An enzyme that catalyses the synthesis of lanthionine in the silkworm, Bombyx mori

Comp. Biochem. PhysioL Vol. 91B, No. 2, pp. 301-307, 1988 Printed in Great Britain

0305-0491/88 $3.00+0.00 © 1988Pergamon Press plc

AN ENZYME THAT CATALYSES THE SYNTHESIS OF LANTHIONINE IN THE SILKWORM, BOMBYX MORI HIROSHI SHINBO The Sericultural Experiment Station, Tsukuba, Ibaraki 305, Japan Scheme I. Proposed reaction of the enzyme in' the silkworm, Bombyx mori. Abstract--l. An enzyme that catalyses the synthesis of lanthionine from cysteine was purified 20-fold from

the larval fat body of the silkworm, Bombyx mori. 2. The pH optimum is 9.0 and pyridoxal phosphate is a necessary cofactor. 3. From the present results, it seemed plausible that the enzyme catalyses the reaction of substitution at the//-carbon atom of cysteine or serine, resulting in formation of cystathionine or lanthionine in the presence of homocysteine or cysteine as a cosubstrate in the second reaction stage in B. mori.

INTRODUCTION

MATERIALS AND METHODS

Lanthionine was first isolated from the acid hydrolysates of wool that had been pretreated with alkali (Horn et al., 1941). It has been later shown that other proteins, if treated first with alkali and then subjected to acid hydrolysis, also give rise to lanthionine (Horn and Jones, 1941; Horn et al., 1941; Du Vigneaud and Brown, 1941; Inoue and Kawaguchi, 1943; Dowling and McCiaren, 1965; Inokuchi, 1972). In these cases it is not usually regarded as a normal constituent of proteins, but rather as a transformation product arising from the alkaline decomposition of cyst(e)ine residues. Sloane and Untch (1966) demonstrated that L-lanthionine is a naturally occurring amino acid; they isolated it in crystalline form from the free amino acid pool of the chick embryo, and also obtained evidence which indicates that L-lanthionine is a constituent of chick embryo protein. Almost simultaneously, Rao et al. (1966, 1967) isolated L-lanthionine from the deproteinized hemolymph of the silkworm, Bombyx mori, and the Japanese oak moth, Antheraea pernyi. In addition, lanthionine has been detected in locust muscle protein (Kermack and Stein, 1959), plant pollen (Rosetti, 1966) and human urine (Wadman et aL, 1978). The investigations on the enzymes involved in the lanthionine metabolism in animals seem to be scanty since lanthionine is often regarded as an artifact, as mentioned above. An enzyme system which catalyses the formation of lanthionine from cysteine has been found in the yolk sac of chicken embryo (Chapeville and Fromageot, 1961; Tolosa et al., 1969). On the other hand, lanthionine has been reported to be a substrate for both fl-cystathionase (Burnell and Whatley, 1977) and 7-cystathionase (Cavallini et aL, 1960; Flavin, 1962). Furthermore lanthionine decarboxylation by animal tissues has been reported (Scandurra et al., 1979). The present paper deals with the occurrence of an enzyme that catalyses the synthesis of lanthionine from cysteine, and also with the possibility of the identity of this enzyme with cystathionine synthetase in the silkworm, Bombyx mori.

Animals and chemicals Hybrid larvae obtained from crosses made betweeen N 137 and C 137 strains ofB. mori were reared on mulberry leaves at 25°C. Fat body tissue was collected according to the procedure reported previously (Shinbo, 1982). OL- and meso-lanthionine and pyridoxal phosphate were purchased from Sigma Chem. Co., USA; L-cystathionine from Calbiochem Behring Corp., USA; cysteine, homocysteine and serine from Nakarai Chem. Co., Japan; DEs2-cellulose from Whatman Chem. Co., USA; Sephadex G-200 from Pharmacia Fine Chemicals AB, Sweden. Other chemicals used were of reagent grade. Enzyme assay methods The enzyme activity was determined by analysis of the lanthionine formed using amino acid analyser; the assay mixture contained 100 #moles of L-cysteine,0.05 gmoles of pyridoxal phosphate, 50/~moles of Tris-HC1 buffer (pH 8.8) and enzyme solution in a total volume of 0.5 ml. The reaction mixture was incubated at 37°C for 40min with shaking, after preincubation for 5 min at the same temperature without cysteine. The reaction was stopped by adding 0.5 ml of 4% sulfosalicylicacid, and the precipitate was removed by centrifugation at 3000g for 10min. The supernatant (0.05 ml) was used for the determination of the lanthionine formed with an automatic amino acid analyser (Hitachi type-835) by stepwise elution of lithium buffers. One unit of enzyme is defined as the amount of enzyme which produces 1 #mole of lanthionine per min under the assay condition described. Specific activity is expressed as units per mg protein. Cystathionine synthetase activity was determined as described (Shinbo, 1982). Purification of enzyme All procedures were carried out at 5°C or in an ice bath. Two hundred grams of fat body were used as starting materials for the purification of the enzyme catalysing the synthesis of lanthionine. Purification procedures through ammonium sulphate fractionation were performed in the same manner described previously (Shinbo, 1982). The enzyme solution fractionated with ammonium sulfate (0.3-0.6 saturation) was applied onto a column of DEsz-cellulose (2.2cm i.d. × 19cm), equilibrated with potassium phosphate buffer (KPB), pH 7.4, and the column was washed with 300 ml of the same buffer. The enzyme was then eluted with a linear gradient of 200 ml of KPB in the mixing

301

302

HIROSHI SHINBO

chamber and an equal volume of KPB containing 0,2 M KCI in the reservoir. The flow rate was 40 ml/hr and 4 ml fractions were collected. The enzyme eluted at approximately 0.08 M KCI, Enzyme rich fractions were pooled and precipitated by addition of ammonium sulfate to a final concentration of 0.9. The precipitate formed was collected by centrifugation at 12,000g for 15 min and dissolved in about 5 ml of KPB containing 0.2 M KC1. The enzyme solution was applied onto a column of Sephadex G-200 (l.7cm i.d. × 88cm), equilibrated with KPB containing 0.2 M KC1, and eluted with the same buffer at a flow rate of 9.2 ml/hr and 3.5 ml fractions were collected. RESULTS

Purification of enzyme Figures 1 and 2 show the elution profile of the enzyme catalysing the synthesis of lanthionine and proteins from the columns of DE52-cellulose and Sephadex G-200, respectively. The enzyme was eluted with the same mobility as cystathionine synthetase from both the columns. The purification and recovery

of the enzyme are summarized in Table 1, The final purification achieved was approximately 20-fold with a specific activity of 150 munits/mg protein and an overall yield of 52%. The purified enzyme exhibited cystathionine synthetase, and the ratio of the enzyme to cystathionine synthetase was almost constant during the purification.

Properties of the enzyme Products from various amino acids by the enzyme reaction. Figure 3 shows the amino acid analyses of the products from various amino acids by the action of the enzyme. Two peaks corresponding to serine (A, 1) and lanthionine (A, 2) were detected in the reaction mixture of cysteine and the enzyme, indicating the formation of serine and lanthionine from cysteine in the enzyme assay. No peak formed by the enzyme reaction was found in the reaction mixture of homocysteine and the enzyme. When cysteine and homocysteine were incubated with the enzyme, a peak corresponding to cystathionine (C, 3) as well as

1.6

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Fraction No.

Fig. 1. DE52-cellulosecolumn chromatography of the enzyme catalysing the synthesis of lanthionine; the enzyme catalysing the synthesis of lanthionine (Q), cystathionine synthetase ( I ) and protein concentration ( - - ) . 800

1.4

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Fig. 2. Sephadex G-200 column chromatography of the enzyme catalysing the synthesis of lanthionine; the enzyme catalysing the synthesis of lanthionine (Q), cystathionine synthetase ( n ) and protein concentration ( ).

Lanthionine synthesizing enzyme in Bombyx mori

303

Table 1. Summary of purification of the enzyme catalysing the synthesis of lanthionine from fat body of the silkworm, Bombyx mori

(1) (2) (3) (4) (5)

Crude extract pH 5.0 treatment (NH4)2SO4 Fr. (0.3~0.6 sat.) DE52-cellulose column chromatography Sephadex G-200 column chromatography

Volume (ml)

Total protein (rag)

Total activity (mU)

Specific activity (mU/mg)

440 460

3077 2384

23,584 29,394

7.66 12.33

62

2015

19,728

9.8

113

195

18,159

61

81

12,157

CTS* Yield (%)

LSf

I 1.6

100 125

2.2 1.9

1.3

84

1.9

93

12.1

77

2.3

150

19.6

52

2.0

Purification

*Cystathionine synthetase. tEnzyme catalysing the synthesis of lanthionine. 3

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Retention time (rain)

Fig. 3. Amino acid analyses of the products from various amino acids by the action of the enzyme. One hundred and thirty-nine u g of the enzyme were incubated with 100 #moles of cysteine (A), 100/~moles of homocysteine (B), 100/~moles of cysteine and 5 pmoles of homocysteine (C), 50,umoles of homocysteine and 25 pmoles of serine (D), 100/~moles of cysteine and 25 pmoles of homoserine (E) for I hr, or 100,umoles of serine and 50 pmoles of N a H S O 3 (F) for 3 hr. (A'), (B'), (C'), (D'), (E') and (F') are controls for (A), (B), (C), (D), (E) and (F), respectively; heat-inactive (100°C, 3 min) enzyme was used instead o f active enzyme. C,BP

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304

HIROSHI SHINBO

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Protein ( Jig )

Time (rain)

Fig. 4. Relationship between the enzyme activity and reaction time or protein concentration. two peaks corresponding to serine (C, 1) and lanthionine (C, 2) were detected in the reaction mixture, indicating the formation of serine, lanthionine and cystathionine from cysteine and homocysteine in the enzyme assay. One peak corresponding to cystathionine (D, 1) was detected in the reaction mixture of homocysteine, serine and the enzyme, indicating the formation of cystathionine from homocysteine and serine in the enzyme assay. Two peaks corresponding to serine (E, 1) and lanthionine (E, 2) were detected in the reaction mixture of cysteine, homoserine and enzyme, indicating the formation of serine and lanthionine from cysteine and homoserine in the enzyme assay. When serine and sodium hydrogen sulfite were incubated with the enzyme, one peak corresponding to cysteic acid (F, 1) was detected in the reaction mixture, indicating the formation of cysteic acid from serine and sodium hydrogen sulfite by the enzyme reaction.

10.8, using Tris-HCl and borate buffers, and found to be highest at about pH 9.0 (Fig. 5).

Effects of pyridoxal phosphate Pyridoxal phosphate at 5 x 10-4M showed 100% stimulation of the enzyme activity. The purified enzyme was strongly inhibited by hydroxylamine and the inhibition was reversed by pyridoxal phosphate (Table 2). Among the several vitamin B6 derivatives listed in Table 3, only pyridoxal phosphate restored the enzyme activity after treatment with hydroxylamine. Figure 6 shows double reciprocal plots of the concentration of pyridoxal phosphate and the activities of the enzyme treated with hydr0xylamine. The apparent Michaelis constant for the coenzyme was estimated to be 2.94 x 10-6M.

Time course and enzyme concentration

Table 2. Effect of hydroxylamine and pyridoxal phosphate on the enzyme activity

As shown in Fig. 4, a linear relation between the rate of lanthionine formation and incubation time or enzyme concentration was observed.

Compound added

pH optimum Enzyme activity was assayed between pH 6.7 and 25

None PALP, 5 NH2OH, NH2OH, NH2OH, PALP, NH2OH, PALP,

× 10 _4 M 10 4M 10 3M |0 4M + 5 × 10 4M 10 3+ 5 × 10-4M

Activity (m units)

% Activity

10. l 20.2 5.5 0.5

100 200 54 5

16.1

160

7.4

73

Incubations were performed as described under enzyme assay with 135#g of enzyme (specific activity 0.15) and hydroxylamine or pyridoxal phosphate. The enzyme was preincubated with hydroxylamine for 3 min and then for 3 rain with pyridoxal phosphate.

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Table 3. Effects of various pyridoxal derivatives on the enzyme activity after treatment with hydroxylamine Compound added

~t

5 0 8

7

8

9

10

11

pH

Fig. 5. Effect of pH on the enzyme activity. Activities were measured under standard conditions except for the buffer; Tris buffer (Q), borate buffer (A).

None Pyridoxal Pyridoxal phosphate Pyridoxamine Pyridoxamine phosphate Pyridoxine

Activity (m units) 0.7 0.7 18.9 1.1 0.8 0.6

An aliquot of enzyme solution (1.33 mg/ml) was dialysed for 40 hr against 500 vols of 10_2 M potassium phosphate buffer (pH 7.4) containing 10 -3 M hydroxylamine and then for 24 hr against two changes of 500 vols of the same buffer without hydroxylamine to remove inhibitor. The enzyme was then preincubated with the pyridoxal derivatives (0.5 mM) indicated for 5 rain.

Lanthionine synthesizing enzyme in Bombyx mori

305

15

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Fig. 6. Effect of the concentration of pyridoxal phosphate (PALP) on the enzyme activity after hydroxylamine treatment. The enzyme preparation used was as described in Table 3.

Effects of various compounds The sulfhydryl reagents tested, p-chloromercuribenzoate, iodoacetate and N-ethylmaleimide, showed no inhibition at the concentrations of 10-4M and 10-3 M. Divalent metal ions, such as Cu (10 -3 or 10 -4 M) and Mn (10-3M) caused approximately 20-50% inhibition of the enzyme whereas Co, Zn, Ba and Hg had no appreciable effect on the enzyme activity. Amino acids, such as serine, taurine, cysteic acid, cysteine sulfinic acid, methionine, homoserine, cystathionine, alanine, threonine and glycine had no effects on the enzyme activity at the concentrations of 10-3 and 5 × 10-3M. Homocysteine, however, showed 14% inhibition of the enzyme (Table 4). Table4. Effectsof aminoacids on the synthesisof lanthioninefrom cysteine by the action of the enzyme Conc. Activity Compound (mM) (m units) 21.2 (100%) None Serine 21.7 (102%) 5 21A (100%) Taurine 20.6 (97%) 5 20.6 (97%) Cysteic acid 20.9 (99%) 5 21.4 (101%) 21.4 (101%) Cysteine sulfinicacid 5 20.4 (96%) Methionine 20.9 (99%) 5 20.6 (97%) Homoserine 20.9 (99%) 5 19.8 (93%) Cystathionine 20.7 (98%) 5 20.4 (96%) Alanine 20.5 (97%) 5 21.5 (101%) Threonine 21.1 000%) 5 20.2 (95%) 20.2 (95%) Glycine 5 19.7 (93%) Homocysteine 20.5 (97%) 17.9 (84%) Incubationswere performed as describedunderenzymeassay with 135#g of enzyme(specificactivity0.15)and aminoacidsshown in the table.

Effects of homocysteine and serine on the formation of lanthionine from cysteine Of the amino acids tested, only 5 mM of homocysteine showed a significant inhibition of the enzyme, as shown in Table 4. More detailed study, therefore, was carried out on the effect of the amino acid on the formation of lanthionine from cysteine by the enzyme reaction (Table 5). The result revealed that the formation of lanthionine from cysteine was inhibited by the addition of homocysteine. In addition, homocysteine caused the inhibition of the formation of serine from cysteine. Although cystathionine as well as lanthionine and serine were formed from cysteine and homocysteine by the enzyme reaction, as seen in Fig. 3, the concentration of cysteine at which the formation of cystathionine from cysteine reached a maximum was lower than that of lanthionine from cysteine. The addition of serine showed a stimulation of the formation of lanthionine from cysteine at low concentrations of cysteine, whereas the amino acid inhibited the formation of lanthionine from cysteine in high concentrations of cysteine. DISCUSSION The present results reveal the occurrence of an enzyme that catalyses the synthesis of lanthionine from cysteine in the fat body of B. mori. The partially purified enzyme was shown to exhibit cystathionine synthetase activity which catalyses the synthesis of cystathionine from serine and homocysteine. In B. mori, cystathionine synthetase has recently been purified and characterized (Shinbo, 1982). Both enzyme activities were eluted with the same mobility from DE52-cellulose and Sephadex G-200 columns and the ratio of lanthionine synthesizing and cystathionine synthesizing activities remained almost constant during the purification, suggesting that the syntheses of lanthionine and cystathionine are catalysed by the same enzyme protein in the fat body of

B. mori.

HIROSHI SHINBO

306

Table 5. Effects of homocysteine and serine on the synthesis of lanthionine from cysteine by the action of the enzyme Lanthionine Cystathionine Serine (nmoles/min/0.1 ml enzyme) I Cysteine (6.25 mM) 0.76 2.33 2 Cysteine (12.5 mM) 1.63 2.59 3 Cysteine (25 raM) 3.72 2.75 4 Cysteine (50 mM) 7.67 2.91 5 Cysteine (100 mM) 14.06 2.95 6 Cysteine (200mM) 20.71 2.81 7 Cysteine (6.25 mM)+ homocysteine (10 mM) 0.06 (7.9%) 13.29 0.06 8 Cysteine (12.5 raM) + homocysteine (10 mM) 0.17 (10.4%) 18.22 0.14 9 Cysteine (25 mM) + homocysteine (10 mM) 0.67 (18.0%) 21,11 0.41 10 Cysteine (50 mM) + homocysteine (10 mM) 2.93 (38.2%) 23,87 1.05 I 1 Cysteine (100 mM) + homocysteine (10 mM) 8.22 (58.5%) 25.85 1.74 12 Cysteine (200mM) + homocysteine (10 mM) 14.07 (67.9%) 23.86 1.96 13 Cysteine (6.25 mM) + homocysteine (5 mM) 0.14 (18.4%) 11.40 0.28 14 Cysteine (12.5 mM) + homocysteine (5 mM) 0.53 (32.5%) 12.45 0.74 15 Cysteine (25 mM) + homocysteine (5 mM) 1.61 (43.3%) 13.11 I. 15 16 Cysteine (50 mM) + homocysteine (5 mM) 4.47 (58.3%) 13.47 1.69 17 Cysteine (100 mM)+ homocysteine (5 mM) 10.21 (72.6%) 13.90 2.15 18 Cysteine (200mM)+ homocysteine (5 mM) 16.63 (80.3%) 13.08 2.30 19 Cysteine (6.25 mM) + serine (50 mM)) 0.92 (121.1%) 20 Cysteine (12.5 mM) + serine (50 mM) 1.93 (118.4%) 21 Cysteine (25 mM) + serine (50 raM) 3.62 (97.3%) 22 Cysteine (50 mM)+ serine (50 raM) 7.18 (93.6%) 23 Cysteine (100 mM) + serine (50 mM) 12.41 (88.3%) 24 Cysteine (200 mM) + serine (50 raM) 17.90 (86.4%) Incubations were performed as described under enzyme assay with 135 pg of enzyme (specificactivity 0.15) and cysteine, homocysteine or serine. The synthesis of lanthionine from cysteine is catalysed by cysteine lyase which is a pyridoxal phosphate-dependent enzyme discovered in the yolk sac of developing chicken embryo (Chapeville and Fromageot, 1961; Tolosa et al., 1969). The enzyme has not yet been found in any other biological material. Cysteine lyase, which catalyses reactions of substitution at the fl-carbon atom of cysteine, is capable of utilizing, as a cosubstrate in the second reaction stage, certain thiol compounds and sulfite as well as a second molecule of cysteine (Tolosa et al., 1969). On the other hand, the synthesis of cystathionine from serine and homocysteine is catalysed by cystathionine synthetase, which is also a pyridoxal phosphate-dependent enzyme (Greenberg, 1975). Cystathionine synthetase has been reported to possess serine sulfhydrylase activity, which catalyses the synthesis of cysteine from serine and H:S. The reaction is reversible (Braunstein et al., 1971 ; Kraus et al.,

1978); it has been suggested that cystathionine synthetase and serine sulfhydrylase activities are properties of a single enzyme catalysing fl-substitution reactions between either serine or cysteine and certain thiol compounds, resulting in formation of the corresponding thioethers of cysteine. However, Braunstein et al. (1969) reported that the enzyme was incapable of utilizing, as a cosubstrate, a second molecule of cysteine or sulfite, in contrast to cysteine lyase. Thus, it has been established that cysteine lyase and cystathionine synthetase (serine sulfhydrylase) are distinct and separate proteins. On the basis of the results of the experiments in which reactions catalysed by the partially purified enzyme and the effects of serine and homocysteine on the formation of lanthionine from cysteine were examined, the proposed enzyme reaction is represented in Scheme 1. The enzyme seems to catalyse the reaction of substitution at the fl-C atom of cysteine

HO-CH2-CH(NH2)-COOH ~ (Serine)

~

HS-CH~-CH(NH2)-COOH

~

(Cysteine)

H~O 4"'-

~

~

H2S

V

CH2- C(NH2)-COOH

HS-CH2- CHz-CH(NH2)-COOH ~ (Homocysteine) ~ CIH2-CH2-CH(NH2)-COOH J S CH2- CH(NH21-COOH (Cystathionine)

~

HS - CH2- CH(NH2)-COOH (Cysteine)

"''-------._~ CIH2-CH(NH2)-COOH S h CH2-CH(NH2)-COOH (Lanthionine)

Scheme 1. Proposed reaction of the enzyme in the silkworm, Bombyx mori.

Lanthionine synthesizing enzyme in Bombyx mori or serine, resulting in formation of cystathionine or lanthionine in the presence of homocysteine or cysteine as a cosubstrate in the second reaction stage in B. mori; it seems plausible that the enzyme has both the function o f cysteine lyase and serine sulfhydrylase. However, further study is required on the purification and homogeneity of the enzyme to establish this view unequivocally, since partially purified enzyme preparation was used for the present study.

Acknowledgements--The author wishes to express his thanks to Drs G. M. Happ, T. Ito, T. Inokuchi, H. Ooi and H. Takizawa for their valuable advice and critical reading of the manuscript.

REFERENCES

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307

of an enzymatic disulfide elimination reaction. J. biol. Chem. 237, 768-777. Greenberg D. M. (1975) Metabolic Pathways, 3rd Edn, Vol. 7, pp. 514-518. Academic Press, New York. Horn M. J. and Jones D. B. (1941) The isolation of lanthionine from human hair, chicken feathers, and lactalbumin. J. biol. Chem. 139, 473. Horn M. J., Jones D. B. and Ringel S. J. (1941) Isolation of a new sulfur-containing amino acid (lanthionine) from sodium carbonate-treated wool. J. biol. Chem. 138, 141-149. Inokuchi T. (1972) Isolation and determination of lanthionine and its metabolism in the silkworm, Bombyx mori. Bull. Ser. Exp. Stn, Japan 25, 169-197. Inoue Y. and Kawaguchi S. (1943) On the sulfur amino acids in the silkworm egg shell. Nogeikagakkai-shi, Japan 19, 653-657. Kermack W. O. and Stein J. M. (1959) Nitrogenous constituents of the thoracic muscle of the African migratory locust (Locusta migratoria migratorioides). Biochem. J. 71, 648~54. Kraus J., Packman S., Fowler B. and Rosenberg L. E. (1978) Purification and properties of cystathionine fl-synthase from human liver. J. biol. Chem. 253, 6523~5528. Rao D. R., Ennor A. H. and Thorpe B. (1966) The occurrence of L-lanthionine in the amino acid pool of insects. Biochem. biophys. Res. Commun. 22, 163-168. Rao D. R., Ennor A. H. and Thorpe B. (1967) The isolation and identification of L-lanthionine and L-cystathionine from insect haemolymph. Biochemistry 6, 1208-1215. Rosetti V. (1966) Free amino acids in the pollen of Lillium candidum. Ann. Chim. (Rome) 56, 935-945. Scandurra R., Conaslvi V., De Marco C., Politi L. and Cavallini D. (1979) Lanthionine decarboxylation by animal tissues. Life Sci. 24, 1925-1930. Shinbo H. (1982) Purification and properties of cystathionine synthetase in the silkworm, Bombyx mori. Insect Biochem. 12, 571-577. Sloane N. H. and Untch K. G. (1966) Studies on amino acids in embryonic tissue. I. L-Lanthionine, a naturally occurring amino acid in the chick embryo. Biochemistry 5, 2658-2665. Tolosa E. A., Chepurnova N. K., Khomutov R. M. and Severin E. S. (1969) Reactions catalysed by cysteine lyase from the yolk sac of chicken embryo. Biochim. biophys. Acta 171, 369-371. Wadman S. K., de Bree P. K. and Kamerling J. P. (1978) Lanthionine detected in human urine. Clin. chim. Acta 82, 281-284.