Survey of a Tryptic Digestive Enzyme in Various

7/27/2019 Survey of a Tryptic Digestive Enzyme in Various

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Comp. Bioehem. PhysioL, 1967, VoL 21, p p . 541 to 546.Pergamon Press Ltd . Printed in Great Bri tain

SURVEY OF A TRYPTIC DIGESTIVE ENZYME IN VARIOUSSPECIES OF CRUSTACEA*

E D W A R D D p V IL L E Z t a nd 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.

( R e c e i v e d 19 D e c e m b e r 1966)A b s t r a c t - - 1 . Th e distribution of a tryptic digestive enzyme was investigatedin gastric juice and digestive organ extracts of a number of Crustacea. Trypticactivity was based on the alkaline hydrolysis of casein and TAME as well as theinhibition of TAME activity in the presence of DFP and/or ovomucoid.2. Trypti c activity was found in all amphipod, isopod and decapod extractstested and could be isolated as a single component by acrylamide electrophoresis.3. A TAME-specific component could not be found in gastric juice extractsof B a l a n u s n u b i lu s and attempts to demonstrate the presence of such a componentin digestive organ extracts were inconclusive.

INTRODUCTIONPROTEOLYTIC digestive enzymes have been studied extensively in Crustacea,especially decapods (Vonk, 1960a). Crustacea and other invertebrates havegenerally been considered to be able to hydrolyze protein with an enzyme com-plement similar to that of vertebrates except for the Jack of a peptic enzyme(Vonk,1960b). Degkwitz (1957) has suggested that crabs and amphipods use cathepticand tryptic enzymes whereas shrimps and barnacles have only catheptic com-ponents. Results were based on the use of digestive gland extracts and charac-terization of enzymes was based primarily on pH optima. More recently, threeproteolytic enzymes have been isolated by acrylamide gel electrophoresisfrom thegastric juice of the crayfishOrconectes virilis (DeVillez, 1965). A trypsin, carboxy-peptidase and a protease of unknown specificity were suggested on the basis ofassays conducted on specific synthetic substrates. The present investigation wasundertaken in order to assess the distribution of the tryptic component inCrustacea.

MATERIALS AND METHODSSpecimenswere collected n the vicinityof San Juan Island of the Puget Sound

region and maintained in aquaria with running sea water. The isopodLirceus sp.*Abbreviations: p-toluenesulfonyl-L-arginine methyl ester (TAME), diisopropyl-fluorophosphate (DFP).t Present address: Department of Zoology and Physiology, Miami University, Oxford,Ohio.Present address: 25 Golfcrest Road, Islington, Toronto, Ontario, Canada.

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542 EDWARD DEVILLEZ AND KAREN BUSCHLENa n d t h e a m p h i p o d Synure l la s p . w e r e co l l ec t ed p e r i o d i ca l l y f r o m a co l d - w a t e rs p r i n g a t A c t o n L ak e , O x f o r d , O h i o , an d m a i n t a i n ed i n n a t u r a l s p r i n g w a t e r a t 4 C .G as t r i c ju i ce w as co l l ec t ed w i t h a f i re - p o l i sh ed g l a ss can n u l a i n t r o d u ced i n to t h es t o m a c h f r o m t h e m o u t h a c c o r d i n g t o t h e m e t h o d o f J o r d a n a s d e s c ri b e d b y V o n k(1960a) . In the case of Balanus nubi lus , t h e i n t e s ti n e w a s u n f o l d e d a n d p u n c t u r e dw i t h a c an n u l a i n o r d e r t o r emo v e t h e f l ui d . Ex t r ac t s o f v a ri o u s d i g est iv e o rg an sw e r e h o m o g en i zed in co l d d i s ti ll ed w a t e r. A l l ex t r ac ts w e r e cen t r i f u g ed f o r 20 mi na t 17 ,000g in t he co ld .P r o t eo l y t i c ac t i v i t y w as d e t e r mi n ed b y t h e A zo cas e i n ( M an n ) co l o r i me t r i cm e t h o d d e s c r i b ed b y C h a r n ey & To m ar e l l i (1 94 7) o r th e ca s e in d ig es t iv e m e t h o d( K u n i t z , 1 94 7) u s in g ca s e i n o f t h e H am m er s t e n q u a l i t y ( N .B . C o . ) an d t h e b o r a t e -p h o s p h a t e - c i t r a t e u n i v e r s a l b u f f e r d e s c r i b ed b y N o r t h r o p ( 19 22 ). T r y p t i c ac t iv i t yw a s d e t e r m i n e d b y t h e s p e c t r o p h o t o m e t r i c m e t h o d o f H u m m e l ( 1 9 5 9 ) u s i n g1 0 - 3 M T A M E ( N .B . C o . ) p r ep a r ed i n u n i v e r s a l b u f fe r . A u n i t o f t r y p t i c ac ti v i tycau s ed an ab s o r b en cy i n c r ea s e o f 0 - 0 0 1 / r a i n a t 2 5 C w h en t h e s p ec t r o p h o t o me t e rwas set a t 247 m/~.S t o c k s o lu t io n s o f 1 0 - 2 M D F P ( M a n n ) w e r e p r e p a r e d i n a n h y d r o u s i so -p r o p an o l . En zy m e p rep a r a t i o n s w e r e d i lu t ed i n 0.0 5 M Tr i s b u f f e r a t p H 8 .1 .A n eq u a l v o l u me o f i s o p r o p an o l o r i n h i b i t o r s o l u t i o n w as t h en ad d ed an d t h emi x t u r e s w e r e a s s ay ed f o r en z y m e ac t iv i t y a f t e r eq u i l i b ra t i o n f o r 4 h r a t r o o mt em p er a t u r e . S t o ck so l u t io n s o f 7 0 / zg / ml o v o mu co i d ( M an n ) , 1 0 - 3 M g l u t a t h i o n ean d 1 0 . 3 M cy s t e i n e w e r e p r ep a r ed i n 0.0 5 M Tr i s b u f f e r a t p H 8 .0 . T h e P C M Bs t o ck s o l u t io n w as p r ep a r ed i n 0 -0 2 M g l y cy l - g l y c i n e b u f f e r a t p H 7 - 6 . En zy m ep r ep a r a t i o n s w e r e d i l u t ed w i t h ap p r o p r i a t e b u f f e r. A n eq u a l v o l u me o f b u f f e r o rs t o ck s o l u t i o n w as t h en ad d ed an d t h e mi x t u r e s w e r e a s s ay ed f o r en zy me ac t i v i t ya f t e r e q u il ib r a ti o n a t r o o m t e m p e r a t u r e f o r 3 0 m i n ( o v o m u c o i d - - w i t h i n 1 0 m i n ) .P r ep a r a t i v e a c r y l am i d e g e l e lec t r o p h o r e s i s w as c o n d u c t e d a s p r ev i o u s lyd es c r i b ed ( D eV i l lez , 19 64 ). A d i s co n t i n u o u s T r i s - c i t r a t e - b o r a t e - L i O H b u f f e rw as u s ed ( B a r r e t t et al. , 1 96 2). P r o t e i n co m p o n e n t s w e r e e l u t ed b y d i f fu s i o n o rd e t ec t ed b y s t a i n i n g t h e g e l i n s a t u r a t ed A m i d o B l ack 1 0 B .

R E S U L T SI t m ay b e s een i n Tab l e 1 t h a t a ll ex tr ac t s t e s t ed d em o n s t r a t ed a lk a l in e p r o t eo -ly t i c ac t i v i t y as ind i ca t ed by case in hydro lys i s . Ga s t r i c j u i ce an d d iges t i ve g l and

ex t r ac t s o f t h e b a r n ac l e d em o n s t r a t e d l es s ac t iv i t y i n an a l k a l in e me d i u m t h a n t h eam p h i p o d , i s o p o d o r d ecap o d ex t r ac ts . G as t r i c j u i ce ex t rac t s o f t h e b a r n ac l eh y d r o l y z ed cas e i n a t a p H o p t i m u m o f 4 .0 w h e r eas a ll o t h e r g a s t r ic j u i ce ex t r ac t sd em o n s t r a t ed a t l e a st o n e o p t i m u m ab o v e p H 7.0 . A l l ex t r ac t s ex cep t th o s e o fBalanus nubi lus h y d r o l y z e d T A M E a t p H 8 .1 . E i g h t g a s tr ic j u i c e e xt ra c t s o f t h eb a r n a c le w e r e a s s a ye d f o r T A M E a c t iv i ty b e t w e e n p H 4 .6 a n d 8 .9 . N o n e o f t h e s eg a s t ri c j u i c e e x tr a c ts d e m o n s t r a t e d T A M E a c t iv i ty i n t h e ra n g e t es t ed . T w od i g es ti v e g l an d ex t r ac t s ( fi ve t e s t ed ) o f t h e b a r n ac l e d em o n s t r a t ed T A M E ac t iv i t ya t p H 5 - 2 b u t n o t a t p H 6 .0, 7 .0 o r 8 .0 . O v o m u co i d h ad n o i n h i b i t o r y e ff ec t o nt h e T A M E ac t i v it y o f t h e s e d i g est iv e g l an d ex t rac t s . C o n t r a s t i n g ly , o v o m u co i d h ad


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SURVEY OF A TRYPTIC DIGE STIV E ENZYME I N CRUSTACEA 5 43a n i n h i b i t o r y e f f e c t o n t h e T A M E a c t i v i ty o f g a s t r ic 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 et 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 a l l o t h e r s p e c i e s t e s te d .S i m i l a r i n h i b i t i o n w a s o b s e r v e d w h e n s u c h e x t ra c t s w e r e a s s a y e d i n t h e p r e s e n c e o fD 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 e f f e c t 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 e ff e c t o n t h e T A M Ea c t i v i t y o f 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 C a n c e r p r o d u c t u s .

TABL E 1 -- Su Rv EY OF A TRYPTIC DIGESTIVE ENZYME IN CRUSTACEA*

S u b s t r a t e I n h i b i t o r sC a se in T A M E D F P O v o m u c o idA n i m a l E x t r a c t ( p H 7 "5 ) ( p H 8 "1 )

Pandalus platyceros g . j . + +Munida quadrispina g . j . + +Paguristes turgidus g . j . + +Pagurus tenuimanus g . j . + +Pagurus kennelyi g . j . + +Cancer productus g . j . + + + +d . g . + +Cancer magister g . j . + + + +Cancer gracilis g . j . + + + +d .g . + + + +i .c . + + + +Cancer oregonensis g . j . + +

Telmessus cheiragonus g . j . + +Hemigrapsus nudus g . j . + +Pinnixa littoralis d . t . + +d .g . + +Pug ett ia productus g . j . + +H y a s l y ra tu s g . j . + +Idothea wosnesenskii d . t . + + + +i .c . + + + +d .g . + +i . + + + +Idothea resecada d .g . + +i . + +

Exosphaeroma oregonensis d .g . + +i . + +Lirceus s p . d . t . + + +Limnoria l ignorum d . t . + +S y n u r e l l a s p . d . t . + + +i . e . + +Balanus nubilus g . j . + -d .g . + + (p H 5"2 ) -* g . j . = g a s t r i c j u i c e ; d . g . = d i g e s t i v e g l a n d ; i .c . = i s o l a t e d c o m p o n e n t ; d . t . = d i g e s t i v eg l a n d a n d i n t e s t i n e ; i . = i n t e s t i n e ; + , p o s i t iv e r e a c t i o n o r i n h i b i t i o n ; - , n o r e a c t i o n o r n oi n h i b i t i o n .


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544 E D W A R D D E V I L L ~ . Z A N D K A R E N B U S C H L E NG l u t a t h i o n e o r c y s t e i n e d i d n o t i n d u c e T A M E a c t iv i t y i n g a st r ic j u i c e e x tr a c ts o ft h e b a r n a c le w h e n a s sa y s w e r e c o n d u c t e d a t p H 4 . 6 -8 . 9 .

T h e T A M E s p e ci fi c e n z y m e w a s is o l at e d as a s in g le c o m p o n e n t f r o m d e c a p o d ,i s o p o d a n d a m p h i p o d e x t r a c t s ( F i g . 2) . S i m i l a r e l e c t r o p h o r e t i c m o b i l it i e s w e r eo b s e r v e d i n all ex t ra c ts t e s te d . T h e T A M E s p ec if ic c o m p o n e n t w a s a n io n i c a tp H 8 .0 a n d f o u n d t o b e p r o t e o l y ti c as ev i d e n c e d b y c as e in h y d ro l y si s . T h e p Ho p t i m u m f o r p r o t e o l y ti c a c t iv i ty b y i s o la t ed c o m p o n e n t s w a s b r o a d ( p H 6 . 5 -8 . 0 ).I t s h o u l d b e m e n t i o n e d t h a t th e T A M E c o m p o n e n t i s ol a te d f r o m Synurella w asn o t a s s a y e d f o r c a s e i n h y d r o l y s i s d u e t o l o w c o n c e n t r a t i o n s o f t h e c o m p o n e n t a f t ere l e c t r o p h o r e s i s a n d e l u t i o n .

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3 0 6 0 9 0 1 2 0T i m e , s e cFIo. 1. Th e effect of D F P and ovomucoid on the TA M E activity of extracts ofdigestive organs of Crustacea; O , control, O, inhibitor. Fo r abbreviations, seefootnote Table 1.

T h e e f fe c t o f p H o n t h e T A M E a c ti v it y o f g a s tr ic j u i c e a n d d i ge s ti v e gl a nde x t r a c ts o f C r u s t a c e a i s s h o w n i n F i g . 3 . A n a l k a li n e p H o p t i m u m w a s o b s e r v e dr a n g i n g f r o m p H 8 . 0 - 8 .3 d e p e n d i n g o n t h e e x t r a c t t e s t e d b u t a s in g l e a l k al in eo p t i m u m w a s o b s e r v e d w h e t h e r t h e e x t r ac t w a s g a st ri c j u i c e o r p r e p a re d f r o ms o m e d i g e s t i v e o r g a n .


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[ \ l " " \ . - ' - . . . . . . " " ' - . " ~ . . / "' ~ f ~ I o - o ~ o - o " e - o e ~ e Je: " ~ i ] L I i ~ I0 5 no L5 20 25 50 55 40F r a c t i o n nu m be r~ 0 - S c m g e l s e g m e n t s( - )FIG. 2. Representative patterns of acrylamide gel electrophoresis of digestiveo r g a n e x t r a c t s o f C r u s t ac e a ; p H 8 '0 , 0 C , 1 6 h r , T r i s - c i t r a t e - b o r a t e - L i O H g e l s ,3 0 0 V , a v e ra g e c u r r en t = 2 0 m A . T A M E a c t iv e c o m p o n e n t s d e s ig n a t e d b y a r ro w s .

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FIG. 3. T he effect of pH on the T A M E activity of gastric juice and digestive organextracts of Crustacea. @, SynureUa (d.t .); A, Lirceus (d.t .); m, Cancer (g.j.).For abbreviations see footnote, Table 1.

D I S C U S S I O NT h e c l as s if i ca t io n o f i n v e r t e b ra t e p ro t e o l y t i c d i g e s ti v e e n z y m e s h a s o f t e n b e e nb a s e d o n s t u d i e s o f p H o p t i m a w h e r e c r u d e e x t ra c ts o f a l i m e n t a r y t is s u e s h a v eb e e n e m p l o y e d . S u c h a n a p p r o a c h g i v e s l it tl e i n f o r m a t i o n c o n c e r n i n g t h e n a t u r e

o f t h e p r o t e o l y t i c c o m p o n e n t s i n v o l v e d i n p r o t e i n d i g e s t i o n s i n c e p r o t e o l y t i ce n z y m e s a r e o f u n iv e r s a l d i st r i b u t io n i n li v in g ce ll s a n d p H o p t i m u m a l o n e i s aw e a k c r i t e r i o n o f c la s si fi c at io n . G a s t r i c j u i c e e x t r a c ts w e re u s e d i n t h e p r e s e n ti n v e s t i g a t i o n w h e re v e r t h e si z e o f t h e a n i m a l p e rm i t t e d . I n t h o s e c a s e s w h e ret i ss u e h o m o g e n a t e s h a d t o b e u s e d , a si n g le t r y p t ic c o m p o n e n t w a s i s ol a te d b ya c r y l am i d e e l e c tr o p h o r e s is a n d d e m o n s t r a t e d p r o p e r t i e s s i m i la r to t h e c o m p o n e n ti s o l a te d f ro m g a s t r ic j u i c e e x t r a c t s o f l a rg e r s p e c i e s.

T r y p t i c a c ti v it y w a s b a s ed o n t h e a l ka l in e h y d r o ly s i s o f T A M E a n d c a se i n a n dt h e i n h ib i ti o n o f T A M E a c ti v it y i n t h e p r e se n c e of D F P a n d / o r o v o m u c o i d . T h er e s u l t s r e p o r t e d h e r e i n d i c a t e t h a t t r y p s i n i s f o u n d i n t h e a m p h i p o d , i s o p o d a n dd e c a p o d . D e g k w i t z (1 9 57 ) h a s s u g g e s t e d t h e p r e s e n c e o f t r y p s i n i n a m p h i p o d sa n d d e c a p o d s e x c e p t i n g Cragon cragon a n d Palaemon serratus. In t h e s e s p e c i e sd i g e s ti o n w a s a t t r ib u t e d t o c a th e p t i c en z y m e s . T h e t w o c a t h e p s i n i n h i b i t o r sP C M B a n d i o d o a c e t a t e a s w e l l a s t h e t w o a c t i v a t o r s g l u t a t h i o n e a n d c y s t e i n e h a dn o e ff ec t o n t h e T A M E c o m p o n e n t o f Cancer productus i n t h e p r e s e n t in v e s t i g a t i o nw h i c h f u r t h e r s u g g es ts t h a t t h e T A M E c o m p o n e n t is n o t a c a th e p t ic - ty p e e n z y m e .A t r y p t i c c o m p o n e n t c o u l d n o t b e d e m o n s t r a t e d f or Balanus nubilus i n t h ep re s e n t i n v e s t ig a t i o n . T h i s i s c o n s i s t e n t w i t h t h e l a ck o f a n a l k a li n e o p t i m u m fo rc a s e in h y d ro l y s i s b y g a s t r i c j u i c e e x t r a c ts . D i g e s t i v e g l a n d e x t r a c ts d e m o n s t r a t e dT A M E a c ti v it y o n l y i n c o n s i st e n t ly a n d t h e n o n l y i n a n a ci d m e d i u m . O v o m u c o i d


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546 EDWARD DEVILLEZ AND KAREN BUSCHLENw a s w i th o u t e f fe c t o n T A M E a c ti v it y. T h i s w o u l d s u g g e s t a c a t h e p t i c -t y p e e n z y m ei n g l a n d e x t r a c t s b u t d o e s n o t n e c e s s a r i l y m e a n t h a t t h e e n z y m e is i n v o l v e d i nd i g e s ti o n . F u r t h e r m o r e , c a t h e p t i c a c t iv a t o r s h a d n o i n f lu e n c e o n T A ~V IE a c t iv i tyi n g a s t r ic j u i c e e x tr a c ts . T h e p o s s i b i li t y t h a t T A M E a c t iv i ty w a s in h i b i t e d b ys o m e i n t e r f e r i n g s u b s t a n c e i n t h e e x t r a c t s c a n n o t b e e x c l u d e d .

Ac k n o wl e d g e m e n t s - - Th e f i r s t au t h o r ap p rec i a t e s t h e a s s i s t an ce p ro v i d ed b y D r . R o b e r tL . F e r n a ld , D i r e c t o r o f t h e F r i d a y H a r b o r M a r i n e L a b o r a t o r ie s o f t h e U n i v e r s it y o fW a s h i ng t o n . T h e r e s e a rc h w a s s u p p o r t e d b y t h e U n i t e d S t at es N a t i o n a l Sc ie n c e F o u n d a -t i o n (G B -7 4 7 ) an d b y t h e F acu l t y R esea rch F u n d o f M i am i U n i v e r s i t y .R E F E R E N C E S

BARRETT R. J . , FRIESEN H . & ASTWOOD E. B. (1962) Ch arac teriza t ion of p i tui ta ry andp ep t i d e h o rm o n es b y e l ec t ro p h o re s i s i n s t a r ch g e l s. f t . biol . Chem. 237, 432.CHARNEY J . & TOMARELLI R . M . (1947) A co lor im et r i c m eth od for t he de t e rm ina t ion oft h e p ro t eo l y t ic a c t i v it y o f d u o d en a l ju i ce . J . biol . Chem. 171, 501-505.DECKWXTZ E. (1957) E in B ei t rag zur N atu r d er Pro t eo ly t i c Ve rdau ung sferm ente be iv e r sch i ed en en C ru s t aceen a r t en . Ver6ff. Inst. Meeresforsch. Bremerh. 5, 1-13 .DEVILLEZ E. J . (1964) Prep ara t ive ac ry l a rn ide ge l e l ec t ropho res i s wi th the conven t iona ls t a r ch -g e l ap p a ra t u s . Annls B iochem. , N.Y . 9, 485-486 .DEVILLEZ E. J . (1965) I so l a t ion o f t he pro t eo ly t i c d iges t ive enzy me s f rom the gas t r i c j u i ceof the c rayf i sh Orcon ectes virilis. Com p. Biochem. Physiol. 14, 577-586.H UM M EL B . C . W . (19 59 ) A m o d i f i ed sp ec t ro p h o t o m e t r i c d e t e rm i n a t i o n o f ch y m o t ry p s i n ,t r y p s in a n d t h r o m b i n . Can . J. Biockem. Physiol. 37, 1393-1400.K U N IT Z M . (19 47 ) C ry s t a l l in e so y b ean t ry p s i n i n h i b i t o r - - I I . G en e ra l p ro p e r t ie s , f t. biol.Chem. 30, 291-310 .NORTHROP J . H . (1922) T h e me cha ni sm of t he in f luence of ac ids and a lka li on the d iges t iono f p ro t e i n s b y p ep s i n an d t ry p s i n . J. gen. Physiol. 5, 263-274 .VONK H . J . (1960a) Dige s t ion and metab ol i sm . In The Physiology of Crustacea (E d i t ed b yWATERMAN T . H . ) V ol . 1 , pp . 291-316 . Ac adem ic Press , New Yo rk .V O N K H . J . ( 1 9 6 0 b) C o m p ara t i v e b i o ch e m i s t ry o f d i g e s ti v e m ech an i sm s . In ComparativeBiochemistry (Edi t ed by FLORKIN M . & MASON H. S . ) Vol . 6, pp . 347-392 . Ac adem icP r e ss , N e w Y o r k .

Survey of a Tryptic Digestive Enzyme in Various

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