Amylase and trypsin- and chymotrypsin-like proteases from Actinia equina L.; Their role in the nutrition of this sea anemone

Conl\‘.Bio~lw~. Ph!~\io/.Vol. 72A. No. 3. pp. 52.3 10 528. 19x2

O300-9619 82~030523-06803.00 0 0 1982 Pergamon Press Ltd

Printed in Great Britain.

AMYLASE AND TRYPSINAND CHYMOTRYPSIN-LIKE PROTEASES FROM ACTlNIA EQUINA L.; THEIR ROLE IN THE NUTRITION OF THIS SEA ANEMONE MICHEL

VAN PRAET

Laboratoire de Biologie des InvertCbrCs marins et Malacologie. Mu&urn National d’Histoire naturelle, 57 rue Cuvier. 75005 Paris. France (Rrceiurd

6 Nocrmhrr

1981)

Abstract-l.

Amylase and two proteases extracted from tissue of the intertidal sea anemone Actiniu vquina L. were studied. Some properties-autoactivation. pH and temperature of maximum activity. thermal inactivation. effect of inhibitors, PM of the two studied proteases-are extremely similar to those of mammalian trypsin and chymotrypsin. 2. Their K, values at different temperatures indicate an optimum of enzyme-substrate affinity around physiological temperatures. 3. Seasonal and experimental variations of the amylase. two proteases and their ratio were studied. 4. The important trypsin and chymotrypsin activities in the lower convoluted part of mesenteries were correlated with the presence of many zymogen cells (pDMAB positive) in their mesenteric filaments. 5. These trypsin- and chymotrypsin-like proteases are responsible for the extracellular digestion of prey. The particles produced by this extracellular digestion or collected in the sea environment are digested in phagocytic endodermal cells. 6. Increase of the amylase activity during June and July is correlated with the capacity for phagocytosis and the contribution of the phytoplankton to the diet of the omnivorous sea anemone. Actinia eyuinu L

We conclude from these results that the particulate organic matter, e.g. microalgae, could constitute a source of food for some intertidal, and perhaps some deep-sea, species of sea anemone (Van Prai;t, 1980, 1981). In this article we indicate some characteristics of amylase and two proteases of Acrinitr cyinu L. and

IlVTRODUCTlOlV Protease activity in sea anemones was detected by Krukenberg as early as 1880. Krijgsman & Talbot (1953) and Nicol (1959) demonstrated how these proteases act during the first stage of digestion of prey: an extracellular digestion in contact with mesenteric filaments (enteroids). Gibson & Dixon (1969) isolated three serine proteases from extracts of mesenteric filaments of the sea anemone Met&urn smile. One of these has properties similar to those of mammalian chymotrypsinogen and a-chymotrypsin. These authors also indicated the existence of a trypsin-like peptidase. In other coelenterates, Coan & Travis (1970) and Tiffon & Bouillon (1975) have also concluded the existence of trypsinand chymotrypsin-like proteases in zymogen form. Amylolytic activities have been less extensively studied; Sawano (1932), Krijgsman & Talbot (1953), Van PraEt (1981) indicate their existence in extracts of sea anemones but no secretion has ever been detected in the coelenteron. The extracellular digestion of prey produces amino acids (Murdock. 1971) which are mainly absorbed by the cnidoglandular tracts of the enteroids (Van Praet. 1980). as well as macromolecules and particles of a few micrometers size. Their digestion entails a second stage: an intracellular digestion in the endodermal phagocytic cells. These cells also ensure the digestion of particles and macromolecules collected from the sea environment, which are pulled toward the coelenteron by tentacles and ciliary currents.

their

seasonal

and

experimental

variations.

MATERIALS AiYD METHODS The organisms were collected during low tide in the Brest Channel, Each sea anemone was ground In distilled water in a Potter homogenizer. After centrifugation, the enzymatic activities were measured in the supernatant and compared to the protein level (Lowry-Folin test with a Technicon. according to the method of Samain er crl.. 1977).

The amylase activities were calculated as measurement of starch hydrolysis (soluble starch. Merck) on a Technicon. according to the method of Samain et a/. (1977). Protease activities were calculated by comparison of speed of hydrolysis (at 4OOnm. 50°C) of synthetic substrates: the substrate N-acetyl-L-tyrosine paranitroanilide (ATPNA, Merck) was used for the chymotrypsin and the substrate L-benzoyl-arginine-paranitroanilide hydrochloride (L-BAPA, Merck) was used for trypsin. The kinetics were compared to standard curves with r-chymotrypsin (Merck; 45 mI.U./mg) for the ATPNA and pancreatic trypsin (Merck 8214) for L-BAPA. ATPNA was dissolved in pure methanol (25 mgiml) and then diluted to I.5 x 10m3M with Tris buffer (0.1 M). CaClz (0.04M. pH 8). L-BAPA was directly dissolved (1.74 mgiml: 4 x 10m3 M) in an identical Trls buffer. The 523

fable Mid-June

Early October

0.070 * 0.004

0.031 f- 0.01

I. Seasonalcanat~onsof amylase Early

I

February

0.020 * 0.006

Using the F test. the values for June are significantly

Table

7. Enzyme

activities

Early

different

27 * 14 45

34

dissolvmg of substrate was facilitated by a pre-heating of the buffer (to 50 C). The study of Michaelis constants (K,) was made on the supernatants of extracts of lower convoluted parts of mesenteric filaments (mesenteric pellets) after a partial purification on a column of Sephadex G-75 [SO mm x 10 mm, eluted with Tris buffer (0.1 M). CaCI, (0.04 M. pH 8) Fig. I]. For the representation of the Michaelis equation we use Hanes’ method: S/V varies as S.

0.05

0.064 f 0.025

from those for October.

in different

I:arlk Octc+
Mid-Jul)

Mid-June

May

0.025 + 0.006

Amylase activity (October) Whole adults Mesenteric pellets Upper part of enteroids Tentacles

levels. I.U., mg protein

tissues (mI.U.:‘mg

1 t 0.031

February

0.027* 0.01:

and May:

P < 0.01

protein1

Trypsin activity (annual mean)

Chymotrypsln activity (annual mean)

x.4 * 2.1 44.5

1.06 F 0.40 9.15

7.5

0.99

9.2

1.44

Soybean trypsin inhibitor entirely Inhibits the hydrolysla of L-BAPA and that of ATPNA IS weakly diminished. ketone L-Tosylamido-2-phenylethyl-chloromethyl (TPCK), an inhibitor of chymotrypsin (specific substrate analogue), completely inhibits the hydrolysis of ATPNA after 45 min of enzyme-inhibitor pre-incubation and reduces it by 50% after I2 min. However, the hydrolysis kinetic of L-BAPA was not changed under the same conditions. Nistochemistry

Benzamidine. a competitive inhibltor of trypsin, does not change the hydrolysis kinetic of ATPNA. whereas with L-BAPA the kinetic is reduced by 90”,, after one minute.

The Actiniu rquinu were fixed rn 7’:;, formalin m sea water. Sections were stained by the p-dimethylaminobenzaldehyde reaction (pDMAB) specific to the tryptophanrich proteins and considered as a indicator of zymogen cells iGabe, 1968).

Fig. I. Elution diagram of mesenterlc pellet supernatant on Sephadex G-75. ch (---): chymotryptic activity; tr (......): tryptic activity; (-) fractions pooled for the studies of trypsin- and chymotrypsin-like K,: am: amylolytic activity. Other enzymatic activities detected (the thickness of lines is proportional to the activity). 1: 3.2.1 20.. r-glucosidase: II: 3 2.1.24.. r-mannosidase: 111: 3.2.1.31.. P-glucuromdase: IV: 321.51.. r-fucosidase: V’ 3.2.1.30.. N-acetyl-/?-glucosidase: VI : 3.4. I I .I ,, leucine arylamidase: VII : 3.4.11.3.. cystine arylamidase: VIII: 3.2.1.23.. /Ggalactosidase: IX: 3.1.3.1.. alkaline phosphatase: X: 3.1.3.2.. acid phosphatase: XI: 3.9.1.1.. phosphoamidase: XII: 3.1.1.1.. esterases (C, and CH).

Amylase

and trypsin-

and chymotrypsin-like

Fig. 2. Initial/maximum speed as a function of the temperature. ch (--): chymotryptic activity; tr (.=): tryptic activity. Residual activity for 25 minutes of preincubation. ch’ (--): residual chymotryptic activity; tr’ (..,-,.): residual tryptic activity.

RESULTS

Amylase The search ence of quite

for the pH optimum reveals the a large curve between pH 6 and

Tr-l@n-like. The pH optimum is at 8; the initial maximum speed of L-BAPA hydrolysis increases with

temperature up to 60°C [Fig. 2, tr (....,.)I. However at this temperature, denaturation processes disturb the kinetics after a few minutes. Thermal inactivation is shown by pre-incubation of the enzymatic extract for 25 min (35”-60°C) and then, after cooling the extract,

of levels of trypsin

and chymotrypsin

Mid-June Trypsin activity (mI.U./mg protein) Chymotrypsin activity (ml.U./mg protem) Amylase,Ichymotrypsin

from Acrinia rquinu

525

Fig. 3. Micrography of section stained with pDMAB. Zones of phagocytosis (P) appear in black in this sea anemone placed in china ink several hours before its tixation. The zymogen cells (---) stained with pDMAB are located at the level of the enteroid sectioned in its lower portion (L) but are absent at the level of the enteroid sectioned in its upper portion (U). m: mesentery; c: coelenteron.

exist7. At

pH 6.8 and 25”C, the K, value is 0.7 & 0.2 mg/ml [mg of soluble starch per ml of phosphate buffer (0.1 M), NaCl (0. I M)]. After fasts of 4-7 days, the amylase levels do not vary significantly; on the other hand, seasonal variations exist between lots of specimens collected on the sea shore (Table I). The important standard deviation of mid-July may of microenvironment in part reflect differences between organisms. Sea anemones living in rock pools at the highest intertidal level have a weaker amylolytic activity (0.035 f 0.014 mI.U./mg protein) than those organisms living at lower levels (0.065 f 0.037 mI.U./mg protein) (F test, significant difference P < 0.05). In mesenteric pellets isolated by dissection we have detected the highest amylase levels (Table Z), but they are not significantly different to those existing in extracts of whole organisms.

Table 3. Variations

proteases

measurement of the hydrolysis kinetics at 50°C [Fig. 2, tr’ (*a....)]. In these conditions inactivation begins between 45” and 50°C and seems complete beyond 60°C. In the study of K, at different temperatures (14’-50cC) the maximum enzyme-substrate affinity (K, minimum) seems to be located in the range of physiological temperatures (sea anemones which have lived at 18°C for several weeks). The wide range of standard deviation at 14°C is caused by difficulties in the measurement of weak kinetic curves at this temperature (Fig. 3). After 7 days of fasting, the levels of tryptic activity from increase Thus they slightly. increase 8.0 & l.OmI.U./mg of protein to 11.3 & 3.0mI.U./mg of protein (F test, significant difference, p < 0.05). Tryptic activity was measured in extracts of whole organisms and in different tissues isolated by dissection (Table II). It appears to be particularly strong in extracts of mesenteric pellets. In organisms collected on the sea shore (Table 3), an increase in the level of tryptic activity was measured in the specimens collected on 4 May, but was accompanied by an increase in the value of the standard deviation. Chymotrypsin-like The pH optimum

activities, of a year

Early October

is located

between

and the amylase/chymotrypsin

Early

February

Early May

8 and

7.6; the

ratio, over the course

Mid-June

6.7 & 0.5

8.0 k 1.1

7.3 * 1.7

10.2 + 2.4

1.02 f 0.13

0.95 * 0.12

0.83 + 0.29

1.54 _t 0.39

0.84 & 0.27

67 + 8

32 + 9

17 + 6

78 f 27

25 f

10

K,at

5O”C=68+_1

3

35”C=l.8tO5 22”C=

IO-‘M D4M

I 3 ?O

14”C=20+_1

5

@M

a

10-4M

Fig. 4. Michaelis constant as a function of the temperature. L-BAPA hydrolysis: tryptic activity. initial maximum speed of ATPNA hydrolysis increases with temperature up to 55 C (Fig. 2. ch). The decrease of residual activity becomes evident beyond 50°C [Fig. 2. ch’ (-)I. As with the trypsin-like protease, the maximum of enzyme-substrate affinity (K, minimum) seems to be located in the range of physiological temperatures (Fig. 4). The measurement of this activity in different tissues leads us to consider it as particularly abundant in the mesenteric pellets (Table 2). The activity appears to have increased after 7 days of experimental fasting. Thus it increases from 0.95 + 0.12 to 1.38 + 0.20mI.U./mg of protein (F test, significant difference, p < 0.01). In these organisms collected on the sea short (Table 3) higher levels of chymotryptic activity were measured in the specimens of 4 May. Hisrochemicc~l

ohsercutions

(Fig. 3)

After staining with pDMAB the granules of certain glandular cells turn blue. These cells with pDMAB positive granules are quite abundant at the level of cnidoglandular tracts of the enteroids of mesenteric pellets. On the other hand, they are extremely rare in the upper portion of these enteroids (between the throat and the mesenteric pellets). Some positive glandular cells are equally visible at the level of the endoderm of mesenteries and of the ectoderm of tentacles.

Quantltati\e mcasurc‘mcnts of pi.otcasc ;IcI~\~tic\ and histochemlcal observations ~~~nlirm out. prcccdlng observations (Van PrGt. 197X1 on the distinction that must hc made betucen the upper and the to\+clportmns of enteroid\. In the upper portion. thclr cnidoglandular tract\ IXIVC tCv /)DVAB p~~sitl\e glandulur cells (Fig. 3) and little protcoi>tlc activity (Tahlc 2). On the other hand. in the louver convoluted portion. dular

at the

level

tracts

of

pDMAB

trypsin-like

und c,h!motr!,psin-/ik~,

The two activities studied in relation to the hydrolysis of synthetic substrates (I.-BAPA and ATPNA) appear to be mainly located in the same tissue: mesenteric pellets. Their physico-chemical characteristics are similar and their levels evolve similarly during experimental fasting and in the different seasonal lots (Table 3). However, the specific nature of substrates, the effect of inhibitors (benzamidine, soybean trypsin inhibitor. TPCK) and the successive elution of two peaks of maximum activity (trypsin then chymotrypsin) confirm the observation of Gibson & Dixon (1969) relative to the existence of two different proteases having

enteroids

pcllet5 shoL\

positive cell concentration

Km atSO”C=43+i3 3O”C=l

I Ot02 I I +O

16” C= /5+02 14”C=lE+l8

IO-“M 3

cnidoglanthe

highest

:tnd considerable

10”M

I6t_O4 C= C=

the

both

IO-M 7?04

29°C; 21” 19”

Proteases:

of mesenteric

IO-“M 10m4M 10”M IO’M /-

Amylase

and trypsin-

and chymotrypsin-like

trypsin and chymotrypsin activities (Fig. 3, Table 2). The results of protease measurements and histochemical observations definitely confirm that the lower convoluted portion of the enteroids, at the level of the mesenteric pellets, constitute the privileged zone of extracellular digestion of prey. The presence of some pDMAB positive glandular cells in the ectoderm of tentacles and the detection of trypsin and chymotrypsin activities in the extracts from isolated tentacles does not allow us to consider as likely an extra-body digestion of large prey. The products of these cells may. with the other mucous secretions, contribute to bacterial protection and ectoderm cleaning. Variations

of the tryptic

and chymotryptic

uctit.Gties

In previous studies of the proteases of coelenterates. the authors have reported an increase in the specific activity of enzymes as the extract ages and have concluded that a zymogen exists. According to Gibson & Dixon (1969), the enzymes are completely activated after 1 hr 40min at 20°C and pH 8 or, according to Tiffon & Bouillon (1975) after 1 hr at 25 ‘C, pH 8.2. Under our own extraction conditions in distilled water, this autocatalytic process is only sensitive during the first 40 min which follow the beginning of grinding and ends when measurements are done (at least 90 min later). The increase in the levels of protease activity after fasts of 4--7 days can be explained by taking into account the observation, by electron microscopy, of numerous zymogen cells in the synthesis stage, in sea anemones fixed at low tide (Van Praet, 1981). The increase of protease activities thus corresponds to the accumulation of zymogen in these cells. Seasonal variations seem slight, and the increase of mean levels (lots of May) is accompanied by a variability which increases according to each sea anemone (Table 3). Only studies taking place over several years will allow us to understand their eventual significance. 4 mvlase In the absence of similar studies on other coelenterates. it is difficult to compare the characteristics of this enzyme. We note nevertheless that for similar conditions of measurement, Van Wormhoudt (1981) obtained a K, value varying from 0.5 to 2 mg/ml with the crustacean Puluemon serrutus. Contrary to that of the proteases studied, amylolytic activity appears to be slightly higher in the extracts of mesenteric pellets than in those of whole organisms (Table 2). If this enzyme intervenes essentially during digestion of microalgae, after phagocytosis of the latter, which was our hypothesis from the results of experimental nutrition with [‘4C]Cyanophyceae (Van Praet, l980), amylase could be in all phagocytic cells distributed in the whole endoderm of mesenteric pellets of the column and of the tentacles.

Srcrsonal variations

in amylase

lecels

In the past 50 years there have been many studies on protease and amylase secretions of the pancreas of mammals and their regulation according to the diet (see the review of Case, 1978). The same type of research has been undertaken on digestive enzymes of sea invertebrates, essentially

proteases

from Actinia rquina

527

crustaceans: Penueus juponicus and Palaemon serratus (Van Wormhoudt et ul., 1980, 1981), Penaeus ,japonicus (Laubier-Bonnichon et al., 1977), Artemia salina (Samain er al., 1980). All these studies show, first, that the regulation of protease synthesis and that of amylase are independent and second, that the latter is correlated with the amount of carbohydrate and/or microalgae in the diet. In an ecophysiological study of different zooplankton species, Boucher et ul. (1976) showed that the amylase level and the amylaselprotease ratio are characteristics of their trophic situation and especially of the richness of microalgae in their diet. The diet of sessile coelenterates is a less rigid concept than with mobile animals, and the respective part constituted by the prey, the particulate organic matter and the dissolved molecules (Schlichter, 1975) are not well known. Coelenterates appear nonetheless, to be well adapted for the capture and phagocytosis of particulate organic matter: macromolecules, bacteriae, microalgae etc. (Di Salvo, 1971; Tiffon & Boutibonnes, 1976; Van Praet, 1980). We have thus looked for the existence of seasonal variations in the amylase levels and their eventual correlation to the diet of Actiniu

equinu.

The results (Tables I and 3) show, for 2 years in succession, the increase of the amylase levels in June, as well as the amylase/chymotrypsin activity ratio (A/C). The values seem to diminish in summer and reach their lowest level in winter. These variations parallel those of microalgae in the sea environment and confirm our hypothesis on the role of the latter in the diet of Actinia equinu. This sea anemone. as well as numerous others, should no longer be considered as a strictly carnivorous species. The increase of individual variations in June and July, both for the amylase activity level and for the A/C ratio (Table 3) led us to look for the eventual existence of microenvironments. The results of collections, on one hand in permanent rock pools at the high tide mark, and on the other hand at the low tide mark, seem to indicate the role of several factors. The amylolytic activities of sea anemones collected in rock pools at the higher levels are more homogeneous and significantly ditferent from those of organisms collected at lower levels (0.035 f 0.014 compared to 0.065 k 0.037 I.U./mg of protein). However, this does not yet allow us to explain the extreme individual variations observed between organisms living at the lower level. Other factors than the presence of abundant microalgae may have a role in the regulation of amylase levels in sea invertebrates, as is becoming evident with crustaceans (Samain et al., 1980; Van Wormhoudt, 1981). Ac,kno~clrdgrmrnrs- The author wishes to thank Mr J. F. Samain (Centre Octanologique de Bretagne, Brest) and Mr C. Milet (Laboratoire de Physiologie du Museum. Paris) for their valuable suggestions and for providing laboratory facilities.

REFEREh’CES BOUCHER J., LAUREC’ A., SAMAIN J. F. & SMITH S.

Etude de la nutrition,

du regime

et du rythme

L. (1976) alimen-

taire du looplancton dans It’s condltlons naturclles par la mesure des activitCs enqmatiques digestives. Pro<. 1Otl1 Europeun Sytnposirrnt of1 Mtrrrw Hwlo~j?.~ &tend. Belgium, Vol. 2. pp. 123-138. CASE M. R. (197X) Synthesis. Intracellular transport and discharge of exportable protelny in the pancrcatlc acinar ccl1 and other cells. Bwl. Rc,r. 53, 21 I 354. CO.AN M. H. & TRAVIS J. (1970) Comparattvc bwchemistq of proteases from a coelenterate. C~~wr~~. Niw /I~I. P/~j-w)/. 32, 127 139. DI SALVO L. H. (1971) Ingestion and asslmllation of bacteria by two scleractinian coral species. In E\pr~rrrwnrtrl Coelrnrrrcrtr Bioloy) (edited by LI.NHOFF H. M.. MLIS(.ATN L. & DACIS L. V.) pp. 129 lib. Ilnivcrsit! of Hawaii Press. GABE M. (1968) Technique? Hi\to/o(/l~/lrc.\. Masson, Paris. GABON D. & DIXON G. H. (1969) Chymotrypsin-like prw teases from the sea anemone. ,21erriclrwn .wni/c .Vc~ruw 222.

153

76.

KRIJGSMAN B. J. & TALHOT F. H. (lY53) Experlmentb on digestion in sea anemones. ilrc,ll,\ 1~11. ~‘l,~~.si~~/.61. 277- 29 I. KRI:KENRERG C. (1880) Ueber den Verdauungsmodus der Actinien. l’qyl. Ph!sio/. Srcrtl. I, 33 56. LAURIER-BONI(.HON A., VAN WORMHOIII)T .A & S~LLOS II. (19771 Croissance larvaire contrblie de Prwrcw ,~!P,Inicxs; enzymes digestives et changemcnt de regime alimentalre. P~rhl. CNEXO ilc,tcc\ Co/h/~\, 4, 203 71 7. MLIR~ITK G. R. (1971) The formatlon and assimilation of alcohol-soluble protems during intracellular digestlon by Hydrrr lirrortrlis and Aipro.sio sp. In E\pwimcnrtrl Corlcw feraf~ Biology (edited by LENHOFF H. M.. MCIS~ATIN~ L. & DAVIS L. V.) pp. 137-145. University of Hawaii Press. NI(.OL J. A. (1959) DigestIon In sea anemones J. nwr. hioi. 4w l’.K. 38, 469-476. SAMAIN J. F., DAVIFL J Y. & Lt Car J. R. (lY77) Trypsmc. amylase et protCmes du rooplancton: dosage automatlque et manuel. J. rup. ww. BIO/. Ec~ol. 29, 279 28’). SAMAIN J. F.. MOAL J.. DA~ITL. J. Y.. LI Co/ J. R & J~rr:~r FL M. (1980) The digestlbc cnqmes amylasr and

VA> PRATT M. (19X0) Absorption des auhstanccs dlssoutes dans le milieu des particules et des produits de la digestion extracollutxire chez ~C,I~IIICICY,I,JM~L. Rr~prod. Ylrtr. D~~cdop. 20. I393 1399. VAU PRAY? M. (19X1) C’omparalson des taux d‘actlvitt: amylasique. trypsque et chymotrypsque ainsi que des types ccllulaircs intervenant dans la digestion cher les actinies littorales et abyssales. O~~&r,~i.s,4cru hiocl~im. wtii-. Iin press). VAV WORMIIOI’III (19X0) Adaptation de la teneur en cwymes digestives de I‘ht;patopancrCas de Ptrltrrmo~r ,wrrrrtus 3 la composition d’aliments expCrimentaux. 4quc1<,~r/rw<,21. 63 7X. V;~N WORMHOIIII~T(19x1) Regulation d‘actlvitC de I‘r-amylxx ;l diff&rentes tempkratures d’adaptation et en fonction de I’ablation des pCdoncules oculaires et du stade de mue chef

I93 203

P~~/w~twrt .wrw~~rv. Hicrhcw.

Sysr.

Eud.,

8.