine and its derivatives were located on the chromatograms as described in an earlier study (Murdock et al., 1970a). Phenylalanine was detected with ninhydrin, hippuric acid by viewing under filtered ultraviolet light (254 m/z), and synthetic 14C-benzoic acid by radiochromatogram scanning. RESULTS

14CO2 production from L-phenylalanine-l-14C with age Previously we have shown that 14CO2 i s produced m u c h more extensively when L-tyrosine-l-l*C is injected just after ecdysis than in older, fully tanned cockroaches ( M u r d o c k et al., 1970a). T o determine if a parallel increase occurs with laCOz f r o m the 1-carbon of the phenylalanine side chain, similar experiments were p e r f o r m e d by injecting teneral and m a t u r e adult males with 1-6/zg phenylalanine1 J * C per insect. T h e latter group was 7 or m o r e days past imaginal ecdysis. Results showed that oxidation of the phenylalanine carboxyl carbon was greater shortly after ecdysis; 14CO~ production reached an earlier peak and remained higher t h r o u g h o u t the experimental period, c o m p a r e d with tanned insects (Fig. 1). I n that phenylalanine, the immediate precursor of tyrosine, showed similar decarboxylation patterns in relation to cuticular tanning, apparently N - a c e t y l d o p a mine produced after ecdysis is biosynthesized not only f r o m the pre-existing tyrosine pool, but also f r o m tyrosine newly synthesized f r o m phenylalanine.

Biosynthesis of tyrosine F o r direct evidence of tyrosine biosynthesis f r o m phenylalanine in Periplaneta, we injected a group of tanned adult males, which had passed the imaginal ecdysis

538

L . L . MURDOCK, T. L. HOPKINS AND R. A. WIRTZ

7 or more days earlier, with L-phenylalanine-l-14C; extracted after 12 hr; the extract was hydrolyzed to liberate peptide-incorporated or bound activity. Paper chromatograms were then prepared. The 1J4C-labelled precursor was particularly useful because preliminary experiments had shown that injection of L-tyrosine-l14C did not result in any radiolabeled metabolites in male cockroaches; only tyrosine, both free and peptide incorporated, was recovered. Therefore, if tyrosine is formed from phenylalanine-l-x*C, it alone except for the injected phenylalanine should show a radioactivity area on the chromatograms. Additional peaks would represent alternate metabolites of phenylalanine retaining the carboxyl carbon. 3.01

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TOTAL %

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2.0

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 MINUTES (x 100)

FIG. 1. x4CO=production from adult male P. americana injected with g-phenylalanine-l-14C shortly after ecdysis (0-(3) and after maturity (0-0). Vertical lines are _+ 1 S.E. The only radioactive compound, other than that of the injected phenylalanine, corresponded in R 1 value to tyrosine by several paper chromatographic systems. The amount of radioactive tyrosine was slightly greater than phenylalanine 12 hr after injection, suggesting extensive biosynthesis. The absence of other radioactive compounds indicates that the only 3-carbon side-chain metabolite of phenylalanine in fully tanned cockroaches is tyrosine. Before the extract was hydrolyzed, chromatograms exhibited radioactivity near the origin. Presumably this reflected only the labeled amino acids incorporated into peptides, because the peaks disappeared after hydrolysis, with a concomitant increase of the tyrosine and phenylalanine peaks; no new peaks appeared. To verify further the identity of the amino acids, extracts from 24-day-old cockroaches were chromatographed and the radioactive zones corresponding to

539

P H E N Y L A L A N I N E M E T A B O L I S M I N COCKROACHES

phenylalanine and tyrosine were cut out, eluted with methanol, and cochromatographed individually (by several systems) with nonlabeled, standard amino acids. In all cases the radiolabeled compounds corresponded to the colored zones of the standards (Table 1). TABLE 1--CocHROMATOGRAPHY OF EXTRACTS FROM PHENYLALANINE-U-14C INJECTED ADULT MALE

P. americana

W I T H P H E N Y L A L A N I N E AND TYROSINE STANDARDS

Chromatographic system (Rt value)*

Radioactive zone (1) Tyrosine standard Radioactive zone (2) Phenylalanine standard

2

3

7

0"36 0"34 0"56 0"55

0"24 0.26 0"55 0-54

0"39 0"39 0-53 0-53

* See Materials and Methods for the composition of the solvent systems. To investigate the metabolism of phenylalanine to tyrosine and other compounds in insects of known age, groups of cockroaches from newly ecdysed to 30 days after imaginal ecdysis were injected with L-phenylalanine-U-14C. After 24 hr extracts of the insects were prepared, portions were hydrolyzed, and then the radiolabeled compounds were separated on paper chromatograms. Tyrosine was synthesized from phenylalanine at all ages and in both teneral and fully tanned insects (Table 2). TABLE 2--BIOSYNTHESIS OF TYROSINE FROM PHENYLALANINE-U-14C FOLLOWING ECDYSIS IN ADULT MALE

P. americana

Relative percentage after 24 hours Age after ecdysis (days)

Tyrosine

Phenylalanine

1 15-16 30

28.4 56.5 58.6

71.6 43.5 41.4

In the period of cuticular tanning during the first day after ecdysis, the relative percentage of tyrosine found after 24 hr was only one-fourth that of phenylalanine. This apparently reflects the rapid utilization of tyrosine for N-acetyldopamine biosynthesis necessary for sclerotization. At 15-30 days after ecdysis the relative percentage of tyrosine was greater than that of phenylalanine by about 15 per cent. Other radiolabeled metabolites also were present on the chromatograms, because of the uniformly labeled precursor; but they separated distincly from phenylalanine and tyrosine by several chromatographic systems. The nonmobile material at

540

L.L. MUROOCK,T. L. HOPKINSANDR. A. WIRTZ

the origin appeared to be phenylalanine and tyrosine incorporated in peptides and proteins, as was previously shown by acid hydrolysis. The data indicate that a phenylalanine hydroxylase system is active in adult cockroaches from ecdysis through maturity for the biosynthesis of tyrosine.

Biosynthesis of benzoyl-fl-glucoside To determine if metabolites other than tyrosine were being produced directly from phenylalanine, we injected insects with either 14C-ring-labeled or uniformly labeled phenylalanine. Chromatograms prepared with unhydrolyzed extracts of adult male cockroaches that had passed the imaginal ecdysis 15 days earlier showed 3 major radioactive areas (Fig. 2). By system 2 the peaks at R! = 0.34 and R! = 0.51 were identified as tyrosine and phenylalanine, respectively. The third peak, ]7i = 0.68, did not appear in extracts of insects injected with L-tyrosine-U-14C, DL-tyrosine-lJ4C, DL-tyrosine-2-x4C or DL-tyrosine-3-14C, suggesting that this metabolite was not derived from t, frosine synthesized from the phenylalanine

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FiG. 2. Paper chromatography (system 2) of extracts of adult male P. americana injected with phenylalanine-UJ4C are held for 24 hours. Upper trace, unhydrolyzed extract; lower trace, HCI hydrolyzed extract. BA, benzoic acid; BBG, benzoyl-~-glucoside; PHA, phenylalanine; TYR, tyrosine.

PHENYLALANINE

METABOLISM

541

IN COCKROACHES

precursor. Therefore, it appeared likely that the unknown contained an unhydroxylated phenyl ring. Furthermore, in that the injection of L-phenylalanine-114C did not produce the radioactive area at R 1 0-68, the unknown did not contain the original 1-carbon of phenylalanine. Two possibilities were benzoic acid and phenylacetic acid. However, the R 1 of the unknown did not correspond to either of those compounds, so the possibility of a conjugate was considered. To test that hypothesis, we subjected samples of the extract to mild acid hydrolysis and examined the hydrolysate by chromatography (Fig. 2). The unknown at R I = 0.68 disappeared upon hydrolysis and a new peak appeared at R / = 0-90 by system 2, showing that the unknown was in fact an acid-labile conjugate. Chromatography of the hydrolysate by several systems showed the radio-activity to have the same R t as benzoic acid in every case: 0.94 (sys. 1), 0.90 (sys. 2), 0.89 (sys. 3), 0-39 (sys. 4), 0-93 (sys. 5), 0.89 (sys. 6). For confirmation that the substance was benzoic

CONTROL

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FIG. 3. Paper chromatography (system 2) of extracts of aposymbiotic adult male P. americana injected with phenylalanine (1-14C-ring labeled) and held 12 hours.

Upper trace, unhydrolyzed; lower trace,/3-glucosidase hydrolyzed. BA, benzoic acid; BBG, benzoyl-/8-glucoside; PHA, phenylalanine; TYR, tyrosine.

542

L. L. Mum~ocK, T. L. HOPKINSAND R. A. WIRTZ

acid, the radioactive areas from several chromatograms prepared with the hydrolysate were cut out and the radioactivity eluted with methanol. The eluate was then mixed with authentic benzoic acid; the mixture was dried and sublimed 3 times. After each sublimation a sample was taken, weighed, and assayed for radioactivity to determine its specific activity. The specific activities found were, respectively: 10.7, 11.1 and 11-2 cpm/mg, confirming the metabolite to be benzoic acid. At first it appeared that the unknown conjugate might be hippuric acid, the glycine conjugate of benzoic acid, because some insects are known to detoxify exogenous benzoic acid in that way (Casida, 1955; Friedler & Smith, 1955; Shyamala, 1964). Cochromatography of hippuric acid and the unknown radioactive conjugate showed that the unknown was not hippuric acid. Quilico et al. (1959) reported isolating benzoyl-fl-D-glucose from Periplaneta; so, we treated portions of the extract with fl-glucosidase. The enzyme rapidly hydrolyzed the unknown, releasing benzoic acid (Fig. 3), which confirmed the unknown to be benzoyl-flglucoside.

Phenylalanine metabolism in aposymbiotic insects In studying phenyl-ring catabolism in normal and aposymbiotic P. americana (Murdock et al., 1970b), we examined the tissues of phenylalanine-l-ringj4C injected male cockroaches for tyrosine and benzol-fl-glueoside biosynthesis. Extracts of the aposymbiotie insects contained both tyrosine and benzoyl-/3glucoside, indicating the respective metabolic pathways to be of insect origin and not dependent on intra-cellular symbionts (Fig. 3). Hydrolysis of the glucoside by either HC1 or fl-glucosidase yielded benzoic acid. Metabolism of phenylpyruvic acid-U-14C In preliminary experiments, to elucidate the pathway of benzoic acid biosynthesis, we injected adult male cockroaches (22 days after ecdysis) with phenylpyruvic acid-U-t4C, which we extracted 10 hours later. In addition to protein-associated radioactivity at the origin, radioactive areas were observed at RF-0.31 ; RF--0.49; R/--0.91 by system 2. Elution of each of these areas and rechromatography showed them to be, respectively, tyrosine, phenylalanine, and phenylpyruvic acid (Fig. 4). No benzoic acid or its glucoside was found, indicating the presence of a potent transaminase in the insect tissues with equilibrium lying in the direction of phenylalanine. Absence of benzoic acid or its glucoside in the extracts could have resulted in part from the shorter duration of the experiment or the site of injection, although phenylpyruvic acid may not be an intermediate in the benzoic acid pathway. This aspect is undergoing further study. DISCUSSION As demonstrated in other insect species, a major metabolite of phenylalanine in P. americana is tyrosine. In this study phenylalanine was converted to tyrosine both in newly ecdysed adults and in those that had undergone a period of maturation,

543

P H E N Y L A L A N I N E METABOLISM I N COCKROACHES

indicating an active phenylalanine hydroxylase system throughout adult life. In tanned adults more than half of the radioactivity from L-phenylalanine-l-14C or U-I4C appeared in tyrosine after 24 hr, so conversion was extensive. Because part of the tyrosine side chain is known to be oxidized to CO2 (Murdock et al., 1970a), the biosynthesis was even more extensive than was indicated by the chromatographic experiments. The relative percentage of tyrosine was only one-fourth that of phenylalanine in the teneral period, reflecting the rapid turnover of tyrosine for the biosynthesis of N-acetyldopamine for cuticular tanning.

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0

I

TYR

I

PHA

PPA

1.0

FIG. 4. Paper chromatography (system 2) of extracts of adult male P. americana injected with phenylpyruvic acid-U-x4c and held 10 hours. PHA, phenylalanine; PPA, phenylpyruvic acid; TYR, tyrosine.

During the teneral period, when hardening and darkening of the cuticle takes place, there was increased COs production from the phenylalanine-l-x4c administered, undoubtedly reflecting the greatly increased utilization of tyrosine for production of the two carbon side-chain metabolite N-acetyldopamine (Murdock et al., 1970a). Both aposymbiotic (lysozyme-treated) and control cockroaches produced tyrosine from phenylalanine, demonstrating that, apart from the symbionts, the cockroach is capable of hydroxylating the aromatic ring. This symbiont-independent hydroxylating capability is in accord with the observation that Blatella germanica, when deprived of its symbionts, acquired a light cuticle, but upon being fed a diet rich in phenylalanine, it regained the normal brown coloration (Henry & Cook, 1964). Benzoyl-fl-glucoside was the other major metabolite of phenylalanine in fully tanned male P. americana; small quantities of free benzoic acid also were present in the extracts. However, the glucoside was not produced in the first 24 hr following

544

L.L. MURDOCK,T. L. HOPKINSANDR. A. WIRTZ

ecdysis. Pavan (1954) reported that the blue fluorescingsecretion of the cervical glands identified as benzoyl-fl-glucose (Quilico et al., 1959) was not seen just after ecdysis. N o hippuric acid, the glycine conjugate of benzoic acid reported in certain other insect species, was found in P. americana. Like tyrosine, benzoyl-fl-glucoside was synthesized in aposymbiotic as well as normal cockroaches, indicating no significant symbiont involvement in this pathway. T h e biosynthetic pathway of benzoic acid is unknown. Attempts to show that phenylpyruvic acid was an intermediate only demonstrated that the tissues of the cockroach contained an active transaminase, in which phenylalanine and tyrosine are rapidly synthesized. T h e action of this transaminase may explain our repeated failure to find in the cockroach the hydroxyphenylcarboxylic acids derived from tyrosine that have been reported in other insects (Sekeris & Karlson, 1962; Karlson & Herrlich, 1965; Sekeris & Herrlich, 1966). In P. americana phenylcarboxylic acids, other than benzoic acid, could be transient intermediates in the pathway not readily detected by in vivo methods. REFERENCES BEI..ZECKAK., LASKOWSKAT. & MOCHNACKAI. (1964) Hydroxylation of phenylalanine in insects and some invertebrates. Actabiochem.polon. 11,191-196. BRICTEAtrX-Gx~cOIRB S., VERLY W. G. & FLORKIN M. (1956) Utilization of the carboxyl carbon of L-phenylalanine for the synthesis of the amino acids of silk by Bombyx mori. Nature, Lond. 177, 1237-1238. CASIDAJ. E. (1955) Toxicity of aromatic acids to the larvae of the mosquito Aedes aegypti L. and the counteracting influence of amino acids. Biochem.jY. 59, 216-221. FRn~LER L. & SMITHJ. N. (1955) Hippuric acid formation in adult locusts. Biochem.ff. 57, 396--400. FUKUDAT. (1956) Conversion of phenylalanine into tyrosine in the silkworm larva (Bombyx mori). Nature, Lond. 177, 429-430. HENRY S. M. & COOK T. W. (1964) Amino acid supplementation by symbiotic bacteria in the cockroach. Contrib. Boyce Thompson lnst. 22, 507. House H. L. (1965) Insect nutrition. In Physiology of Insecta (edited by ROCKSTEINM.), Vol. 2, pp. 769-813. Academic Press, New York and London. ISHII S. & HIRANO C. (1958) Biosynthesis of tyrosine from C14-phenylalanine in the larva of the rice stem borer, Chilo suppressalis Walker. Proc. lOth Intern. Congr. Entomol., Montreal, 1956, 2, 295-298. KAm~ON P. & I-IERRLICrIP. (1965) Der Tyrosinstoffwechsel der Heuschrecke Schistocerca gregaria Forsk. ft. lnsect Physiol. 11, 79-89. KARLSON P., SE~maIS C. E. & SErmRI K. E. (1962) Identifizierung von N-Acetyl-3,4dihydroxy-fl-phen~ithylamin (N-Acetyl-dopamin) als Tyrosinmetabolit. Z. physiol. Chem. 327, 86-94. 1VI~ISTER A. (1957) Preparation of 0t-keto acids. In Methods in Enzymology (edited by COLOWICKS. P. & KAPLANN. O.), Vol. 3, pp. 404-414. Academic Press, New York. MURDOCK L. L., HOPKINS T. L. & WIRTZ R. A. (1970a) Tyrosine metabolism in vivo in teneral and mature cockroaches, Periplaneta americana, ft. lnsect Physiol. 16, 555-560. MtmDOCK L. L., HOPKINST. L. & WmTZ R. A. (1970b) Phenylalanine metabolism in cockroaches, Periplaneta americana: intracellular symbionts and aromatic ring cleavage. Comp. Biochem. Physiol. 34, 143-146.

P H E N Y L A L A N I N E M E T A B O L I S M I N COCKROACHES

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PAU R. N. & ACHESONR. M. (1968) The identification of 3-hydroxy-4-O-/~-D-glucosidobenzylalcohol in the left coUeterial gland of Blaberus discoidalis. Biochim. biophys. Acta 158, 206-211. PAVAN M. (1954) Un nuovo organo cervicale in Blatta orientalis e Periplaneta americana produttore di secreto aUa luci di Wood. Boll. Soc. ital. Biol. sper. 30, 873-875 (cited in GUTHmE D. M. & TINDALLA. R., The Biology of the Cockroach, p. 72). Arnold, London, 1968. PRICE G. M. (1965) Aspects of amino acid and nucleic acid metabolism in the larva of the blowfly, Calliphora erythrocephala. Biochem.jY. 95, 39-40P. QUILICO A., PlozzI F., PAVANM. & MANTICAE. (1959) The structure of periplanetin. Tetrahedron 5, 10-14. SEKERIS C. E. & HERRLICI-IP. (1966) Der Tyrosinstoffwechsel yon Tenebrio molitor and Drosophila melanogaster. Z. physiol. Chem. 344, 267-275. SEKERISC. E. & KARLSONP. (1962) Der katabohsche Abbau des Tyrosins und die Biogenese der Slderotisierungssubstanz, N-Acetyl-dopamin. Biochim. biophys. Acta 62, 103-113. gHYAMALAM. B. (1964) Detoxication of benzoate by glycine conjugation in the silkworm, Bombyx mori L. ~. Insect Physiol. 10, 385-391.

Key Word Index--Phenylalanine ; tyrosine; benzoyl-fl-glucoside; N-acetyldopamine; benzoic acid; transaminase; insect biochemistry.