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

Printed in Great Britain

SURVEY OF A TRYPTIC DIGESTIVE ENZYME IN VARIOUS SPECIES OF CRUSTACEA*

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

542

EDWARD DEVILLEZ AND KAREN BUSCHLEN

and the amphipod Synurella sp. were collected periodically from a cold-water spring at Acton Lake, Oxford, Ohio, and maintained in natural spring water at 4C. 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). In the case of Balanus nubilus, the intestine was unfolded and punctured with a cannula in order to remove the fluid. Extracts of various digestive organs were homogenized in cold distilled water. All extracts were centrifuged for 20 min at 17,000g in the cold. Proteolytic activity was determined by the Azocasein (Mann) colorimetric method described by Charney & Tomarelli (1947) or the casein digestive method (Kunitz, 1947) using casein of the Hammersten quality (N.B. Co.) and the boratephosphate-citrate universal buffer described by Northrop (1922). Tryptic activity was determined by the spectrophotometric method of Hummel (1959) using 10 -3 M T A M E (N.B. Co.) prepared in universal buffer. A unit of tryptic activity caused an absorbency increase of 0-001/rain at 25C when the spectrophotometer was set at 247 m/~. Stock solutions of 10 -2 M DFP (Mann) were prepared in anhydrous isopropanol. Enzyme preparations were diluted in 0.05 M Tris buffer at pH 8.1. 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. Stock solutions of 70/zg/ml ovomucoid (Mann), 10 -3 M glutathione and 10 .3 M cysteine were prepared in 0.05 M Tris buffer at pH 8.0. The PCMB stock solution was prepared in 0-02 M glycyl-glycine buffer at pH 7-6. Enzyme preparations were diluted with appropriate buffer. 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). Preparative acrylamide gel electrophoresis was conducted as previously described (DeVillez, 1964). A discontinuous Tris-citrate-borate-LiOH buffer was used (Barrett et al., 1962). Protein components were eluted by diffusion or detected by staining the gel in saturated Amido Black 10 B. RESULTS It may be seen in Table 1 that all extracts tested demonstrated alkaline proteolytic activity as indicated by casein hydrolysis. Gastric juice and digestive gland extracts of the barnacle demonstrated less activity in an alkaline medium than the amphipod, isopod or decapod extracts. Gastric juice extracts of the barnacle hydrolyzed casein at a pH optimum of 4.0 whereas all other gastric juice extracts demonstrated at least one optimum above pH 7.0. All extracts except those of Balanus nubilus hydrolyzed T A M E at pH 8.1. Eight gastric juice extracts of the barnacle were assayed for T A M E activity between pH 4.6 and 8.9. None of these gastric juice extracts demonstrated T A M E activity in the range tested. Two digestive gland extracts (five tested) of the barnacle demonstrated T A M E activity at pH 5-2 but not at pH 6.0, 7.0 or 8.0. Ovomucoid had no inhibitory effect on the T A M E activity of these digestive gland extracts. Contrastingly, ovomucoid had

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 , d i g e s t i v e g l a n d , 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 . Similar inhibition was observed when such extracts were assayed in the presence of D F P . 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 . 1). G l u t a t h i o n e , c y s t e i n e , 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.

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.j. g.j. g.j. g.j. g.j. g.j. d.g. g.j. g.j. d.g. i.c. g.j. g.j. g.j. d.t. d.g. g.j. g.j. d.t. i.c. d.g. i. d.g.

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. Limnoria lignorum Synurella sp. Balanus nubilus

i.
d.g.

+
+

+
+

i.
d.t. d.t. d.t. i.e. g.j. d.g.

+
+ + + + +

+
+ + + + + (pH 5"2) + + + -

* g.j. = gastric juice; d.g. = digestive gland; i.c. = isolated c o m p o n e n t ; d.t. = digestive gland and intestine; i. = intestine; + , positive reaction or inhibition; - , no reaction or no inhibition.

544

EDWARD DEVILL~.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 4.6-8.9. The T A M E specific enzyme was isolated as a single component from decapod, isopod and amphipod extracts (Fig. 2). Similar electrophoretic mobilities were observed in all extracts tested. The T A M E specific component was anionic at pH 8.0 and found to be proteolytic as evidenced by casein hydrolysis. The pH optimum for proteolytic activity by isolated components was broad (pH 6.5-8.0). 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.

8r
>., ),

I.. wosnesenskii DFP (i.c.)

20C

Synure//o sp. Ovomucoid (cl.l.)

60

- - ~
I Time, 2
rain

W I--

40 20 0
~ 4

1 5 C I O C 5 C/
0

~1

Time, rain

C. mogister DFP (g.j.)

200
:~ 150
I00 I--

--

20C 15C
IO( 5o

C. productus Ovomucoid (l.c~

- - i ~

50
30

Time,

60

90
sec

120

30

Time,

60

90
sec

120

FIo. 1. The effect of DFP and ovomucoid on the TAME activity of extracts of digestive organs of Crustacea; O, control, O, inhibitor. For abbreviations, see footnote Table 1. The effect of pH on the T A M E activity extracts of Crustacea is shown in Fig. 3. An ranging from pH 8.0-8.3 depending on the optimum was observed whether the extract some digestive organ. of gastric juice and digestive gland alkaline pH optimum was observed extract tested but a single alkaline was gastric juice or prepared from

(o) Sample slot

Cancer

gracilis

iOC i~

:': %"

II .": %"

ee ": "

" " %"

l(. = ~)1 I I[.*.

(b)

Zdofheo

wosnesenskii

"" S'"'= :." =. ..... .=

":)

III

(c)

5ynurella

sp.

>, 20 -u LL) tO J'~ f ~ I 0

/ ~

[\ l"
o-o

~o-o"

e-o

"\.-'-. .... .""'-."~../"

L
25 50 0-Scm gel segments

e~eJe 55

:"

no

L5 20 Fraction number~

40

(-)

(+)

FIG. 2. Representative patterns of acrylamide gel electrophoresis of digestive

organ extracts of Crustacea; pH 8'0, 0C, 16 hr, Tris-citrate-borate-LiOH gels, 300 V, average current = 20 mA. T A M E active components designated by arrows.

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!

6.0

7.0
pH

8.0

~0

FIG. 3. The effect of pH on the TAME activity of gastric juice and digestive organ extracts of Crustacea. @, SynureUa (d.t.); A, Lirceus (d.t.); m, Cancer (g.j.). For abbreviations see footnote, Table 1. 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. 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. Gastric juice extracts were used in the present investigation wherever the size of the animal permitted. In those cases where tissue homogenates had to be used, a single tryptic component was isolated by acrylamide electrophoresis and demonstrated properties similar to the component isolated from gastric juice extracts of larger species. 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. T h e results reported here indicate that trypsin is found in the amphipod, isopod and decapod. Degkwitz (1957) has suggested the presence of trypsin in amphipods and decapods excepting Cragon cragon and Palaemon serratus. In these species digestion was attributed to catheptic enzymes. 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. A tryptic component could not be demonstrated for Balanus nubilus in the present investigation. This is consistent with the lack of an alkaline optimum for casein hydrolysis by gastric juice extracts. Digestive gland extracts demonstrated T A M E activity only inconsistently and then only in an acid medium. Ovomucoid

546

EDWARD DEVILLEZ AND KAREN BUSCHLEN

was without effect on T A M E activity. 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. F u r t h e r m o r e , catheptic activators had no influence on TA~VIE activity in gastric juice extracts. T h e possibility that T A M E activity was inhibited by some interfering substance in the extracts cannot be excluded.

Acknowledgements--The first author appreciates the assistance provided by Dr. Robert L. Fernald, Director of the Friday Harbor Marine Laboratories of the University of Washington. The research was supported by the United States National Science Foundation (GB-747) and by the Faculty Research Fund of Miami University.
REFERENCES BARRETT R. J., FRIESEN H. & ASTWOOD E. B. (1962) Characterization of pituitary and peptide hormones by electrophoresis in starch gels. ft. biol. Chem. 237, 432. CHARNEY J. & TOMARELLI R. M. (1947) A colorimetric method for the determination of the proteolytic activity of duodenal juice. J. biol. Chem. 171, 501-505. DECKWXTZ E. (1957) Ein Beitrag zur Natur der Proteolytic Verdauungsfermente bei verschiedenen Crustaceenarten. Ver6ff. Inst. Meeresforsch. Bremerh. 5, 1-13. DEVILLEZ E. J. (1964) Preparative acrylarnide gel electrophoresis with the conventional starch-gel apparatus. Annls Biochem., N . Y . 9, 485-486. DEVILLEZ E. J. (1965) Isolation of the proteolytic digestive enzymes from the gastric juice of the crayfish Orconectes virilis. Comp. Biochem. Physiol. 14, 577-586. HUMMEL B. C. W. (1959) A modified spectrophotometric determination of chymotrypsin, trypsin and thrombin. Can. J. Biockem. Physiol. 37, 1393-1400. KUNITZ M. (1947) Crystalline soybean trypsin inhibitor--II. General properties, ft. biol. Chem. 30, 291-310. NORTHROPJ. H. (1922) The mechanism of the influence of acids and alkali on the digestion of proteins by pepsin and trypsin. J. gen. Physiol. 5, 263-274. VONK H. J. (1960a) Digestion and metabolism. In The Physiology of Crustacea (Edited by WATERMANT. H.) Vol. 1, pp. 291-316. Academic Press, New York. VONK H. J. (1960b) Comparative biochemistry of digestive mechanisms. In Comparative Biochemistry (Edited by FLORKINM. & MASON H. S.) Vol. 6, pp. 347-392. Academic Press, New York.