Comp. Bioehem. PhysioL, 1967, VoL 21, pp. 541 to 546. Pergamon Press Ltd.

Printed in Great Britain

E D W A R D D p V I L L E Z t and K A R E N B U S C H L E N ~ : Friday Harbor Laboratories, University of Washington, Seattle, Washington, U.S.A., and Department of Zoology and Physiology, Miami University, Oxford, Ohio, U.S.A.
(Received 19 December 1966)

A b s t r a c t - - 1 . The distribution of a tryptic digestive enzyme was investigated in gastric juice and digestive organ extracts of a number of Crustacea. Tryptic activity was based on the alkaline hydrolysis of casein and TAME as well as the inhibition of TAME activity in the presence of DFP and/or ovomucoid. 2. Tryptic activity was found in all amphipod, isopod and decapod extracts tested and could be isolated as a single component by acrylamide electrophoresis. 3. A TAME-specific component could not be found in gastric juice extracts of Balanus nubilus and attempts to demonstrate the presence of such a component in digestive organ extracts were inconclusive.

INTRODUCTION PROTEOLYTIC digestive enzymes have been studied extensively in Crustacea, especially decapods (Vonk, 1960a). Crustacea and other invertebrates have generally been consideredto be able to hydrolyze protein with an enzyme complement similarto that of vertebrates except for the Jackof a peptic enzyme(Vonk, 1960b). Degkwitz(1957) has suggested that crabs and amphipods use catheptic and tryptic enzymes whereas shrimps and barnacles have only catheptic components. Results were based on the use of digestive gland extracts and characterization of enzymes was based primarily on pH optima. More recently, three proteolytic enzymeshave been isolated by acrylamidegel electrophoresisfrom the gastric juice of the crayfish Orconectes virilis (DeVillez,1965). A trypsin, carboxypeptidase and a protease of unknown specificitywere suggested on the basis of assays conducted on specific synthetic substrates. The present investigationwas undertaken in order to assess the distribution of the tryptic component in Crustacea. MATERIALS AND METHODS Specimenswere collectedin the vicinityof San Juan Island of the Puget Sound region and maintainedin aquaria with running sea water. The isopod Lirceus sp.
*Abbreviations: p-toluenesulfonyl-L-arginine methyl ester (TAME), diisopropylfluorophosphate (DFP). t Present address: Department of Zoology and Physiology, Miami University, Oxford, Ohio. Present address: 25 Golfcrest Road, Islington, Toronto, Ontario, Canada. 541

0. The PCMB stock solution was prepared in 0-02 M glycyl-glycine buffer at pH 7-6. Proteolytic activity was determined by the Azocasein (Mann) colorimetric method described by Charney & Tomarelli (1947) or the casein digestive method (Kunitz. RESULTS It may be seen in Table 1 that all extracts tested demonstrated alkaline proteolytic activity as indicated by casein hydrolysis. Extracts of various digestive organs were homogenized in cold distilled water. All extracts were centrifuged for 20 min at 17. Enzyme preparations were diluted with appropriate buffer.0. 10 -3 M glutathione and 10 .6 and 8. An equal volume of isopropanol or inhibitor solution was then added and the mixtures were assayed for enzyme activity after equilibration for 4 hr at room temperature. the intestine was unfolded and punctured with a cannula in order to remove the fluid.) prepared in universal buffer.0.9.05 M Tris buffer at pH 8. Eight gastric juice extracts of the barnacle were assayed for T A M E activity between pH 4.1.) and the boratephosphate-citrate universal buffer described by Northrop (1922).1. Tryptic activity was determined by the spectrophotometric method of Hummel (1959) using 10 -3 M T A M E (N. 1964). Co. 7.3 M cysteine were prepared in 0.000g in the cold. 1947) using casein of the Hammersten quality (N. An equal volume of buffer or stock solution was then added and the mixtures were assayed for enzyme activity after equilibration at room temperature for 30 min (ovomucoid--within 10 min).05 M Tris buffer at pH 8. 1962).0. Two digestive gland extracts (five tested) of the barnacle demonstrated T A M E activity at pH 5-2 but not at pH 6. All extracts except those of Balanus nubilus hydrolyzed T A M E at pH 8..542 EDWARD DEVILLEZ AND KAREN BUSCHLEN and the amphipod Synurella sp. Stock solutions of 10 -2 M DFP (Mann) were prepared in anhydrous isopropanol. Protein components were eluted by diffusion or detected by staining the gel in saturated Amido Black 10 B. None of these gastric juice extracts demonstrated T A M E activity in the range tested. Stock solutions of 70/zg/ml ovomucoid (Mann). In the case of Balanus nubilus. A discontinuous Tris-citrate-borate-LiOH buffer was used (Barrett et al.0 whereas all other gastric juice extracts demonstrated at least one optimum above pH 7. Ovomucoid had no inhibitory effect on the T A M E activity of these digestive gland extracts. Contrastingly. Gastric juice extracts of the barnacle hydrolyzed casein at a pH optimum of 4.B. Enzyme preparations were diluted in 0. Preparative acrylamide gel electrophoresis was conducted as previously described (DeVillez.0 or 8. Gastric juice and digestive gland extracts of the barnacle demonstrated less activity in an alkaline medium than the amphipod. and maintained in natural spring water at 4°C. Gastric juice was collected with a fire-polished glass cannula introduced into the stomach from the mouth according to the method of Jordan as described by Vonk (1960a). ovomucoid had . were collected periodically from a cold-water spring at Acton Lake.B. A unit of tryptic activity caused an absorbency increase of 0-001/rain at 25°C when the spectrophotometer was set at 247 m/~. isopod or decapod extracts. Co. Oxford. Ohio.

d. d. g.j. g. = digestive gland and intestine..c. i. positive reaction or inhibition. Balanus nubilus i. d.g.j.j. d. i. g. d. g. + + + + i.j.g.j.j. i. = gastric juice.g.e. . .g. G l u t a t h i o n e . Similar inhibition was observed when such extracts were assayed in the presence of D F P . d. d. g. i.t.j. d. d.g. g.t. TABLE 1--SuRvEY OF A TRYPTIC DIGESTIVE ENZYME I N CRUSTACEA* Substrate Casein (pH 7"5) + + + + + + + + + + + + + + + + + + + + + + + TAME (pH 8"1) + + + + + + + + + + + + + + + + + + + + + + + DFP Inhibitors Ovomucoid Animal Extract g. d i g e s t i v e g l a n d . + .t. Pandalus platyceros Munida quadrispina Paguristes turgidus Pagurus tenuimanus Pagurus kennelyi Cancer productus Cancer magister Cancer gracilis Cancer oregonensis Telmessus cheiragonus Hemigrapsus nudus Pinnixa littoralis Pugettia productus Hyas lyratus Idothea wosnesenskii + + + + + + + + + + + + + + + + Idothea resecada Exosphaeroma oregonensis Lirceus sp.t. c y s t e i n e . g.g. g. d. no reaction or no inhibition. 1). g. = isolated c o m p o n e n t . g. i. d.j. d i g e s t i v e t r a c t o r e x t r a c t s o f t h e i s o l a t e d T A M E c o m p o n e n t i n all o t h e r s p e c i e s t e s t e d . d. T h e t y p e o f e x t r a c t u s e d h a d n o i n f l u e n c e o n t h e effect o f t h e i n h i b i t o r s ( F i g .c. d.j.j. = intestine. g.j.g. Limnoria lignorum Synurella sp. = digestive gland.SURVEY OF A TRYPTIC DIGESTIVE ENZYME I N CRUSTACEA 543 a n i n h i b i t o r y effect o n t h e T A M E a c t i v i t y o f g a s t r i c j u i c e .t.j.t. i.j. d. g. i o d o a c e t a t e a n d P C M B h a d n o effect o n t h e T A M E a c t i v i t y of g a s t r i c j u i c e e x t r a c t s a n d t h e i s o l a t e d c o m p o n e n t o f Cancer productus.j.j. g.c.g. + + + + + + + + + + + + (pH 5"2) + + + - * g.

For abbreviations. O. of gastric juice and digestive gland alkaline pH optimum was observed extract tested but a single alkaline was gastric juice or prepared from . control.544 EDWARD DEVILL~. 60 90 sec 120 FIo.0).. The effect of DFP and ovomucoid on the TAME activity of extracts of digestive organs of Crustacea.l. 2). 1.. I. ).0 and found to be proteolytic as evidenced by casein hydrolysis.c.Z AND KAREN BUSCHLEN Glutathione or cysteine did not induce T A M E activity in gastric juice extracts of the barnacle when assays were conducted at pH 4. 2 rain W I-- 40 20 0 ~ 4 1 5 C I O C 5 C/ 0 ~1 I Time.i ~ 50 30 Time..5-8. The pH optimum for proteolytic activity by isolated components was broad (pH 6.c~ . 60 90 sec 120 0 30 Time. 3. rain 2 I 3 I 4 I C. It should be mentioned that the T A M E component isolated from Synurella was not assayed for casein hydrolysis due to low concentrations of the component after electrophoresis and elution. The effect of pH on the T A M E activity extracts of Crustacea is shown in Fig. productus Ovomucoid (l. mogister DFP (g.3 depending on the optimum was observed whether the extract some digestive organ.) 60 . 8°r >.) 200 :~ 150 I00 I-- -- 20C 15C IO( 5o C.) 20C Synure//o sp.~ I Time.9. The T A M E specific component was anionic at pH 8.j. An ranging from pH 8.0-8. see footnote Table 1. Similar electrophoretic mobilities were observed in all extracts tested. Ovomucoid (cl. O.. isopod and amphipod extracts (Fig. wosnesenskii DFP (i. inhibitor. The T A M E specific enzyme was isolated as a single component from decapod.6-8.

.. Representative patterns of acrylamide gel electrophoresis of digestive organ extracts of Crustacea.""'-. Tris-citrate-borate-LiOH gels.(o) Sample slot Cancer gracilis I iOC i~ ¸¸ D :': %" II .. T A M E active components designated by arrows.. . (b) Zdofheo wosnesenskii 0 I "" S°'"'= :.. pH 8'0.= ":) III (c) 5ynurella sp. 2./" • L 25 50 0-Scm gel segments e~eJe 55 :" 5 ~ no i L5 20 Fraction number~ ] I i ~ 40 I (-) (+) FIG. 16 hr.": %" ee ": " • " "• %" l(." =.-'-. 20 -u LL) tO J'~ f ~ I 0 / ~ [\ l" o-o ~o-o" e-o "\. . . 0°C. l >. average current = 20 mA.. = ~)1 I I[.*... 300 V.. ."~.

In these species digestion was attributed to catheptic enzymes. Ovomucoid . This is consistent with the lack of an alkaline optimum for casein hydrolysis by gastric juice extracts.).0 I 7. The effect of pH on the TAME activity of gastric juice and digestive organ extracts of Crustacea. 3. Tryptic activity was based on the alkaline hydrolysis of T A M E and casein and the inhibition of T A M E activity in the presence of D F P and/or ovomucoid. Digestive gland extracts demonstrated T A M E activity only inconsistently and then only in an acid medium.). In those cases where tissue homogenates had to be used.j. Gastric juice extracts were used in the present investigation wherever the size of the animal permitted.t.0 pH I 8.SURVEY O F A T R Y P T I C D I G E S T I V E E N Z Y M E I N CRUSTACEA 140 545 120 - 8C-6C-- 4C-- o! 6. Degkwitz (1957) has suggested the presence of trypsin in amphipods and decapods excepting Cragon cragon and Palaemon serratus.). Lirceus (d. a single tryptic component was isolated by acrylamide electrophoresis and demonstrated properties similar to the component isolated from gastric juice extracts of larger species. m. @. A tryptic component could not be demonstrated for Balanus nubilus in the present investigation. SynureUa (d. Cancer (g.t. Such an approach gives little information concerning the nature of the proteolytic components involved in protein digestion since proteolytic enzymes are of universal distribution in living cells and pH optimum alone is a weak criterion of classification. Table 1. isopod and decapod. DISCUSSION The classification of invertebrate proteolytic digestive enzymes has often been based on studies of pH optima where crude extracts of alimentary tissues have been employed. The two cathepsin inhibitors PCMB and iodoacetate as well as the two activators glutathione and cysteine had no effect on the T A M E component of Cancer productus in the present investigation which further suggests that the T A M E component is not a catheptic-type enzyme. For abbreviations see footnote.0 I ~0 I FIG. T h e results reported here indicate that trypsin is found in the amphipod. A.

263-274. M. B. Biochem. 5.546 EDWARD DEVILLEZ AND KAREN BUSCHLEN was without effect on T A M E activity. H. J. Biockem. catheptic activators had no influence on TA~VIE activity in gastric juice extracts. Academic Press. Academic Press. VONK H. In Comparative Biochemistry (Edited by FLORKINM. DEVILLEZ E. (1965) Isolation of the proteolytic digestive enzymes from the gastric juice of the crayfish Orconectes virilis. The research was supported by the United States National Science Foundation (GB-747) and by the Faculty Research Fund of Miami University. 347-392. 485-486.) Vol. New York. 9. J. In The Physiology of Crustacea (Edited by WATERMANT. biol. 37. Comp. (1947) A colorimetric method for the determination of the proteolytic activity of duodenal juice. VONK H. FRIESEN H. 171. Can. W. Chem. . biol. 1-13. J. J. (1960b) Comparative biochemistry of digestive mechanisms. HUMMEL B. 237. NORTHROPJ. CHARNEY J. H. New York. (1964) Preparative acrylarnide gel electrophoresis with the conventional starch-gel apparatus. 14. DEVILLEZ E. biol. 1393-1400. N . T h e possibility that T A M E activity was inhibited by some interfering substance in the extracts cannot be excluded. 577-586. Fernald.) Vol. trypsin and thrombin. Physiol. J. (1962) Characterization of pituitary and peptide hormones by electrophoresis in starch gels. pp. F u r t h e r m o r e . Chem. (1957) Ein Beitrag zur Natur der Proteolytic Verdauungsfermente bei verschiedenen Crustaceenarten. C. ft. Director of the Friday Harbor Marine Laboratories of the University of Washington. Physiol. Chem. J. Y . 291-316. KUNITZ M. 6. Acknowledgements--The first author appreciates the assistance provided by Dr. Bremerh. pp. 30. Annls Biochem. 1. 5. J. 501-505. Meeresforsch. T h i s would suggest a catheptic-type enzyme in gland extracts but does not necessarily mean that the enzyme is involved in digestion. (1947) Crystalline soybean trypsin inhibitor--II. REFERENCES BARRETT R. Inst. S. & TOMARELLI R. DECKWXTZ E. Robert L.. 291-310. Ver6ff. (1922) The mechanism of the influence of acids and alkali on the digestion of proteins by pepsin and trypsin. ft. J. 432. (1959) A modified spectrophotometric determination of chymotrypsin. & ASTWOOD E. gen. (1960a) Digestion and metabolism. & MASON H. Physiol.. General properties.

Sign up to vote on this title
UsefulNot useful