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Phenoloxidase activity and its rôle in cuticular sclerotization in a mole crab Emerita asiatica Milne Edwards
J. Exp. Mar. Biol. Ecol., 1982, Vol. 61, pp. 75-83 Elsevier Biomedical Press
75
PHENOLOXIDASE ACTIVITY AND ITS ROLE IN CUTICULAR SCLEROTIZATION IN A MOLE CRAB EMERITA
K. NELLAIAPPAN,
ASIATICA
Mike Edwards
T. THANGARAJ
and K. RAMALINGAM
~e~a~rn~n~ of Zoology, UniygT~i~~ of Madras, Mad~as~~
005. India
Abstract: Phenoloxidase has been localized in the epicuticle, exocuticle, and epidermal cells of the mole crab, Emerita asiatica Milne Edwards. The enzyme activity in different moulting stages is in the order of freshmoult > premoult > intermoult = postmoult. The phenoioxidase of the freshmoult cuticle oxidizes pyrogallol and epinephrine (adrenaline) more effectively than the other phenols studied. There is no monophenolase activity. The possible metabolic pathway has been suggested based on the specificity of the enzyme and the chromatographic identification of the extracted phenols. Phenoloxidase shows different pH optima in different buffers. The protein patternin the various moulting stages of the cuticle differs and the results are discussed in relation to sclerotization.
INTRODUCTION
It is well known that the protein of most of the arthropod cuticle is stabilized by quinone. Many workers characterized the precursors of the sclerotin such as phenol, phenoloxidase, and protein in a number of insects (see Andersen, 1979; Brunet, 1980, for reviews). The chemical aspects of the cuticle of arthropods other than insects have, however, been largely neglected. Although phenoloxidase and phenols have been reported in crustacean cuticle (for a review see Hackman, 1971; Vacca & Fingerman, 1975) and in the cement gland of Lepas ~~c~c~~ffr~ (Barnes & Blackstock, 1976), studies regarding its nature and characterization have only been carried out in a few crustaceans (Stevenson & Adomako, 1967; Summer, 1967). The present study deals with the cuticular sclerotization and characterization of phenoloxidase of the mole crab Eme~ta asiuticu Milne Edwards.
MATERIAL
AND METHODS
E. asiatica were collected from the intertidal region of Madras beach opposite Madras University. The moult stages such as fresh-, post-, inter- and premoult were identified as described by Drach (1939). The carapace cuticles (2-2.5 cm) were removed manually after washing the animals in running tap water to remove the sand particles. To localize the enzyme, the cuticles in various stages of development were fixed in 70% alcohol for 30 min and washed in distilled water for 15 min. The cuticles were incubated 0022”0981/82/00~-0~0/$02.75
0 1982 Elsevier Biomedical Press
16
K.NELLAIAPPAN ETAL.
in 0.1 y0 catechol solution at 37 “C for 1 h as described by Smyth (1954). Subsequently, the cuticles were sectioned (10-15 pm thick) with a Cryocut microtome (American Opticals) at -20 “C. For the enzyme assay, 25 cuticles were homogenized in 5 ml of ice-cold distilled water in a mortar and pestle. The homogenate was centrifuged at 2000 g for 20 min and the clear supernatant containing soluble phenoloxidase was used as the enzyme source. The activity measured in the sediment showed the presence of an insoluble particulate form of the enzyme, and was found to have a low activity (0.001 OD * mg protein-’ * min-’ ). The enzyme assay was carried out as described by Nellaiappan & Ramalingam (1980) with a few modifications. One ml of 0.01 M substrate and 1 ml of phosphate buffer (0.25 M) at pH 7.5 were taken in a l-cm cuvette. Increase in optical density (OD) was recorded in Bausch & Lomb Spectronic 21 Spectrophotometer at 420 nm immediately after the addition of 0.2 ml of the enzyme. For the control, 0.2 ml of distilled water was used instead of enzyme. The results are expressed as OD * mg protein - ’ . min - ‘. 0.25 M Tris-HCl, phosphate, and citrate buffers were used at 0.5 pH intervals to study the pH optimum. Protein content of the sample was determined by the method of Lowry et al. (1951) using bovine albumin as standard. The Student-Newmann-Keuls procedure as detailed by Sokal dz Rohlf (1969) was followed to test the differences between means. 5 % polyacrylamide gel was used to study the protein pattern of the cuticle in different stages as described by Smith (1968). 0.02 M Tris-glycine at pH 8.3 and 0.3 M Tris-HCl at pH 8.9 were used as tank and gel buffers, respectively. About 200 pg protein were directly applied over the running gel and 3 mA current was supplied to each gel tube. The gels were stained in 0.1% amido black in 7% acetic acid and de-stained in 7% acetic acid. The stained gels were scanned in a Carl Zeiss ERI-10 recording densitometer. The phenols were extracted by homogenizing 50 cuticles in 20 ml of 95% alcohol in 0.01 N HCI and centrifuging at 1000 g for 5 min. The supernatant was evaporated to dryness and the residue was taken up in 10% isopropanol and spotted on Whatman No. 1 chromatogram paper. Butanol : acetic acid : water in the ratio of 12 : 3 : 5 was used as solvent and descending chromatography was carried out for 17 h. The phenols were localized by spraying with ferric chloride-ferricyanide reagent, because it detects < 1 pg of phenols (Hathway, 1969). The various phenols were identified by using the standards and R, values.
RESULTS
In the Cryocut sections, epicuticle, exocuticle, and endocuticle layers could easily be seen. In the fresh moult cuticle the epidermal cells are polygonal in shape and are secretory in nature. In the subsequent stages the volume of the cells is reduced and the thickness of the cuticle increases. Similar morphological changes during the moult cycle by the integument of the land crab Gecarcinus lateralis have been reported by Skinner
PHENOLOXIDASE
ACTIVITY IN EMERITA ASZATICA
71
(1962). After incubation in catechoi, epicuticle, exocuticle, and epidermal cells of the freshmoult show an intense brown colour. In other stages exocuticle and epidermal cells show only a faint positive reaction. The extractable cuticular phenol oxidase activity of Emerita asiatica on epinephrine (adrenaline) in different moult stages is given in Table I. In the freshmoult cuticle the TABLE
I
Cuticular phenoloxidase activity (4 SD) of Emerita usiutica in different moult stages on the substrate, epinephrine (AOD . mg protein- ’ min-‘): mean activity, average of five readings; ns., not significant; *, significant at the 5% level. Stages
Mean activity
Postmoult Intermoult Premoult Freshmouh
0.0065 + 0.00030 n.s. 0.007 1 f 0.00034 n.s. 0.0092 k 0.00020* 0.0192 f 0.00030*
enzyme shows more activity when compared with other stages. In the postmoult stage the activity is moderate. Intermoult and premoult cuticles show less activity and the difference in the activity of these two stages is statistically non-significant. The electrophoretic pattern of the cuticular protein varies in the different moult stages. In fr~shmoult and premoult stages the protein pattern is identical; these two show five protein fractions. In intermoult and postmoult stages ten protein fractions are, however, separated in the gel (Fig. 1). As the freshmoult cuticle shows more activity, the characterization of the enzyme was carried out in freshmoult cuticle. The activity of the enzyme in the freshmoult cuticle on different substrates is given in Table II. The enzyme activity is more than other substrates when pyrogallol or epinephrine is used and the mean values of activity in these two substrates are statistically non-si~i~c~t. Catechol is oxidized less TABLE II Cuticular
activity (f SD) of Emerita ask&a on various substrates (AOD . mg promean activity, average of live readings; ns, not significant; *, significant at the 5% level.
phenoloxidase
tein-’ . mm’):
Substrate
Mean activity
Tyrosine Tyramine Phenol Hydroquinone Dopamine Dopa Catechol Epinephrine Pyrogallol
No activity No activity No activity No activity 0.0096 + 0.00 15n.s. 0.0106 & 0.0011 ns. 0.0160 + 0.0020* 0.0203 + 0.0028 ns. 0.0203 f 0.0020 ns.
78
K. NELLAIAPPAN
ET AL.
effectively than pyrogallol and epinephrine. The enzyme shows less activity to DOPA and dopamine when compared with catechol, pyrogallol, and epinephrine. The inefficiency of the enzyme in oxidizing hydroquinone suggests that the enzyme is not a lactase type. The enzyme shows no activity when monophenols are used. In view of the high activity of the enzyme on epinephrine, this substrate was used for characterization of the enzyme.
A
Fig. 1. Scanned 5% polyacrylamide gel electrophoretic pattern of the cuticular protein of Emerita asiatica: the gels stained in 0.1 y0 amido black in 7% acetic acid and de-stained in 7% acetic acid were scanned in a Carl Zeiss ERI-10 recording densitometer; A, premoult; B, freshmoult; C, intermoult; D, postmoult.
PHENOLOXIDASE
ACTIVITY
IN EMERITA
19
ASIATICA
The pH optimum of the phenoloxidase of freshmoult cuticle differs in different buffers as shown in Fig. 2. In Tris buffer, the enzyme shows more activity at pH 8.5.
0.030 0.026 w
4
5
6
7
7
8
8
9
5
6
7
8
PH A
B
C
Fig. 2. Phenoloxidase activity of the freshmoult cuticle of Emerita asiatica in different buffers (0.25 M) at various pH levels: epinephrine was used as substrate; increase in optical density was recorded in Bausch & Lomb Spectronic 21 spectrophotometer at 420 nm immediately after the addition of 0.2 ml of the enzyme; A, citrate buffer; B, Tris buffer; C, phosphate buffer.
Above this pH the activity is completely destroyed. The enzyme activity is also high at pH 8.5 in phosphate buffer. In citrate buffer it shows much lower activity with a peak at pH 5.0. The difference in activity seen with citrate and Tris buffer may be caused by inhibition by the citrate anion. The pH activity curve was re-determined in 0.05 M citrate buffer and found not to differ significantly from that in 0.25 M citrate, suggesting that citrate inhibition is not the cause of this difference. Barrett & Andersen (1981) observed the phenoloxidase of ‘A’ type from Calliphora vicina showing optimum activity at pH 4.5. The phenoloxidase in the periostracum of the marine bivalve Modiolus demissus shows maximum activity at pH 8.0-8.5 (Waite & Wilbur, 1976). Chromatographic analysis shows that the cuticle of Emerita asiatica contains at least five different phenols viz. dopamine, norepinephrine (adrenaline), normetaepinephrine and N-acetyl norepinephrine. The DOPA spot appears to be very faint (Fig. 3). As several phenols can easily travel at the same rate in the chromatogram (S. 0. Andersen, pers. comm.), it is essential to confirm the results before coming to a firm conclusion.
80
K. NELLAIAPPAN
r
ET AL.
Origin
DOPA
Noradrenaline
Dopamine
Normetadrenaline N-Acetyl
noradrenaline
Fig. 3. Chromatogram showing phenols extracted from 50 freshmoult cuticles of Emerita asiatica in 20 ml of 95% alcohol in 0.01 M HCl and evaporated after centrifugation: the residue was dissolved in 10% isopropanol and spotted on Whatman No. 1 chromatogram paper; butanol: acetic acid: water in the ratio of 12 : 3 : 5, was used as solvent; ferric chloride-ferricyanide reagent was used to localize the phenols.
PHENOLOXIDASE ACTIVITY IN EMERITA ASZATICA
81
DISCUSSION The present study shows that the precursors of the tanning of crustacean cuticle such as protein, phenol, and phenoloxidase are distributed in the epicuticle and exocuticle as observed by Stevenson & Adomako (1967). The preponderance of diphenoloxidase activity and the absence of monophenolase in the freshmoult cuticle of E. asiatica suggest that the diphenol which is involved in the sclerotin formation is synthesized elsewhere. Summer (1967) observed both monophenolase and diphenoloxidase activity in the blood of Uca pugnax and reported a decrease in the blood phenoloxidase in the freshmoult stage, suggesting the sequestration of the enzyme into cuticle where the tanning process takes place. Incidentally, Vacca & Fingerman (1975) have reported the presence of diphenol in the blood cells of U. pugilutor. These observations envisage that the monophenolase in the blood hydroxylates the monophenol to diphenol and subsequently the cuticular phenoloxidase converts the diphenol to quinone rendering the protein hard and rigid. Although the phenoloxidase oxidizes a number of phenols, epinephrine and pyrogallol are oxidized more effectively. It is noted, however, that the maximum absorptivity of the oxidation products of the various diphenols used as substrate will vary and that product instability may become a significant factor at above pH 7.0 (Waite, 1976), so that the selective differences in the activities of the di- and triphenol substrates used in these studies may be a fortuitous consequence of the standard enzyme assay conditions chosen. The indication that the soluble cuticle enzyme has no monophenoloxidase activity is not affected, however, by these considerations. Occurrence of substantial amounts of dopamine, norepinephrine, and N-acetyl norepinephrine in in situ cuticle and more activity of the cuticular enzyme on epinephrine suggest that the derivative of epinephrine may be the immediate precursor for tanning. This possibility is supported by the observation of Vacca & Fingerman (1975) who have reported that the accumulation of labelled N-acetyl norepinephrine subsequent to incubation of 14C dopamine in the blood and their transportation to the cuticle of U. pugilutor by a carrier protein. These results suggest that DOPA may be decarboxylated into dopamine which may be further hydroxylated by dopamine /I-hydroxylase to norepinephrine. A similar metabolic route has been suggested by Marmaras et al. (1971) in the midgut gland of decapod crustacean Upogebia littoralis. The significance of normetaepinephrine in the cuticle of Emerita asiatica deserves further study. Characterization of the enzyme associated with sclerotization other than phenoloxidase in crustaceans has received little attention. In addition to quantitative changes, cuticular phenoloxidase in the same animal also showed qualitative changes (Nellaiappan et al., 1980). These authors observed two identical fractions in premoult and postmoult and five fractions in the intermoult suggesting the multiple forms of the enzyme. The presence of multiple forms of the enzyme is evidenced by the occurrence of more than one pH optima in the freshmoult cuticle. The peak activity at pH 5.0 was not observed, when phosphate buffer was used, suggesting that the enzyme may be sensitive to phosphate ions. Occurrence of more
82
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ET AL.
than one pH optima in parasitic phosphatase has been attributed to the existence of the enzyme in multiple forms (Nellaiappan & Ramalingam, 1981). In addition to phenoloxidase, other protein fractions show heterogeneity in different moult stages. The identical five protein fractions in freshmoult and premoult and the ten fractions in intermoult and premoult suggest protein polymorphism or addition of different proteins during cuticle synthesis. Protein polymorphism is not uncommon in insect cuticle (Hackman & Goldberg, 1976).
ACKNOWLEDGEMENTS
The critical comments of Professor S.O. Andersen are gratefully acknowledged. Thanks are due to University Grants Commission, New Delhi, for financial support.
REFERENCES
ANDERSEN,SO., 1979. Biochemistry of insect cuticle. Annu. Rev. Entomol., Vol. 24, pp. 29-61. BARNES,H. & J. BLACKSTOCK,1976. Further observations on the biochemical composition of the cement of Lepusfasciculuris Ellis 8c Solander; electrophoretic examination of the protein moieties under various conditions. J. Exp. Mar. Biol. Ecol., Vol. 25, pp. 263-271. BARRETT,F.M. & S. 0. ANDERSEN,1981. Phenol oxidases in larval cuticle of the blow fly Calliphora vicina. Insect Biochem., Vol. 11, pp. 17-23. BRUNET,P. C. J., 1980. The metabolism of the aromatic amino acids concerned in the cross linking of insect cuticle. Insect Biochem., Vol. 10, pp. 467-500. DRACH,P., 1939. Mue et cycle d’intermue chez les Crustacts decapodes. Ann. Inst. Ockanogr. (Paris), N.S., Vol. 19, pp. 103-391. HACKMAN,R. H., 1971. The integument of Arthropoda. In, Chemical zoology, Vol. VI, Arthropoda, Part B, edited by M. Florkin & B.T. Scheer, Academic Press, New York, pp. l-62. HACKMAN,R. H. & M. GOLDBERG,1976. Comparative chemistry of arthropod cuticular proteins. Comp. Biochem. Physiol., Vol. 55B, pp. 201-206. HATHWAY,D. E., 1969. Plant phenols and tannins. In, Chromatographic and electrophorefic techniques. Vol. I, edited by 1. Smith, William Heinemann Medical Books Ltd., London, 2nd edition, pp. 390-436. LOWRY,O.H., N.J. ROSEBROUGH,A.L. FARR & R. J. RANDALL,1951. Protein measurement with Folin phenol reagent. J. Biol. Chem., Vol. 193, pp. 265-275. MARMARAS,V.J., E.G. FRAGOULIS& A. CHRISTOPHORATOU MANIOTI, 1971. The metabolism of “‘C dopamine in the decapod crustacean Upogebia littoralis. Comp. Gen. Pharmacol., Vol. 2, pp. 377-382. NELLAIAPPAN,K. & K. RAMALINGAM,1980. Specificity of the enzyme phenoloxidase and possible metabolic pathway of sclerotization in Paraplerwus sauridae. J. Parasitol., Vol. 66, pp. 217-219. NELLAIAPPAN,K. & K. RAMALINGAM,1981. Studies on the activity of phosphatase in Puruplerurus sauridae. Comp. Physiol. Ecol., Vol. 6, pp. l-9. NELLAIAPPAN,K., K. MANIMOZHI& K. RAMALINGAM,1980. Iso-enzyme pattern of phenol oxidase in Emerita asiatica. Comp. Physiol. Ecol., Vol. 5, pp. 3 18-3 19. SKINNER,D.M., 1962. The structure and metabolism of a crustacean integumentary tissue during a molt cycle. Biol. Bull. (Woods Hole, Mass.), Vol. 123, pp. 635-647. SMITH, I., 1968. Acrylamide gel disc electrophoresis. Section I: techniques of disc electrophoresis. In, Chromatographic and electrophoretic technique, Vol. II, edited by I. Smith, William Heinemann-Medical Books Ltd., London, 2nd edition, pp. 365-385. SMYTH, J.D., 1954. A technique for the histochemical demonstration of polyphenol oxidase and its application to egg shell formation in helminth and byssus formation in Mytilus. Q. J. Microsc. Sci., Vol. 95, pp. 139-152.
PHENOLOXIDASE
ACTIVITY
IN EMERITA ASIATICA
83
SOKAL, R. R. & F. J. ROHLF, 1969. Biometry - the principles and practice of statistics in biological research. W.H. Freeman & Co., San Francisco, California, pp. 776. STEVENSON, J.R. & T.Y. ADOMAKO, 1967. Diphenol oxidase in the crayfish cuticle. Localization and changes in activity during the moulting cycle. J. Insect Physiol., Vol. 13, pp. 1803-1811. SUMMERS, N.M., 1967. Cuticle sclerotization and blood phenol oxidase in the fiddler crab, Uca pugnax. Comp. Biochem. Physiol., Vol. 23, pp. 129-138. VACCA, L. L. & M. FINGERMAN, 1975. The mechanism of tanning in the fiddler crab, Uca pugilator. II. The cyclic appearance of tanning agents and attached carrier proteins in the blood during the molting cycle. Comp. Biochem. Physiol., Vol. 51B, pp. 483-487. WAITE, J. H., 1976. Calculating extinction coefftcients for enzymatically produced 0-quinones. Anal. Biochem., Vol. 75, pp. 211-218. WAITE, J. H. & K. M. WILBUR, 1976. Phenoloxidase in the periostracum of the marine bivalve Modiolus demissus Dillwyn. J. Exp. Zool., Vol. 195, pp. 359-368.
75
PHENOLOXIDASE ACTIVITY AND ITS ROLE IN CUTICULAR SCLEROTIZATION IN A MOLE CRAB EMERITA
K. NELLAIAPPAN,
ASIATICA
Mike Edwards
T. THANGARAJ
and K. RAMALINGAM
~e~a~rn~n~ of Zoology, UniygT~i~~ of Madras, Mad~as~~
005. India
Abstract: Phenoloxidase has been localized in the epicuticle, exocuticle, and epidermal cells of the mole crab, Emerita asiatica Milne Edwards. The enzyme activity in different moulting stages is in the order of freshmoult > premoult > intermoult = postmoult. The phenoioxidase of the freshmoult cuticle oxidizes pyrogallol and epinephrine (adrenaline) more effectively than the other phenols studied. There is no monophenolase activity. The possible metabolic pathway has been suggested based on the specificity of the enzyme and the chromatographic identification of the extracted phenols. Phenoloxidase shows different pH optima in different buffers. The protein patternin the various moulting stages of the cuticle differs and the results are discussed in relation to sclerotization.
INTRODUCTION
It is well known that the protein of most of the arthropod cuticle is stabilized by quinone. Many workers characterized the precursors of the sclerotin such as phenol, phenoloxidase, and protein in a number of insects (see Andersen, 1979; Brunet, 1980, for reviews). The chemical aspects of the cuticle of arthropods other than insects have, however, been largely neglected. Although phenoloxidase and phenols have been reported in crustacean cuticle (for a review see Hackman, 1971; Vacca & Fingerman, 1975) and in the cement gland of Lepas ~~c~c~~ffr~ (Barnes & Blackstock, 1976), studies regarding its nature and characterization have only been carried out in a few crustaceans (Stevenson & Adomako, 1967; Summer, 1967). The present study deals with the cuticular sclerotization and characterization of phenoloxidase of the mole crab Eme~ta asiuticu Milne Edwards.
MATERIAL
AND METHODS
E. asiatica were collected from the intertidal region of Madras beach opposite Madras University. The moult stages such as fresh-, post-, inter- and premoult were identified as described by Drach (1939). The carapace cuticles (2-2.5 cm) were removed manually after washing the animals in running tap water to remove the sand particles. To localize the enzyme, the cuticles in various stages of development were fixed in 70% alcohol for 30 min and washed in distilled water for 15 min. The cuticles were incubated 0022”0981/82/00~-0~0/$02.75
0 1982 Elsevier Biomedical Press
16
K.NELLAIAPPAN ETAL.
in 0.1 y0 catechol solution at 37 “C for 1 h as described by Smyth (1954). Subsequently, the cuticles were sectioned (10-15 pm thick) with a Cryocut microtome (American Opticals) at -20 “C. For the enzyme assay, 25 cuticles were homogenized in 5 ml of ice-cold distilled water in a mortar and pestle. The homogenate was centrifuged at 2000 g for 20 min and the clear supernatant containing soluble phenoloxidase was used as the enzyme source. The activity measured in the sediment showed the presence of an insoluble particulate form of the enzyme, and was found to have a low activity (0.001 OD * mg protein-’ * min-’ ). The enzyme assay was carried out as described by Nellaiappan & Ramalingam (1980) with a few modifications. One ml of 0.01 M substrate and 1 ml of phosphate buffer (0.25 M) at pH 7.5 were taken in a l-cm cuvette. Increase in optical density (OD) was recorded in Bausch & Lomb Spectronic 21 Spectrophotometer at 420 nm immediately after the addition of 0.2 ml of the enzyme. For the control, 0.2 ml of distilled water was used instead of enzyme. The results are expressed as OD * mg protein - ’ . min - ‘. 0.25 M Tris-HCl, phosphate, and citrate buffers were used at 0.5 pH intervals to study the pH optimum. Protein content of the sample was determined by the method of Lowry et al. (1951) using bovine albumin as standard. The Student-Newmann-Keuls procedure as detailed by Sokal dz Rohlf (1969) was followed to test the differences between means. 5 % polyacrylamide gel was used to study the protein pattern of the cuticle in different stages as described by Smith (1968). 0.02 M Tris-glycine at pH 8.3 and 0.3 M Tris-HCl at pH 8.9 were used as tank and gel buffers, respectively. About 200 pg protein were directly applied over the running gel and 3 mA current was supplied to each gel tube. The gels were stained in 0.1% amido black in 7% acetic acid and de-stained in 7% acetic acid. The stained gels were scanned in a Carl Zeiss ERI-10 recording densitometer. The phenols were extracted by homogenizing 50 cuticles in 20 ml of 95% alcohol in 0.01 N HCI and centrifuging at 1000 g for 5 min. The supernatant was evaporated to dryness and the residue was taken up in 10% isopropanol and spotted on Whatman No. 1 chromatogram paper. Butanol : acetic acid : water in the ratio of 12 : 3 : 5 was used as solvent and descending chromatography was carried out for 17 h. The phenols were localized by spraying with ferric chloride-ferricyanide reagent, because it detects < 1 pg of phenols (Hathway, 1969). The various phenols were identified by using the standards and R, values.
RESULTS
In the Cryocut sections, epicuticle, exocuticle, and endocuticle layers could easily be seen. In the fresh moult cuticle the epidermal cells are polygonal in shape and are secretory in nature. In the subsequent stages the volume of the cells is reduced and the thickness of the cuticle increases. Similar morphological changes during the moult cycle by the integument of the land crab Gecarcinus lateralis have been reported by Skinner
PHENOLOXIDASE
ACTIVITY IN EMERITA ASZATICA
71
(1962). After incubation in catechoi, epicuticle, exocuticle, and epidermal cells of the freshmoult show an intense brown colour. In other stages exocuticle and epidermal cells show only a faint positive reaction. The extractable cuticular phenol oxidase activity of Emerita asiatica on epinephrine (adrenaline) in different moult stages is given in Table I. In the freshmoult cuticle the TABLE
I
Cuticular phenoloxidase activity (4 SD) of Emerita usiutica in different moult stages on the substrate, epinephrine (AOD . mg protein- ’ min-‘): mean activity, average of five readings; ns., not significant; *, significant at the 5% level. Stages
Mean activity
Postmoult Intermoult Premoult Freshmouh
0.0065 + 0.00030 n.s. 0.007 1 f 0.00034 n.s. 0.0092 k 0.00020* 0.0192 f 0.00030*
enzyme shows more activity when compared with other stages. In the postmoult stage the activity is moderate. Intermoult and premoult cuticles show less activity and the difference in the activity of these two stages is statistically non-significant. The electrophoretic pattern of the cuticular protein varies in the different moult stages. In fr~shmoult and premoult stages the protein pattern is identical; these two show five protein fractions. In intermoult and postmoult stages ten protein fractions are, however, separated in the gel (Fig. 1). As the freshmoult cuticle shows more activity, the characterization of the enzyme was carried out in freshmoult cuticle. The activity of the enzyme in the freshmoult cuticle on different substrates is given in Table II. The enzyme activity is more than other substrates when pyrogallol or epinephrine is used and the mean values of activity in these two substrates are statistically non-si~i~c~t. Catechol is oxidized less TABLE II Cuticular
activity (f SD) of Emerita ask&a on various substrates (AOD . mg promean activity, average of live readings; ns, not significant; *, significant at the 5% level.
phenoloxidase
tein-’ . mm’):
Substrate
Mean activity
Tyrosine Tyramine Phenol Hydroquinone Dopamine Dopa Catechol Epinephrine Pyrogallol
No activity No activity No activity No activity 0.0096 + 0.00 15n.s. 0.0106 & 0.0011 ns. 0.0160 + 0.0020* 0.0203 + 0.0028 ns. 0.0203 f 0.0020 ns.
78
K. NELLAIAPPAN
ET AL.
effectively than pyrogallol and epinephrine. The enzyme shows less activity to DOPA and dopamine when compared with catechol, pyrogallol, and epinephrine. The inefficiency of the enzyme in oxidizing hydroquinone suggests that the enzyme is not a lactase type. The enzyme shows no activity when monophenols are used. In view of the high activity of the enzyme on epinephrine, this substrate was used for characterization of the enzyme.
A
Fig. 1. Scanned 5% polyacrylamide gel electrophoretic pattern of the cuticular protein of Emerita asiatica: the gels stained in 0.1 y0 amido black in 7% acetic acid and de-stained in 7% acetic acid were scanned in a Carl Zeiss ERI-10 recording densitometer; A, premoult; B, freshmoult; C, intermoult; D, postmoult.
PHENOLOXIDASE
ACTIVITY
IN EMERITA
19
ASIATICA
The pH optimum of the phenoloxidase of freshmoult cuticle differs in different buffers as shown in Fig. 2. In Tris buffer, the enzyme shows more activity at pH 8.5.
0.030 0.026 w
4
5
6
7
7
8
8
9
5
6
7
8
PH A
B
C
Fig. 2. Phenoloxidase activity of the freshmoult cuticle of Emerita asiatica in different buffers (0.25 M) at various pH levels: epinephrine was used as substrate; increase in optical density was recorded in Bausch & Lomb Spectronic 21 spectrophotometer at 420 nm immediately after the addition of 0.2 ml of the enzyme; A, citrate buffer; B, Tris buffer; C, phosphate buffer.
Above this pH the activity is completely destroyed. The enzyme activity is also high at pH 8.5 in phosphate buffer. In citrate buffer it shows much lower activity with a peak at pH 5.0. The difference in activity seen with citrate and Tris buffer may be caused by inhibition by the citrate anion. The pH activity curve was re-determined in 0.05 M citrate buffer and found not to differ significantly from that in 0.25 M citrate, suggesting that citrate inhibition is not the cause of this difference. Barrett & Andersen (1981) observed the phenoloxidase of ‘A’ type from Calliphora vicina showing optimum activity at pH 4.5. The phenoloxidase in the periostracum of the marine bivalve Modiolus demissus shows maximum activity at pH 8.0-8.5 (Waite & Wilbur, 1976). Chromatographic analysis shows that the cuticle of Emerita asiatica contains at least five different phenols viz. dopamine, norepinephrine (adrenaline), normetaepinephrine and N-acetyl norepinephrine. The DOPA spot appears to be very faint (Fig. 3). As several phenols can easily travel at the same rate in the chromatogram (S. 0. Andersen, pers. comm.), it is essential to confirm the results before coming to a firm conclusion.
80
K. NELLAIAPPAN
r
ET AL.
Origin
DOPA
Noradrenaline
Dopamine
Normetadrenaline N-Acetyl
noradrenaline
Fig. 3. Chromatogram showing phenols extracted from 50 freshmoult cuticles of Emerita asiatica in 20 ml of 95% alcohol in 0.01 M HCl and evaporated after centrifugation: the residue was dissolved in 10% isopropanol and spotted on Whatman No. 1 chromatogram paper; butanol: acetic acid: water in the ratio of 12 : 3 : 5, was used as solvent; ferric chloride-ferricyanide reagent was used to localize the phenols.
PHENOLOXIDASE ACTIVITY IN EMERITA ASZATICA
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DISCUSSION The present study shows that the precursors of the tanning of crustacean cuticle such as protein, phenol, and phenoloxidase are distributed in the epicuticle and exocuticle as observed by Stevenson & Adomako (1967). The preponderance of diphenoloxidase activity and the absence of monophenolase in the freshmoult cuticle of E. asiatica suggest that the diphenol which is involved in the sclerotin formation is synthesized elsewhere. Summer (1967) observed both monophenolase and diphenoloxidase activity in the blood of Uca pugnax and reported a decrease in the blood phenoloxidase in the freshmoult stage, suggesting the sequestration of the enzyme into cuticle where the tanning process takes place. Incidentally, Vacca & Fingerman (1975) have reported the presence of diphenol in the blood cells of U. pugilutor. These observations envisage that the monophenolase in the blood hydroxylates the monophenol to diphenol and subsequently the cuticular phenoloxidase converts the diphenol to quinone rendering the protein hard and rigid. Although the phenoloxidase oxidizes a number of phenols, epinephrine and pyrogallol are oxidized more effectively. It is noted, however, that the maximum absorptivity of the oxidation products of the various diphenols used as substrate will vary and that product instability may become a significant factor at above pH 7.0 (Waite, 1976), so that the selective differences in the activities of the di- and triphenol substrates used in these studies may be a fortuitous consequence of the standard enzyme assay conditions chosen. The indication that the soluble cuticle enzyme has no monophenoloxidase activity is not affected, however, by these considerations. Occurrence of substantial amounts of dopamine, norepinephrine, and N-acetyl norepinephrine in in situ cuticle and more activity of the cuticular enzyme on epinephrine suggest that the derivative of epinephrine may be the immediate precursor for tanning. This possibility is supported by the observation of Vacca & Fingerman (1975) who have reported that the accumulation of labelled N-acetyl norepinephrine subsequent to incubation of 14C dopamine in the blood and their transportation to the cuticle of U. pugilutor by a carrier protein. These results suggest that DOPA may be decarboxylated into dopamine which may be further hydroxylated by dopamine /I-hydroxylase to norepinephrine. A similar metabolic route has been suggested by Marmaras et al. (1971) in the midgut gland of decapod crustacean Upogebia littoralis. The significance of normetaepinephrine in the cuticle of Emerita asiatica deserves further study. Characterization of the enzyme associated with sclerotization other than phenoloxidase in crustaceans has received little attention. In addition to quantitative changes, cuticular phenoloxidase in the same animal also showed qualitative changes (Nellaiappan et al., 1980). These authors observed two identical fractions in premoult and postmoult and five fractions in the intermoult suggesting the multiple forms of the enzyme. The presence of multiple forms of the enzyme is evidenced by the occurrence of more than one pH optima in the freshmoult cuticle. The peak activity at pH 5.0 was not observed, when phosphate buffer was used, suggesting that the enzyme may be sensitive to phosphate ions. Occurrence of more
82
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ET AL.
than one pH optima in parasitic phosphatase has been attributed to the existence of the enzyme in multiple forms (Nellaiappan & Ramalingam, 1981). In addition to phenoloxidase, other protein fractions show heterogeneity in different moult stages. The identical five protein fractions in freshmoult and premoult and the ten fractions in intermoult and premoult suggest protein polymorphism or addition of different proteins during cuticle synthesis. Protein polymorphism is not uncommon in insect cuticle (Hackman & Goldberg, 1976).
ACKNOWLEDGEMENTS
The critical comments of Professor S.O. Andersen are gratefully acknowledged. Thanks are due to University Grants Commission, New Delhi, for financial support.
REFERENCES
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SOKAL, R. R. & F. J. ROHLF, 1969. Biometry - the principles and practice of statistics in biological research. W.H. Freeman & Co., San Francisco, California, pp. 776. STEVENSON, J.R. & T.Y. ADOMAKO, 1967. Diphenol oxidase in the crayfish cuticle. Localization and changes in activity during the moulting cycle. J. Insect Physiol., Vol. 13, pp. 1803-1811. SUMMERS, N.M., 1967. Cuticle sclerotization and blood phenol oxidase in the fiddler crab, Uca pugnax. Comp. Biochem. Physiol., Vol. 23, pp. 129-138. VACCA, L. L. & M. FINGERMAN, 1975. The mechanism of tanning in the fiddler crab, Uca pugilator. II. The cyclic appearance of tanning agents and attached carrier proteins in the blood during the molting cycle. Comp. Biochem. Physiol., Vol. 51B, pp. 483-487. WAITE, J. H., 1976. Calculating extinction coefftcients for enzymatically produced 0-quinones. Anal. Biochem., Vol. 75, pp. 211-218. WAITE, J. H. & K. M. WILBUR, 1976. Phenoloxidase in the periostracum of the marine bivalve Modiolus demissus Dillwyn. J. Exp. Zool., Vol. 195, pp. 359-368.