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Corap. Biochem.Physiol., 1978,VoL59B,pp. 81 to 85. Pergamon Press. Printed in Great Britain

A N E W P R O T E O L YT I C E N Z Y M E I S O L A TE D F R O M

M Y T I L U S G A L L O P R O V I N C I A L I S : M Y T I L I D A S E .

P U R I F I C A T I O N , C R Y S T A L L I Z A T I O N A N D P A R T I A L

C H A R A C T E R I Z A T I O N

IOAN FLOREA DUM ITRU, DAN A IORDACHESCU* AND STE LIAN NICULESCU*

Facul ty o f Bio logy , Bucharest , Romania

(Received 2 March 1977)

Ab st ract - -1 . A procedure fo r the advance d pur i f icat ion of acid p ro tease(s) o b taine d f rom the hepato-pancreas o f the sea mussel , Mytilus galloprovincialis , is described.

2 . P ro teo ly t ic act iv i ties at p H 1 .6 an d 2 .6 cou ld no t be separated , a l though presen t ing d i f ferent op t i -mal ac t i o n p a ramete r s .

3 . The mole cu lar weight o f myt i lidase, determ ined by gel f i l t ra t ion on a Sephade'x" G-200 co lum n,was 36,000 + 600.

INTRODUCTION

T h e e n z y m e s p r e s e n t i n d i f f er e n t t is s u es a n d m o r p h o -

l o g i c a l f o r m a t i o n s o f t h e M y t i l i d a e f a m i l y - - M y t i l u s

edu l is a n d M y t i l u s g a l l o p r o v i n c i a l i s - - h a v e b e e n t h e

s u b j e c t o f s tu d y o f n u m e r o u s i n v e s t ig a t i o n s c a rr i e d

o u t b y S k a r d i v ( 1 9 5 7 ) , M i n a f r a ( 1 9 6 8 ) , G o r o m s o v a

( 1 9 7 1 ) , G o r o m s o v a & S h a p i r o ( 1 9 7 0 ) , D e Z w a n n

(19 7 2) , De Z w an n & Va n M e r rew i jk (1 9 73 ), S t ru s i

(1964) and Reid (1966) .

I s o l a t i o n a n d s t u d y o f t h e p r o t e o l y t i c e n z y m e s o f

i n v e r t e b r a t e s a n d e s p e c i a l ly o f m u s s e l s h a v e r e v e a l e d

t h e p r e s e n c e o f e n z y m e s a t p r e v a l e n t l y n e u t r a l o r

a l k a l i n e p H . T h u s , B u n d y & G u s t a f s o n ( 19 73 ) p u r i fi e d

a p r o t e a s e - l i k e t r y p s i n f r o m t h e s t a r f i s h , K o z l o v s k a y a

& V a s k o v s k y ( 1 9 7 0 ) c a r r i e d o u t a c o m p a r a t i v e s t u d y

o n s o m e e x t r a c ts w i t h a p r o t e o l y t i c a c t i v i t y o b t a i n e d

f r o m 5 0 s p e c i e s o f m a r i n e i n v e r t e b r a t e s , P i g n e r o &

R o c c a ( 1 9 6 9 ) f o u n d e v i d e n c e o f m a r k e d p e p t i d a s e a n d

e s t e r a s e a c ti v it i e s i n th e h e p a t o p a n c r e a s o f Loligo vu l-

garis , a n d R e i d & R a u c h e r t ( 1 9 7 0 ) , w h o s t u d i e d p r o -

t e o l y t i c e n z y m e s i n t h e b i v a l v e m o l l u s c C h la m y s h e r i -

cius , d e m o n s t r a t e d t h e p r e s e n c e o f a n a c t i v it y s i m i l a r

t o t h a t o f t ry p s i n , c h y m o t r y p s i n , c a r b o x i- a n d a m i n o -

p e p t i d a s e i n m a m m a l s . R e i d (1 96 6) s h o w e d t h a t i n

t h e d i g e s t i v e t u b e o f M ya arenar ia , p r o t e o l y s i s t a k e sp l a c e a t a n a c i d p H a n d n e c e s s i t a t e s th e a c t i v a t i o n

o f e n z y m e w i t h a n e n d o p e p t i d a s e a c t i v it y , a n d S t r u s i

(19 6 4) me n t i o n s t h a t t h e d ig es t i b i l i t y o f My t i lu s g a l lo -

prov inc ia l is r e a c he s m a x i m u m v a l u e s i n th e s u m m e r

m o n t h s .

I n a n e a r l i e r w o r k , w e r e p o r t e d o n t h e p r e s e n c e

o f t w o p r o t e o l y t i c a c t i v it i e s a t a c i d p H i n t h e m a r i n e

1 97 5) . S t u d y o f th e d i s t r i b u t i o n o f t h e s e t w o p r o t e o l y -

t i c a c t i v i t i e s i n f i v e m o r p h o l o g i c a l f o r m a t i o n s o f t h e

m o l l u s c s h o w e d t h a t t h e h e p a t o p a n c r e a s i s c h a r a c t e r -

i z e d b y a c c e n t u a t e d p r o t e o l y t i c a c t i v i t y . T w o h y p o t h -

e s e s w e r e d i s c u s s e d : t h e e x i s t e n c e o f t w o d i s t i n c t a c i d

p r o t e a s e s , a n d t h e e x i s t e n c e o f a s i n g l e p r o t e a s e w i t h

t w o o p t i m a l p H v a lu e s . T h e f o r m e r a s s u m p t i o n is s us -

*Laboratory of Enzymology , Inst i tu te o f Bio log icalSciences, Bucharest , Ro m ania .

c.a.P. 59/IB -F

t a i n e d b y c e r t a i n e x p e r i m e n t a l d a t a : a t b o t h o p t i m a l

p H v a l u e s d if f e r e nt s p e ci f ic i ti e s w i th r e g a r d t o n a t u r a l

s u b s t r a t e s , d i f f e r e n t r e q u i r e m e n t s f o r s a t u r a t i o n w i t h

h e m o g l o b i n , d if f e re n t o p t i m a l t e m p e r a t u r e s f o r t h e

r e a c t i o n , e t c. h a v e b e e n r e c o r d e d . T h e b e h a v i o u r o f

t h e t w o p r o t e o l y t i c a c t i v i t i e s s h a r p l y d i f f e r f r o m t h a t

o f p e p s i n i s o l a t e d fr o m t h e s t o m a c h o f sw i n e , w h i c h

d o e s n o t a t t a c k p e p t o n e s .

T h e p r e s e n t p a p e r d e s c r i b e s a p r o c e d u r e f o r t h e

a d v a n c e d p u r i f i c a t i o n o f a c i d p r o t e a se ( s ), w i t h o u t s u c -

c e e d i n g , h o w e v e r , i n t h e i r c h r o m a t o g r a p h i c s e p a r -

a t i o n ; i t l i k e w i s e d e a l s w i t h c e r t a i n p h y s i c o e h e m i c a l

p r o p e r t i e s o f t h e p u r i f i e d e n z y m e , t h e c o n d i t i o n s i n

w h i c h i t c r y s t a l li z e s a n d t h e s h a p e o f t h e c r y s t a l s.

MATERIAL AND METHODS

All o f the reagents p resen ted a h igh degree of pur i ty .Hem oglobin , the F o l in -Cioc~. l teu reagent, abso lu te ethy l ica l co h o l an d a mm o n iu m su lf at e were M erck p ro d u c t s ;po lyacry lamide, myoglob in , cy t rochrom e c were f rom

B.D.H.; Sephadex G-100, SE-Sephadex C-25 Dextran Bluefrom Upsala Pharmacia and Bio-Gel P-100 f rom Bio-Rad .

The m ol luscs were col lected on the Ro m ania n shore o fthe B lack Sea, selecting shellfish 345 cm long. Th e hepa to-

pancre ases were separated, s tored at - 15°C, weighed accu -rately, then t r i tu rated in a Po t ter hom ogenizer (1 g w ettissue/20 ml water). The t issue homogenate kept for extrac-t ion at 4°C for 1 h r was cen t r i fuged at 10 ,000 rev /m in for15m n , the su perna tan t represen t ing the to tal p ro teicextract.

Pro teo ly t ic act iv i ty was determined accord ing to thespect rophotometr ic me thod descr ibed b y An son & Mirsky(1933); the react ion mix ture in a f inal vo lum e of 1 ml con-t a in ed 1 0 mg h emo g lo b in an d 0 .2 -1 0 ~g p ro t e in an d p re -sen ted two d if ferent pH va lue s-- l .6 and 2 .6 .

The determ inat ions were performed at the two op t im alact iv i ty pH's , the value of which var ied wi th in res t r ic tedl imi ts in terms of the month in which the mol luscs werecollected.

P ro t e in co n cen t r a t i o n s were measu red b y t h e m eth o do f Lo wry et al. (1951).

Elect rophoresis in po lyacry lamide gel was carr ied ou taccord ing to the me thod descr ibed by Da vies (1964) , ina buffer g lycocol Tr is-HC 1 s o lu t ion , 1 × 10 -~ M, pH 8.6 ,

81

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82 IOAN FLO REA DUMITRU, DANA IORDACHESCUAND STELIAN NICULESCU

P r o t e i n: = : E nz y m e a t pH 16 . ~

- o - o - o - E n z y m e a t ~ ~ .

p H 2 6

0.81i

0 7

c 0 6

0 .5

~3. 0.4:Z i i~ ~ 1 i

~. 03

~ 2'

O II , , , J l , , , ~ l , , , , I

iO i5 20 2 5 30 35

T u b e n u m b e r

~30tD

I10

c90 ~

so ~

70

5o ~

~o ~

50

0 ~

0

Fig. 1. Elution curve of the enzymatic preparation withacid pH proteolyt ic activi ty f rom a Sephadex G-100medium column (2.8 × 25 cm), equ ilibrated in 0.3% NaC1.The 3 ml f ract ions f rom the e luate obtained with the same

saline solutio n were analy zed from the viewpoint of proteinconcentrat ion and proteolyt ic act ivi ty at pH 1.6 and 2.6.

in 8 x 0.4 cm tubes . Electrophoret ic separat ion wasobtained a t 5 mA /tube and 350 V. Af ter 90 min the electro-pho regra ms were stained with Amido-Schwartz 10 B, pre-pared in a methylic a lcoho l-acet ic acid-w ater mixture(5:1:5), a nd then deeoloured electroph oret ical ly us ing a7% acetic acid solution.

RESULTS

Purification of the enzyme

T h e t o t a l p r o t e i c e x t r a c t w a s a c i d u l a t e d b y t r e a t -m e n t w i t h 0 .5 N H z S O 4 t o p H 3 . P r e c i p i t a t i o n w a s

p e r f e c te d in t h e c o u r s e o f 1 h r a t 4 ° C ; t h e i n e r t c a t a -l y t i c p r o t e i n s w e r e t h e n r e m o v e d b y c e n t r i f u g a t i o na t 10 ,000 r ev /m in f or 15 min .

T h e p r o t e o l y t i c e n z y m e ( s ) i n t h e s u p e r n a t a n t

o b t a i n e d w a s s e p a r a t e d b y f r a c t i o n a l p r e c i p i t a t i o n

w i t h a m m o n i u m s u lf at e. M o s t o f t h e p r o t e o l y t ic a c-

t i v i t y w a s f o u n d i n t h e f r a c t io n p r e c i p i t a t i n g a t 0 . 3 4 ) .7

s a t u r a t i o n i n ( N H 4 ) 2 S O 4 .T h e e n z y m a t i c a l l y a c t i v e fr a c t i o n w a s s u b j e c t e d t o

g e l f i l t r a t i o n o n a S e p h a d e x G - 1 0 0 c o l u m n

( 2.8 x 25 cm) , equ i l ib r a ted in a 0 .3% N aC1 so lu t ion .

E l u t i o n o f t h e p r o t e i n s w a s o b t a i n e d w i t h t h e s a m es a l i n e so l u t i o n , a t a n e l u t i o n r a t e o f 2 0 m l / h r , c o l l e c t -

i n g s a m p l e s o f 3 m l e a c h . F i g u r e 1 g iv e s t h e e l u t i o no f p r o t e i n a n d p r o t e o l y t i c a c t i v i ty . E l u a t e s 2 2 3 4

( p e a k I ) e x h i b i t i n g a r e m a r k a b l e e n z y m a t i c a c t i v i ty ,

w e r e p o o l e d a n d s u b j e c t e d a g a i n t o f r a c t i o n a l p r e c i p i -

t a t i o n w i t h a m m o n i u m s u l f a t e . W i t h i n t h e 0 4 ) . 4 s a t u -r a t i o n r a n g e f o r m e d a p r o t e i c p r e c i p i t a t e t h a t w a s

r e m o v e d b y c e n t r i f u g a t i o n a t 1 0 ,0 0 0 r e v / m i n . T o t h e

s u p e r n a t a n t o b t a i n e d , ( N H 4 ) 2 S O 4 w a s a g a i n a d d e dup to a s a tu r a t io n of 0 .4 and 0 .7 , w hen the pr o te in( s )

w i t h t h e p r o t e o l y t i c a c i v i t y s t u d i e d p r e c i p i t a t e d . T h ep r e c i p i t a t e w a s t h e n d i s s o l v e d in a c o r r e s p o n d i n g

v o l u m e o f 0 .0 05 M N a 2 S O 4 a n d a d j u s t e d t o p H 3

w i t h 0 .5 N H 2 S O 4 .T h e e n z y m a t i c p r e p a r a t i o n , r es u l ti n g f ro m t h e p r i o r

s t ag e , b y p r e c i p i t a t i o n a t a s a t u r a t i o n o f 0. 4 -0 . 7 i n

a m m o n i u m s u lf at e, w a s s e p a r a t e d b y c h r o m a t o g r a p h yon a Bio- G el P - 100 co lu mn ( 2.8 x 15 cm) , p r ev ious ly

e q u i l i b r a t e d w i t h 0. 00 5 M N a 2 S O , , p H 3, c o l l e c ti n g3 m s a m p l e s . A s m a y b e s e e n f r o m F i g . 2 , t h i s c h r o -

m a t o g r a p h i c s e p a r a t i o n g a v e t h r e e p r o t e i n p e a k s ,n o t e d i n c o n t i n u a t i o n I I , I I I a n d I V , m o s t o f t h e e n z y -

m a t i c a c t i v i t y b e i n g f o u n d , h o w e v e r , i n p e a k l I I .

E l u a t e s 1 4 -- 20 w e r e p o o l e d a n d u s e d f o r t h e n e x t p u r i -f i ca t ion s tage .

T h e p a r t l y p u r i f i e d e n z y m a t i c p r e p a r a t i o n w a sa g a i n se p a r a t e d b y c h r o m a t o g r a p h y o n a s t r o n g l y

a c i d io n - e x c h a n g e c o l u m n - - S E - S e p h a d e x C - 25 (1 .8 x

2 3 c m ). T h e i o n e x c h a n g e r w a s e q u i l i b r a t e d i n a

0 . 0 5 M N a 2 S O 4 s o l u t io n , p H 3 ; 3 m l s a m p l e s w e r e

t a k e n u p . E l u t i o n w a s d o n e s t e p w i s e , i n c r e a s i n g t h e

i o n i c s t r e n g t h o f t h e s a l i n e s o l u t i o n , a s i n d i c a t e d i n

F i g . 3 . F o u r p r o t e i n p e a k s w e r e o b t a i n e d , V - V I I I ,

p e a k s V I a n d V I I p r e s e n t i n g a p a r t i c u l a r l y m a r k e d

e n z y m a t i c a c t i v i t y . F o l l o w i n g t h i s p u r i f i c a t i o n p r o -c e d u r e , t h e e n z y m a t i c s a m p l e s p r e s e n t e d a n a c i d - p r o -

t e o l y t i c a c t i v i t y a p p r o x i m a t e l y 6 0 t i m e s t h a t o f t h e

t o t a l p r o t e i c e x t r a c t i n i t i a l l y u se d .T a b l e 1 s u m m a r i z e s t h e r e s u l t s o b t a i n e d i n 2 0 e x -

p e r i m e n t s c o n c e r n i n g s p e c if i c a c t i v i t y a t t w o p H

v a l u e s a n d t h e p u r i f ic a t i o n f a c t o r s re c o r d e d i n t h e

d i f f e r en t s tages of pur i f i ca t ion ,

T h e d a t a i n F i g s 1 - 3 a n d T a b l e 1 d e m o n s t r a t e t h a t

t h e e lu t i o n c u r v e s o f t h e t w o a c i d p H p r o t e o l y t i c a c -t i v i ti e s i n t h e S e p h a d e x G - 1 0 0 , B i o - G e l P - 1 0 0 a n d

S E - S e p h a d e x C - 2 5 c o l u m n s h a v e s i m i l a r p r o f i l e s a n d

t h a t t h e p u r i f i c a t i o n f a c t o r s p r e s e n t i n t h e v a r i o u s

s t a g e s o f t h e p r o c e s s h a v e c l o s e b u t n o t i d e n t i c a l

va lues .F i n a l l y , p e a k V I o b t a i n e d b y s e p a r a t i o n o n t h e S E -

S e p h a d e x C - 2 5 m e d i u m c o l u m n , w i t h a 5 0 - 6 1 - fo l d

e n r i c h e d p r o t e o l y t i c a c t i v i t y , w as c r y s t a l l i z e d i n a n

P r o t e i n ]

- - o - - o - -- E n z y m e 0 t p H 1 6 I

: -- E n z y m e o t p H 2 6 l

/ , 0 0

V / I l l 7 °°

/ i \ t l t \ - I oo :

/ = r i~ ~ -14o ~ .

20" ~

0 c5 I0 15 20 25

T u b e n u m b e r

Fig. 2. Chro mato graphy on Bio-Gel P-100 column(2.8 x 15 cm) equilibrate d in 0.005 M Na2SO 4, pH 3, of apar t ia l ly pur if ied enzymatic preparat ion f rom the h epato-

pancreas of a mar ine mussel, by precipi ta t ion at pH 3,within 0.34) .7 saturat ion range in ammonium sulfate ,chrom atograph y on Sephadex G-100 and renewed precipi-tation with ammonium sulfate (0.4-0.7 saturation). FJutionwas obtain ed with the same saline solution, collecting 3 ml

fractions which were corresp ond ingly analyzed.

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A new proteolytic enzyme isolated from Mytilus galloprovincialis 83

t 0 - - Pro te in t he hepa to panc r ea s o f t he m ar ine m usse l . T he e lu t i onEnzyme ot oH 1.61 curves of the two ac t ivi t ies-- -a t pH 1.6 an d pH 2.6 --

--*-*--*-Enzym e of pH 2.6]6o .~ ob t a ined fo r de t e rm ina t i on o f t he m olecu l a r we igh t ,

.ZM / ,% were superp osable , indica t ing simi lar or a lm os t s imi-

] ~ ~ ~ ' la r mo lecular weights (Fig . 6).0 - - 5 0 o .

0.4M c:

4 0 - - ~ 4 0 ~ D I S C U S S I O N

~- De te rm ina t i on o f t he op t im a l a c t i on pa ram e te r s o f° 3 c ~ l ~ ( ~ i ° ~ th e p ur if ie d p r ot eo ly ti c e nz ym e (s ) f ur ni sh ed d a ta v er y

"~ 2£ ~- 0

5 IO 15 20 25 3 0

Tube number

F i g . 3. E l u t i o n c u r v e o n a S E - S e p h a d e x C -2 5 c h r o m a t o -

g r a p h i c column (1.8 x 23 cm) of the enzymatic preparationobtained by sep aration on Bio-Gel P-100. Elution was per-form ed stepwise by increasing the concentration of theNa2SO 4 solution, p H 3, as show n; 3 ml fractions w ere col-

lected and analyzed.

(a )

Fig. 4. The two types of crystals obtained by crystalliza-tion in absolute ethyl alcohol, at pH 3 and pH 1.5 (a andb, respectively) of a purified enzym atic pr epara tion from

the h epatopancreas of the marine mussel ( x 960).

abso lu t e e thy l i c a l coho l so lu t i on . T wo types o f c rys -ta ls were obta ined (Fig. 4a and b) , a needle- l ike and

a p r i sm a t i c t ype , l end ing suppor t t o t he hypo thes i s

o f two d i f fe ren t enzym es . T he c rys t a l s sepa ra t ed bycen t r i fuga t i on an d t ake n u p i n a sm a l l vo lum e o f d i s -

t il led wa te r , ad jus t ed t o p H 3 wi th a 1 N HC I so lu -

t ion, exhibi ted the same speci f ic ac t ivi ty as the pur i -f i ed p repa ra t i on used fo r c rys t a l l i z a t i on o f t he

enzym e .T he pur i f i c a t i on s t ages we re accom pan ied by an

elec t rophore t ic s tudy (Fig. 5) tha t indica ted a s ingle

p r o t e in b a n d f o r p e a k s V I a n d V I I . T h e a p p e a r a n c eof severa l prote in peaks wi th a ca ta lyt ic ac t ivi ty in

the cour se o f pu r if i c a t ion m igh t be exp l a ined by au to -

d ige s t ion and t he appea ran ce o f c a t a ly t ic a l l y ac t ive

in t e rm ed ia ry p roduc t s , a s r epor t ed by Bohak (1969)fo r peps in i so l a ted f rom the ch i cken s tom ac h m ucosa . (b)

T he m olecu l a r we igh t , de t e rm ined by Whi t ake r ' sm e thod (1963) us ing a Sephadex G-200 co lum n

(2.8 × 23 cm ) w as 36,400 ___ 600 for the sam ple pre-

sen t i ng p ro t eo ly t i c a c t i v i t y a t a c id pH, pu r i f ed f rom

Tab le 1. The purification o f mytilidase from the hepatopancreas of Mytilus 9alloprovincialis

p H 1 .6 p H 2 . 6

P u r i f i ca t i o n Vo l u m e P r o t e i n E n zy m at i c S p ec i f ic P u r i f i ca t i o n E n zy m at i c S p ec i f i c P u r i f i ca t i o n

s t ag e ( m l ) ( m g / m l ) ( m g ) ac t i v i t y ac t i v i t y f ac t o r ac t i v i t y ac t i v i t y f ac t o r

T o t a l ex t r ac t 1 4 0 2 . 2 4 3 1 3 . 6 6 2 2 8 1 4 1 1 8 .3 1

S u p e r n a t a n t a f t e r

p r ec i p i t a t i o n a t

pH 3 135 1 .18 159 .3 79 66 .1 2 .36 44 37 .3 2 .04P r e c i p i t a t e a t

0 . 3 - 0 . 7 sa t u r a t i o n

o f am m o n i u m su l f a t e 5 0 1 .0 2 5 1 . 0 9 9 9 7 3 .4 7 6 7 4 3 .5F r a c t i o n I ,

S ep h ad ex G- 1 0 0 3 5 0 . 6 2 2 1 . 7 1 2 6 2 0 3 . 2 7 .3 8 7 1 4 0 7 . 6P r e c i p i t a t e a t

0 . 4 - 0 . 7 sa t u r a t i o n

in am mo niu m su lfate 20 0 .48 9 .6 128 266 9 .5 79 164 .5 9 .0

F r a c t i o n I I I .

B i o - Ge l P - 1 0 0 2 0 0 . 2 0 4 . 0 1 4 0 7 0 0 2 5 1 2 2 6 1 0 3 3 . 3

F r a c t i o n V I ,

S E - S ep h ad ex C- 2 5 6 0 . 0 5 0 . 3 7 0 1 4 0 0 5 0 5 5 1 1 0 0 6 1F r a c t i o n V I I ,

S E - S ep h ad ex C- 2 5 6 0 . 0 5 8 0 . 3 4 8 4 5 7 7 8 2 8 5 0 8 6 3 5 0

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84 lOAN FLOREA DUMITRU, DANA IORDACHESCU AND STELIAN NICULESCU

o b c d

m

e f g h

Fig. 5. Disc electrophoresis in po lyac rylam ide gel ofsamples collected at different stages of the purification pro -cess. (a) Tota l p roteic extract ; (b) supern atant pH 3; (c)fraction p recip itated in am mo nium sulfate at 0.3~0.7 satu-ration; (d) fraction I; (e) fraction precipitated inammonium sulfate at 0.4-43.7 satur ation ; (f) fraction III;

(g) fraction VI; (h) fraction VII.

c l o se t o t h o s e o f t h e n o n - p u r i f i e d p r e p a r a t io n ( D u m i -

t r u et al. , 1 9 75 ), sa v e f o r th e o p t i m a l p H v a l u e s w h i c h ,

w i t h t h e p u r i f i e d s a m p l e , w e r e s l i g h t l y sh i f te d t o w a r d sm o r e a c i d v a l u e s . T h i s m i g h t b e a c c o u n t e d f o r b y

t h e p r e s e n c e i n t h e n o n - p u r i f i e d e x t r a c t s o f c a t a l y t i -

c a l l y i n e r t p r o t e i n s . T h e e n z y m e o f p H 1.6 h a s a n

o p t i m a l a c t i o n t e m p e r a t u r e o f 37 °C , p r e s e n t s a r e a c -

t i o n r a t e w h i c h i n c re a s e s p r o p o r t i o n a l l y o n l y w i t h i nt h e f i rs t 5 r a i n a n d t h e m a x i m u m r a t e w a s r e c o r d e d

i n t h e p r e s e n c e o f 1 0 m g h e m o g l o b i n . T h e e n z y m eo f p H 2 .6 h a s a n o p t i m a l a c t i o n t e m p e r a t u r e o f 5 0° C,

a p r o p o r t i o n a l i n c r e a s e i n t h e r e a c t i o n r a t e d u r i n g

t h e f i r s t 15 m i n , a n d i s s a t u r a t e d i n t h e s u b s t r a t e i nt h e p r e s e n c e o f 1 4 m g h e m o g l o b i n . D i f f er e n c e s i n t h e

e n z y m a t i c re a c t i o n i n t e r m s o f p H w e r e l ik e w i s e

o b s e r v e d i n t h e p r e s e n c e o f c o p p e r i o n s w h i c h s t i m u -

l a t e t h e i r c a t a l y t i c a c t iv i t y ( u n p u b l i s h e d r e s u l ts ) a n d

w i t h r e g a r d t o d i f f e re n t n a t u r a l s u b s t r a t e s w h i c h t h e yh y d r o l y z e : b o v i n e s e r u m a l b u m i n , o v a l b u m i n , g e l a t i n ,

case ine , pep tone of d i f f e r en t o r ig in , e tc .

A d d i t i o n a l p r o o f t h a t t h e r e a r e t w o p H a c i d p r o -t e o l y t i c e n z y m e s a r e t h e t w o t y p e s o f c r y s t a l so b t a i n e d b y c r y s t a l l i z a t i o n o f t h e p u r i f ie d p r e p a r a t i o n

i n t h e p r e s e n c e o f a b s o l u t e e t h y l i c a l c o h o l . P r e l i m i -

n a r y s t u d i e s , t o b e p u b l i s h e d , s h o w e d t h a t t h e s e a c i dp r o t e a s e s ( p r o t e a s e ) a r e t o b e f o u n d i n t h e c e l l i n

t h e f o r m o f z y m o g e n , a n d t h a t a t p H v a l u e s s m a l l e rt h a n 3 t h e y a r e t r a n s f o r m e d i n t o a c a t a l y t i c a l l y a c t i v e

e n z y m e . T h e a c t i v a t i o n p r o c e s s a p p e a r s t o t a k e p l a c e

e x t r e m e l y s l o w l y , g r a d u a l l y e v o l v i n g a t 4 ° C f o r o n e

w e e k a n d b e i n g c a t a l y z e d b o t h b y t h e m e d i u m p r o -t o n s a n d b y t h e n e w l y f o r m e d a c t i v e e n z y m e .

B y t h e p u r i f i c a t i o n p r o c e d u r e d e s c r i b e d i t w a s n o tp o s s i b l e t o s e p a r a t e t h e t w o p r o t e o l y t i c a c t i v i ti e s , t h e

p u r i f i c a t i o n f a c t o r s p r e s e n t i n g c l o s e b u t n o t i d e n t i c a l

va lues in the va r ious s tages of the pr ocess . I f in thep u r i f i e d e n z y m a t i c p r e p a r a t i o n t h e r e a r e t w o d i f -f e re n t p r o t e o l y t i c e n z y m e s , t h e n t h e i r b e h a v i o u r o n

S e p h a d e x c o l u m n s , a n d i n t h e c a s e o f e l e c t r o p h o r e s i s

i n p o l y a c r y l a m i d e g e l , m i g h t b e a t t r i b u t e d t o t h e i r

i d e n t i c a l o r e x t r e m e l y s i m i l a r m o l e c u l a r w e i g h ts . A

p r o t e o l y t i c e n z y m e l i k e t r y p s i n , i n t h e f o r m o f

z y m o g e n a n d w i t h a m o l e c u l a r w e i g h t e q u a l t o2 6 , 3 0 0 , w i t h p h y s i c o c h e m i c a l p r o p e r t i e s s i m i l a r t o

t h o s e o f t h e e n z y m e ( s ) s t u d i e d b y u s , w a s i s o l a t e db y B u n d y & G u s t a f s o n ( 1 9 7 3 ) f r o m t h e s t a r f i s h . T h e

m o l e c u l a r w e i g h t o f t h e p r o t e o l y t i c e n z y m e ( s) i s o l a t e d

f r o m t h e h e p a t o p a n c r e a s o f t h e m a r i n e m u s s e l i s v e r y

c l o s e t o t h a t o f p e p s i n i s o l a t e d fr o m m a m m a l s b yB o v e y & Y a n a r y ( 1 9 60 ).

S u p p l e m e n t a r y d a t a r e g a r d i n g t h e r a t e o f t h e e n z y -

m a t i c r e a c t i o n i n t h e c a s e o f t h e t w o e n z y m a t i c a c t i v i-

t ie s , i n t h e p r e s e n c e o f a n i o n s a n d c a t i o n s , c o n f i rm st h e e x i s t e n c e o f t w o p r o t e o l y t i c e n z y m e s i n t h e p u r i -

f i e d e n z y m a t i c p r e p a r a t i o n ( u n p u b l i s h e d d a t a ) .

R E F E R E N C E S

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6C

5C

~ 4c

g 2o

-- I0tal

C y t h o c r o m c .

- - I 1 ~ 1 1 j V I i o g i o b i n

~ " . . . ~ , ~ . ~ p r o ea se 5 6 . 4 0 0

~ ' 1 1 Hoemoglobin

~ B l u e dexfran

I [ I I I I I I I I I t l , I4 1 4 2 4 5 4 4 4 5 4 6 4 7 4 8 4 9 5 0 5 1 5 2 5 5 5 4

l o g m o l e c u l o r w e i g h t

Fig. 6. Dete rmin ation of the molecular weight o f the prote olytic enzyme(s) in acid pH , purified from thehepa topancr eas of M ytilu s galloprovincialis , using Sephadex G - 100 column and Wh itaker ' s method (1963) .

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A n e w p r o t e o l y t i c e n z y m e i s o l a t e d f r o m My tilus galloprovincialis 85

o f t h e s e a m u s s e l Myti lus edu lis L . Comp . Biochem. Phy-

siol. 42 , 7 -14 .DE ZWANN A. & VAN MARREWlJK W. J . A . (1973) Int rac el-

l u l a r l o c a l i z a t i o n o f p y r u v a t e c a r b o x y l a s e , p h o s p h o e n o l -p y r u v a t e c a r b o x ik i n a s e a n d " m a l i c e n z y m e " a n d t h e

a b s e n c e o f g l y o x y l a te c yc le e n z y m e s i n t h e s e a m u s s e l

My tilus edulis. Com p. Biochem. Physiol. 44, 1057-1066.

DUMITRU I. F ., NICULESCU S., IORDACHESCU D. & GH I-

TESCU E . (1975) Ev iden c ing som e ac id p ro tea ses in thes e a m u s s e l M ytilus galloprovincialis. Rev. roum . biochim.12, 159-165.

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p h o r y l a s e m u s c u l a i r e c h e z q u e l q u e s i n v e r t e b r a t e s . Z h.evol. Biokhim. Fiziol. Suppl . 3 -5 , 1971 .

GOROMOSOVA S. A. & SHAPIRO A. Z. (1970) Ac tivi t6 d e

r a m y l a s e d a n s l ' h 6 p a t o p a n c r e a s e t l e s m u s c l e s d eMytilus. Biol. Moria 18 , 89-98 .

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t i v e s t u d y o f m a r i n e i n v e r t e b r a t e s . Co mp . B io ch em.Physiol. 34, 137-142.

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R . J . ( 1 9 5 1 ) P r o t e i n m e a s u r e m e n t w i t h t h e F o l i n p h e n o l

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s p e c i e d i m o l l u s c h i m a r i n i . Biologica. lat, 21 , 223-234 .PIGNEgO A. & ROCCA A. (1969) P ro te o ly t i c an d pep t ida s ic

a c t i v it i e s o f a p a r t i c u l a t e f r a c t i o n o f Loli#o vulgaris h e p a -

t o p a n c r e a s , Comp. Biochem. Physiol. 29, 1271-1275.

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Lima hians G m e l i n a n d My a arenaria L . Com p. Biochem.Physiol. 17 , 417-433 .

REID R. G . B . & RAUCHERT K. (1970) P ro teo ly t i c enzym esi n t h e b i v a l v e m o l l u s c Chlamys hericius G o u l d . Comp.Biochem. Physiol. 35 , 689-695 .

SKARDIV M. E . (1957) Su l l ' a t t iv i t a t i amin as ica ne l Myti lusgalloprovincialis. D i s t r i b u z i o n e d e l l a t t i v i t a e n z i m a t i c a

ne i va r i o rgan i . Bol l. Soc. i tal. biol. sperim. 33, 1089-1090.

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(Mytilus galloprovincialis) g r o w n i n M o r P i c c o l o a n d

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WHITAKER J . (1963) De te rm ina t ion o f mole cu la r w e igh tso f p r o t e i n s b y g e l f i l t r a t i o n o f S e p h a d e x . Analyt. Chem.35, 1950-1957.