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Carbohydrases of the crystalline style and hepatopancreas of Strombus gigas linné
Comp. Biochem. PhysioL, 1966, VoL 17, pp. 1189 to 1197. Pergamon Press Ltd. Printed in Great Britain
CARBOHYDRASES OF THE CRYSTALLINE STYLE AND HEPATOPANCREAS OF S T R O M B U S G I G A S LINNI~* S H I R O H O R I U C H I t and C H A R L E S E. LANE Institute of Marine Science, University of Miami, Miami, Florida, U.S.A.
(Received 19 October 1965) Strombus gigas Linn~ carbohydrase activity is present both in the crystalline style and in the hepatopancreas. 2. Only cellulase and maltase were detected in the crystalline style, while extracts of the hepatopancreas contained amylase, eellulase, maltase, ceUobiase, c~-glucosidase, fl-glucosidase, sucrase, galactosidase and trehalase activities. 3. Neither chitin nor pectin was hydrolyzed by extracts from either digestive organ. 4. Some temperature and pH characteristics of enzymes of the crystalline style and the hepatopancreas are described. A b s t r a c t - - 1 . In
INTRODUCTION DIGESTIVE enzymes of animals are closely correlated with food and feeding habits. Herbivorous animals generally have more active carbohydrases than carnivores (Prosser & Brown, 1961). The marine snail, Strombus gigas, is the largest of the herbivorous gastropods, feeding on delicate macroscopic or unicellular algae and on algal detritus (Yonge, 1932a; Robertson, 1961; Randall, 1964). Horiuchi & Lane (1965) described the ceUulase activity of the crystalline style of S. gigas, showing that this activity is not contributed by symbionts in the gut but originates from the style itself. The present study describes the other carbohydrate-splitting activities of the two principal digestive organs of S. gigas, the crystalline style and the hepatopancreas. MATERIALS AND METHODS Specimens of the queen conch, S. gigas Linnt, which is native to southeast Florida and the West Indies, were collected from the shallow water adjacent to Virginia Key, Miami, Florida. They were maintained in sea-water pens on the laboratory grounds until they were used. The animals were separated from the shell, and the crystalline style and hepatopancreas quickly removed. The lyophilized style extracts were prepared as * Contribution No. 661 from The Marine Laboratory, Institute of Marine Science, University of Miami. This study was supported by Grant No. HE-5489 from the Heart Institute of the National Institutes of Health. t Present address: Biological Laboratory, Sophia University, Tokyo, Japan. 1189
1190
SHmOHOmUCHIANDCrL,atLr~E. LANE
previously described (Horiuchi & Lane, 1965). The hepatopancreas was homogenized with 1/15 M phosphate buffer (pH 6.55) made isotonic with sea water by the addition of NaC1. The homogenate was centrifuged at 4°C and 20,200 g for 30 min and the supernatant was lyophilized. The lyophilized powders dissolved readily in Sorensen's phosphate buffer at room temperature. The hepatopancreas extract was dialyzed against distilled water below 5°C, until the outer bath was free of reducing sugar and chloride ions, as shown by the Somogyi-Nelson reagent and AgNO 3 respectively. Precipitates appearing in the solution during dialysis were separated by low speed centrifugation and discarded. Only the clear supernatant solution was used for enzyme assay. Carbohydrase activity was determined both viscosimetrically and by the formation of reducing sugar. The substrates chitin, sodium alginate, carrageenan and native cellulose were hydrolyzed slowly. For these the incubation period was extended to 24 hr under toluene. For other substrates the incubation period was 2 hr. Reducing sugar was estimated by the colorimetric method of Somogyi (1928, 1952) as modified by Nelson (1944). Optical density was measured at 500 m/~ with either the Beckman Model DU spectrophotometer or Coleman Model 6A spectrophotometer. Glucose was determined by the glucose oxidase method (Worthington Biochemical Corporation). Control experiments employed the dialyzed enzyme solution that had been boiled for 15 min. Changes in substrate viscosity were measured in Ostwald viscosimeters. Other procedures were similar to those described previously (Horiuchi & Lane, 1965). Substrates used in the present study were: Soluble potato starch (J. T. Baker Chemical Company); glycogen from shellfish (Sigma Chemical Company); laminarin was kindly given us by Dr. B. J. D. Meeuse, University of Washington, Seattle; sodium carboxymethylcellulose (CMC) of different degrees of polymerization and substitution (Hercules Powder Company); refined carrageenan, Gelcarin H-WG (Marine Colloids, Inc.); purified sodium alginate (Fisher Scientific Company); methyl-~-D-glucopyranoside; p-nitrophenyl-fl-D-glucopyranoside; and cellobiose (California Corporation for Biochemical Research); agar, chitin, inulin, pectin, salicin, raffinose, lactose, maltose, melibiose, sucrose and trehalose. Protein was determined colorimetrically using the Folin-Ciocalteu phenol reagent (Fisher Scientific Company) by the method of Lowry et al. (1951), using crystalline bovine albumin (Nutritional Biochemicals Corporation) as a standard. RESULTS
Various carbohydrase activities of the crystalline style and the hepatopancreas in S. gigas are shown in Table 1 and Figs. 1-5. Among the carbohydrases tested in the present study, amylase was the most active in extracts of both digestive organs. The hepatopancreas amylase was approximately twice as active as that from the crystalline style. About 85 per cent of its activity was lost after prolonged dialysis. Addition of NaC1 (Table 1) restored activity nearly to pre-dialysis levels. The amylase activity for both starch and glycogen was strictly proportional to enzyme protein between 0.025 mg and
CARBOHYDRASI~ OF S T R O M B U S GIGAS LINNE
1191
0.270 mg. The relationship between pH and amylase activity of extracts of style and hepatopancreas is presented in Fig. 1. The style amylase shows two pH optima: the first at pH 5.8-5-9 and the second at pH 6.5-6.7. Activity is somewhat greater in the higher pH range. Starch was more readily hydrolyzed than glycogen at the higher optimum (Fig. 1). The amylase from the hepatopancreas, on the other hand, shows but one pH optimum; between pH 6"3-6"5 (Fig. 1). The pH of gut contents of S. gigas varied between pH 6.25 and pH 6"55. TABLE 1----CARBOHYVP~TEDIGESTION IN Stlrombu$ g/gas*
Enzyme activityt Substrate Crystalline style Starch
1568
Glycogen
1301
Laminarin Inulin Cellulose, CMC 70 low medium high Phosphoric acid swollen Agar Carrageenan Chitin Pectin Sodium alginate Methyl-a-glucoside p-Nitrophenyl-fl-glucoside Salicin Raffinose Cellobiose Lactose Maltose Melibiose Sucrose Trehalose
129 0 -463 I --
Hepatopancreas
1"3014 Before dialysis -~ 472 After dialysis [2681 After dialysis + NaCI ~2351 Before dialysis -{ 352 After dialysis L2349 After dialysis + NaCI 826 0 792 690 256 92
0
15
28 0 0 31 0 0 0 0 22 0 68 0 0 0
66 0 0 70 30 397 70 15 149 25 119 24 42 29
* Enzyme assays conducted at 35°C, pH 6"8-7"2. t Activity is expressed as ftg glucose/hr/mg enzyme protein. During 60 min of amylolysis by extracts of both the crystalline style and the hepatopancreas the absorption spectrum of the reaction mixture with iodine shifted progressively from maximum absorption of 600 m/~ toward 430 mtt (Fig. 2). Amylase from the crystalline style showed optimum activity at 28°C and nearly complete inactivation at 50°C (Fig. 3).
1192
SHIRO HORIUCHI AND CHARLES E. LANE
Laminarin was completely hydrolyzed by hepatopancreas amylase but not by the style enzyme. Inulin was not hydrolyzed by extracts of either organ. Extracts of both hepatopancreas and crystalline style effectively hydrolyze structural
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FIo. 1. Effect of pH on amylase activity of crystalline style and hepatopancreas. Solid line is hepatopancreas, broken lines are crystalline style incubated with starch and glycogen. Reaction mixtures were incubated at 30°C for 60 rain. Hepatopancreas extract contained 0"0046 M NaCI.
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500 600 WAVE LENGTH(n~J.)
FIG. 2. Changes in absorption spectra during amylolysis by the crystalline style. Incubation time in min is shown on the right.
polysaccharides such as cellulose. The cellulase activity of the hepatopancreas is greater than that of the crystalline style (Table 1) and differs slightly from it with respect to different synthetic substrates. Cellulose fibers swollen by exposure to phosphoric acid, were hydrolyzed less readily than the CMC substrate. Native cellulose powder yielded only 3/~g glueose/hr/mg of enzyme protein when incubated with hepatopancreas extract. The viscosity of CMC substrate was reduced
OF STROMBUS
CARBOHYDRASES
GIG, S
1193
LINNE
by enzymes in the hepatopancreas extract in inverse relationship to its degree of substitution (Fig. 4). The cellulase activity of the crystalline style has been described previously (Horiuchi & Lane, 1965). Agar was not attacked by the style extract, and only slightly hydrolyzed by extracts of the hepatopancreas. Carrageenan, which is extracted from certain red marine algae, was readily hydrolyzed by hepatopancreas and less by the style. 15O
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AMYLASE
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Temperature
stahiIhy
28
40 5O TEMPERATURE (%)
of crystalline
60
70
style amy]ase ~nd ce]luIase.
Reaction
mixtures contained 1'8 mg of" lyophil/zed style extract and 0.25 mg of starch or 1.2 mg of CMC 70. I00, ~ . . . . . . . . . . . . . . . . . . . . . . . . . .
BOILED EXTRACT
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INCUBATIONTIME (MINUTES)
FIG. 4. Viscosimetric change in reaction mixtures when hepatopancreas extract was incubated with CMC 70 of different degrees of substitution. Incubation at 35°C; pH 7.2. Final CMC concentration was 0.6%. Sodium alginate, like carrageenan, was digested by both extracts. (Table 1, Fig. 5). Neither chitin nor pectin was hydrolyzed by either enzyme extract, although these substrates were incubated between pH 5.6 and 7" 1 for 24 hr or more. Disaccharidases of both organs are considerably less active than are the polysaccharidases (Table 1). ~-Glucosidase, fl-glucosidase, cellobiase, maltase and
1194
SHIRO HORIUCHI AND CHARLES E . LANE
sucrase activities were found in the hepatopancreas, together with slight galactosidase and trehalase activities. Only cellulase and maltase were detected in the crystalline style (Table 1). I00, --'~'~'&"- " ~
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INcUeATION TIMF (MINUTEs)
Fzo. 5. Viscosimetricchange in reaction mixture of sodium alginate and hepatopancreas extract. Incubation temperature 35°C; pH 7"2; sodium all,hate concentration 0"6%. DISCUSSION Yonge (1932a) originally characterized S. g/gas as the largest herbivorous gastropod, feeding on fine filamentous weeds. In the Miami area S. g/gas is usually found on beds of Thalassia testudinum KSnig and Cymodoceanumatorum Ascherson. Its feeding habit was recently studied by Robertson (1961), who established by field observations that this conch feeds continuously on a filamentous blue-green alga, a fine filamentous green alga and two delicate, branched red algae. These algae were either epiphytic or grew in dumps on sand. Randall (1964) confirmed by examination of stomach contents that the conch feeds principally on plant material. He emphasized that the dominant plants in a particular habitat form the principal food of the conch. Unlike Robertson, Randall found fragments of Thalassia in the stomach, indicating that turtle grass was also digested by the conch. However, the bulk of the plant material ingested by S. g/gas from a bed of Thalassia is algal, particularly epithytic algae. The field observations support the laboratory identification of the enzymes available in the digestive system of the conch, emphasizing the animal's nutritional dependence on both reserve polysaccharides and structural components of the algal cell wall. That carbohydrases are widely distributed in molluscs has been shown by a number of investigators: in Cryptochiton (Meeuse & Fluegel, 1958); in the marine snail, Tegu/a funebra//s (Galli & Giese, 1959); in He~.xpomatia(Myers & Northcote, 1958); in the slug, At/on at~r (Evans & Jones, 1962), arm in a bivalve, Veneru~ phiIippinarum (Horiuchi, 1963). The greatest carbohydrase activity was found to be an amylase in Tegu/a and in Vowrup/s. On the other hand, a cellulase activity w u highest in Helix.
CARBOHYDRASH8 OF 8 T R O M B U S G I G A S I.,INNI~
| 195
In S. g/gas, as well, amylases are the most active carbohydrases. These enzymes occur in the crystallinestyle and in the hepatopancreas. The style amylase shows two pH optima; pH 5.8 and 6.8 (Fig. I). Two pH optima were also described for the style a-amylase of Crassostrea virginica (Black & Pengelley, 1964). These authors suggest that the style of the adult may contain a second amylase that is not present in the larva. During development of the oyster, Black & Pengelley (1964) indicated that amylase reached a high level of activity before the larva begins to feed. The crystallinestyle of the gastropod Cr@idula is the most acid region of the gut at pH 5.8. Stomach contents ranged between pH 5"8 and 6.2. The pH of the gut increased through mid and hind gut to pH 8.3 (Yonge, 1925). The pH of the gut contents of S. gigas varied between pH 5-25 and 6.65. Changes in the absorption spectrum of the iodine--polysaccharide complex during amylolytic hydrolysis reveals the mode of action of the amylases. A rapid color change with iodine occurs during hydrolysis of starch (Hanes & Cattle, 1938), and amylose (Swanson, 1948), with different amylases from a-malt, .4spergillus, saliva and pancreas. On the other hand, this color change never occurs during hydrolysis by fl-malt-amylase, the position of maximum absorption remaining almost constant. Marked changes in the absorption spectrum during hydrolysis strongly suggest that both the crystalline style and the hepatopancreas of S. gigas produce a-amylases. Both the optimum temperature and the thermostability of the style amylase present marked differences from the style cellulase. The latter activity was undiminished up to 45°C (Horiuchi & Lane, 1965). The greatest activity and maximum thermostability of style amylase were found at approximately 28°C. This may be an adaptive response to the average temperature of the littoral environment in the Miami area. In this connection Barrington's comment (1962) that optimum temperatures for enzyme activity in poikilotherms need not always be 37°C should be recalled S. gigas is shown to digest such structural (i.e. cell-wall) polysaccharides as cellulose, carrageenan and sodium alginate. Hashimoto & Onoma (1949) examined the activity of cellulase, alginase, xylanase and amylase in the gastric teeth of the stomach of the gastropod, Dolabella, and found activity similar to that in the crystalline style. Franssen & Jeuniaux (1965) have also described the distribution of alginolytic activity in the style of a bivalve, Tapes, and in 13 different species of gastropods. An alginase seems to be a common constituent of the enzymatic equipment of gastropods. It occurs not only in those phytophagous marine species feeding on brown algae containing alginic acid, but also in marine carnivores, and in phytophagous terrestrial and fresh-water species. It is of interest to compare the digestive activity of the style with that of the hepatopancreas (Table 1). Polysaccharides are reduced to low molecular weight fragments by enzymes liberated in the stomach when the crystalline style dissolves. This digestive process is probably accelerated by the mechanical mixing resulting from style rotation. Further breakdown of partially digested food materials and
1196
SHIRO HORIUCHI AND CHARLES E. LANE
disaccharides occurs in the hepatopancreas. Morton (1951) described a strong amylolytic activity in the style of the gastropod, Struthiolaria. The style of this form hydrolyzed neither disaccharides nor cellulose. Morton ascribes equal importance to the mechanical and enzymatic functions of the style of Struthiolaria. Morton (1952) concluded that the original function of the crystalline style was essentially mechanical trituration. He considered that it developed during evolution from a rotating faecal rod, a so-called "protostyle" devoid of special enzymatic activity. With evolution and dietary specialization the crystalline style acquired its present enzyme activity. Graham (1939), on the contrary, in his comprehensive account of the style-bearing gastropods, described the style as a device to provide digestive enzymes found in a number of rather specialized prosobranchs. Coupin (1900) demonstrated the enzymatic nature of the style in Cardium edule. Yonge (1932b), and Graham (1939), described the crystalline style in microphagous herbivorous gastropods. Mansour (1946) claimed that in the lamellibranchs enzymes are liberated into the stomach by the fragmentation of holocrine digestive cells of the hepatopancreatic diverticula. However, Morton (1951) emphasized that in Struthiolaria such cellular fragments consist principally of excretion products and pass directly to the intestine. Traces of enzymes are probably added to stomach contents by this process. However, this snail generally conforms entirely to Yonge's rule that stylebearers digest intracellularly. In S. gigas, the hepatopancreas seems to be involved in gastric digestion through enzymes produced by ceils of the diverticula, even though the primitive function of these cells was phagocytosis.
REFERENCES BARRINGTONE. J. W. (1962) Digestive enzymes. In Advances in Comparative Physiology and Biochemistry (Edited by LOWENSTEINO.) Vol. 1, pp. 1-65. Academic Press, New York. BLACK R. E. & PENGELLEYE. T. (1964) Alpha amylase development in embryos of Crassostrea virginica. Biol. Bull., Woods Hole 126, 199-204. CouPIN H. (1900) Sur les fonetions de la tige eristalline des Ac~phales. C.r. Acad. Sci., Paris 130, 1214-1216. EVANSW. A. L. & JONESE. G. (1962) Carbohydrases in the alimentary tract of the slug, Arion alter L. Comp. Biochem. Physiol. 5, 149-160. FR~'qSSENJ. & JmrNXAOXCH. (1965) Digestion de l'aeid alginique chez les inverttbrts. Cah. Biol. mar. 6, 1-21. GALLXD. R. & GImE A. C. (1959) Carbohydrate digestion in a herbivorous snail, Tegula funebralis, fl. expl Zool. 140, 415--440. GRAHAMA. (1939) On the structure of the alimentary canal of style-bearing prosobranehs. Proc. zool. Soc. Lond. B 109, 75-112. HANm C. S. & CATTLE M. (1938) Starch-iodine coloration as an index of differential degradation by the amylases. Proc. R. Soc. B 125, 387-414. HASHIMOTOY. & ONOMA K. (1949) On the digestion of higher carbohydrates by moUusca (Do/abella scapu/a and Teredo sp.). Bull. flap. Soc. scient. Fish. 15, 253-258. HoamcHx S. (1963) On the nature of carbohydrases of digestive diverticula in the marine lamellibranch, Venerupis philippinarura. Sci. Pep. Tokyo Kyoiku Da/g., B 11, 133-152.
CARBOHYDRASES
OF S T R O M B U S
G I G A S LINNI~
1197
HORIUCHI S. • LANE C. E. (1965) Digestive enzymes of the crystalline style of Strombus gigas Linn6 I. Cellulase and some other carbohydrases. Biol. Bull., Woods Hole 129, 273-281. LOWRY O. H., ROSENBROUGHN. J., FAm~A. L. &RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent, ft. biol. Chem. 193, 265-275. MANsotm K. (1946) Food and digestive organs of lamellibranchs. Nature, Land. 158, 378. M~USE B. J. D. & FLtmGEL W. (1958) Carbohydrate-digesting enzymes in the sugar-gland juice of Cryptochitan stelleri Middendorff (Polyplacophora, Mollusca). Arch* neerl. Zool. 13, Suppl. 1,301-313. MORTON J. E. (1951) The ecology and digestive system of the Struthiolariidae (Gastropoda). Quart. Jl microsc. Sci. 92, 1-25. MORTON J. E. (1952) The role of the crystalline style. Proc. malac. Soc. Lond. 29, 85-92. MYERS F. L. & NORTHCOT~D. H. (1958) A survey of the enzymes from the gastro-intestinal tract of Helix pomatia. J. exp. Biol. 35, 639-648. NELSON N. (1944) A photometric adaptation of the Somogyi method for the determination of glucose. J. biol. Chem. 153, 375-380. PRoss~a C. L. & BROWN F. A. (1961) In Comparative Animal Physiology (2nd ed.), pp. i 106-134. Saunders Philadelphia, I~NDALr. J. E. (1964) Contributions to the biology of the queen conch, Strombus gigas. Bull. mar. Sci, Gulf Caribb. 14(2), 246-294. ROBERTSON R. (1961) The feeding of Strombus and related herbivorous marine gastropods. Notul. Nat. 343, 1-9. SOMOGYI M. (1928) The distribution of sugar in normal human blood, y. biol. Chem. 78, 117-127. SOMOGYI M. (1952) Notes on sugar determination. J. biol. Chem. 195, 19-23. SWANSON M. A. (1948) Studies on the structure of polysaccharidesMIV. Relation of the iodine color to the structure, ft. biol. Chem. 172, 825-837. YONGE C. M. (1925) The hydrogen ion concentration in the gut of certain lamellibranchs and gastropods, ft. mar. Biol. Ass. U.K. 13, 938-952. YONGE C. M. (1932a) On the size attained by the crystalline style in Tridacna and Strombus. Proc. malac. Soc. Land. 20(1), AA A,5" YONGE C. M. (1932b) Notes on feeding and digestion in Pterocera and Vermetus, with a discussion on the occurrence of the crystalline style in the gastropods. Sci. Rep. Brit. Mus. (Nat. Hist.) (Gt. Barrier Reef Exped.) 1, 259-281.
CARBOHYDRASES OF THE CRYSTALLINE STYLE AND HEPATOPANCREAS OF S T R O M B U S G I G A S LINNI~* S H I R O H O R I U C H I t and C H A R L E S E. LANE Institute of Marine Science, University of Miami, Miami, Florida, U.S.A.
(Received 19 October 1965) Strombus gigas Linn~ carbohydrase activity is present both in the crystalline style and in the hepatopancreas. 2. Only cellulase and maltase were detected in the crystalline style, while extracts of the hepatopancreas contained amylase, eellulase, maltase, ceUobiase, c~-glucosidase, fl-glucosidase, sucrase, galactosidase and trehalase activities. 3. Neither chitin nor pectin was hydrolyzed by extracts from either digestive organ. 4. Some temperature and pH characteristics of enzymes of the crystalline style and the hepatopancreas are described. A b s t r a c t - - 1 . In
INTRODUCTION DIGESTIVE enzymes of animals are closely correlated with food and feeding habits. Herbivorous animals generally have more active carbohydrases than carnivores (Prosser & Brown, 1961). The marine snail, Strombus gigas, is the largest of the herbivorous gastropods, feeding on delicate macroscopic or unicellular algae and on algal detritus (Yonge, 1932a; Robertson, 1961; Randall, 1964). Horiuchi & Lane (1965) described the ceUulase activity of the crystalline style of S. gigas, showing that this activity is not contributed by symbionts in the gut but originates from the style itself. The present study describes the other carbohydrate-splitting activities of the two principal digestive organs of S. gigas, the crystalline style and the hepatopancreas. MATERIALS AND METHODS Specimens of the queen conch, S. gigas Linnt, which is native to southeast Florida and the West Indies, were collected from the shallow water adjacent to Virginia Key, Miami, Florida. They were maintained in sea-water pens on the laboratory grounds until they were used. The animals were separated from the shell, and the crystalline style and hepatopancreas quickly removed. The lyophilized style extracts were prepared as * Contribution No. 661 from The Marine Laboratory, Institute of Marine Science, University of Miami. This study was supported by Grant No. HE-5489 from the Heart Institute of the National Institutes of Health. t Present address: Biological Laboratory, Sophia University, Tokyo, Japan. 1189
1190
SHmOHOmUCHIANDCrL,atLr~E. LANE
previously described (Horiuchi & Lane, 1965). The hepatopancreas was homogenized with 1/15 M phosphate buffer (pH 6.55) made isotonic with sea water by the addition of NaC1. The homogenate was centrifuged at 4°C and 20,200 g for 30 min and the supernatant was lyophilized. The lyophilized powders dissolved readily in Sorensen's phosphate buffer at room temperature. The hepatopancreas extract was dialyzed against distilled water below 5°C, until the outer bath was free of reducing sugar and chloride ions, as shown by the Somogyi-Nelson reagent and AgNO 3 respectively. Precipitates appearing in the solution during dialysis were separated by low speed centrifugation and discarded. Only the clear supernatant solution was used for enzyme assay. Carbohydrase activity was determined both viscosimetrically and by the formation of reducing sugar. The substrates chitin, sodium alginate, carrageenan and native cellulose were hydrolyzed slowly. For these the incubation period was extended to 24 hr under toluene. For other substrates the incubation period was 2 hr. Reducing sugar was estimated by the colorimetric method of Somogyi (1928, 1952) as modified by Nelson (1944). Optical density was measured at 500 m/~ with either the Beckman Model DU spectrophotometer or Coleman Model 6A spectrophotometer. Glucose was determined by the glucose oxidase method (Worthington Biochemical Corporation). Control experiments employed the dialyzed enzyme solution that had been boiled for 15 min. Changes in substrate viscosity were measured in Ostwald viscosimeters. Other procedures were similar to those described previously (Horiuchi & Lane, 1965). Substrates used in the present study were: Soluble potato starch (J. T. Baker Chemical Company); glycogen from shellfish (Sigma Chemical Company); laminarin was kindly given us by Dr. B. J. D. Meeuse, University of Washington, Seattle; sodium carboxymethylcellulose (CMC) of different degrees of polymerization and substitution (Hercules Powder Company); refined carrageenan, Gelcarin H-WG (Marine Colloids, Inc.); purified sodium alginate (Fisher Scientific Company); methyl-~-D-glucopyranoside; p-nitrophenyl-fl-D-glucopyranoside; and cellobiose (California Corporation for Biochemical Research); agar, chitin, inulin, pectin, salicin, raffinose, lactose, maltose, melibiose, sucrose and trehalose. Protein was determined colorimetrically using the Folin-Ciocalteu phenol reagent (Fisher Scientific Company) by the method of Lowry et al. (1951), using crystalline bovine albumin (Nutritional Biochemicals Corporation) as a standard. RESULTS
Various carbohydrase activities of the crystalline style and the hepatopancreas in S. gigas are shown in Table 1 and Figs. 1-5. Among the carbohydrases tested in the present study, amylase was the most active in extracts of both digestive organs. The hepatopancreas amylase was approximately twice as active as that from the crystalline style. About 85 per cent of its activity was lost after prolonged dialysis. Addition of NaC1 (Table 1) restored activity nearly to pre-dialysis levels. The amylase activity for both starch and glycogen was strictly proportional to enzyme protein between 0.025 mg and
CARBOHYDRASI~ OF S T R O M B U S GIGAS LINNE
1191
0.270 mg. The relationship between pH and amylase activity of extracts of style and hepatopancreas is presented in Fig. 1. The style amylase shows two pH optima: the first at pH 5.8-5-9 and the second at pH 6.5-6.7. Activity is somewhat greater in the higher pH range. Starch was more readily hydrolyzed than glycogen at the higher optimum (Fig. 1). The amylase from the hepatopancreas, on the other hand, shows but one pH optimum; between pH 6"3-6"5 (Fig. 1). The pH of gut contents of S. gigas varied between pH 6.25 and pH 6"55. TABLE 1----CARBOHYVP~TEDIGESTION IN Stlrombu$ g/gas*
Enzyme activityt Substrate Crystalline style Starch
1568
Glycogen
1301
Laminarin Inulin Cellulose, CMC 70 low medium high Phosphoric acid swollen Agar Carrageenan Chitin Pectin Sodium alginate Methyl-a-glucoside p-Nitrophenyl-fl-glucoside Salicin Raffinose Cellobiose Lactose Maltose Melibiose Sucrose Trehalose
129 0 -463 I --
Hepatopancreas
1"3014 Before dialysis -~ 472 After dialysis [2681 After dialysis + NaCI ~2351 Before dialysis -{ 352 After dialysis L2349 After dialysis + NaCI 826 0 792 690 256 92
0
15
28 0 0 31 0 0 0 0 22 0 68 0 0 0
66 0 0 70 30 397 70 15 149 25 119 24 42 29
* Enzyme assays conducted at 35°C, pH 6"8-7"2. t Activity is expressed as ftg glucose/hr/mg enzyme protein. During 60 min of amylolysis by extracts of both the crystalline style and the hepatopancreas the absorption spectrum of the reaction mixture with iodine shifted progressively from maximum absorption of 600 m/~ toward 430 mtt (Fig. 2). Amylase from the crystalline style showed optimum activity at 28°C and nearly complete inactivation at 50°C (Fig. 3).
1192
SHIRO HORIUCHI AND CHARLES E. LANE
Laminarin was completely hydrolyzed by hepatopancreas amylase but not by the style enzyme. Inulin was not hydrolyzed by extracts of either organ. Extracts of both hepatopancreas and crystalline style effectively hydrolyze structural
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5.
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pH
FIo. 1. Effect of pH on amylase activity of crystalline style and hepatopancreas. Solid line is hepatopancreas, broken lines are crystalline style incubated with starch and glycogen. Reaction mixtures were incubated at 30°C for 60 rain. Hepatopancreas extract contained 0"0046 M NaCI.
°f°~o 1.0
0
/
/ ~0.5
AI
4 O0
i
A__A_.....A__A~t,~A_ i
i
I
i
i
l
q
'O A 45 6O t
:
500 600 WAVE LENGTH(n~J.)
FIG. 2. Changes in absorption spectra during amylolysis by the crystalline style. Incubation time in min is shown on the right.
polysaccharides such as cellulose. The cellulase activity of the hepatopancreas is greater than that of the crystalline style (Table 1) and differs slightly from it with respect to different synthetic substrates. Cellulose fibers swollen by exposure to phosphoric acid, were hydrolyzed less readily than the CMC substrate. Native cellulose powder yielded only 3/~g glueose/hr/mg of enzyme protein when incubated with hepatopancreas extract. The viscosity of CMC substrate was reduced
OF STROMBUS
CARBOHYDRASES
GIG, S
1193
LINNE
by enzymes in the hepatopancreas extract in inverse relationship to its degree of substitution (Fig. 4). The cellulase activity of the crystalline style has been described previously (Horiuchi & Lane, 1965). Agar was not attacked by the style extract, and only slightly hydrolyzed by extracts of the hepatopancreas. Carrageenan, which is extracted from certain red marine algae, was readily hydrolyzed by hepatopancreas and less by the style. 15O
e --.-.B-,--- e ~
o\
e~
'('-- CELLULASE
~so ~O~o~
'
AMYLASE
~o
~4o >
20
0 F I G . 3.
Temperature
stahiIhy
28
40 5O TEMPERATURE (%)
of crystalline
60
70
style amy]ase ~nd ce]luIase.
Reaction
mixtures contained 1'8 mg of" lyophil/zed style extract and 0.25 mg of starch or 1.2 mg of CMC 70. I00, ~ . . . . . . . . . . . . . . . . . . . . . . . . . .
BOILED EXTRACT
z 50
° ~ ° ~ O ~ °
N
~"0"""
~ • ~ O---O CMC 120 HIGH
0 ~"-"~"- ~-.- 0
CMC 90 HIGH CMC 70 HIGH
,,,,
I
30
,
I
60
*
I
90
i
I
120
INCUBATIONTIME (MINUTES)
FIG. 4. Viscosimetric change in reaction mixtures when hepatopancreas extract was incubated with CMC 70 of different degrees of substitution. Incubation at 35°C; pH 7.2. Final CMC concentration was 0.6%. Sodium alginate, like carrageenan, was digested by both extracts. (Table 1, Fig. 5). Neither chitin nor pectin was hydrolyzed by either enzyme extract, although these substrates were incubated between pH 5.6 and 7" 1 for 24 hr or more. Disaccharidases of both organs are considerably less active than are the polysaccharidases (Table 1). ~-Glucosidase, fl-glucosidase, cellobiase, maltase and
1194
SHIRO HORIUCHI AND CHARLES E . LANE
sucrase activities were found in the hepatopancreas, together with slight galactosidase and trehalase activities. Only cellulase and maltase were detected in the crystalline style (Table 1). I00, --'~'~'&"- " ~
o
~> 5o
,',~
~,.....____
BOILED EXTRACT - &...,..--___._/,,
\o , o
z
.N ,N IIIII
I 30
I 60
I
I
I gO
I llO
INcUeATION TIMF (MINUTEs)
Fzo. 5. Viscosimetricchange in reaction mixture of sodium alginate and hepatopancreas extract. Incubation temperature 35°C; pH 7"2; sodium all,hate concentration 0"6%. DISCUSSION Yonge (1932a) originally characterized S. g/gas as the largest herbivorous gastropod, feeding on fine filamentous weeds. In the Miami area S. g/gas is usually found on beds of Thalassia testudinum KSnig and Cymodoceanumatorum Ascherson. Its feeding habit was recently studied by Robertson (1961), who established by field observations that this conch feeds continuously on a filamentous blue-green alga, a fine filamentous green alga and two delicate, branched red algae. These algae were either epiphytic or grew in dumps on sand. Randall (1964) confirmed by examination of stomach contents that the conch feeds principally on plant material. He emphasized that the dominant plants in a particular habitat form the principal food of the conch. Unlike Robertson, Randall found fragments of Thalassia in the stomach, indicating that turtle grass was also digested by the conch. However, the bulk of the plant material ingested by S. g/gas from a bed of Thalassia is algal, particularly epithytic algae. The field observations support the laboratory identification of the enzymes available in the digestive system of the conch, emphasizing the animal's nutritional dependence on both reserve polysaccharides and structural components of the algal cell wall. That carbohydrases are widely distributed in molluscs has been shown by a number of investigators: in Cryptochiton (Meeuse & Fluegel, 1958); in the marine snail, Tegu/a funebra//s (Galli & Giese, 1959); in He~.xpomatia(Myers & Northcote, 1958); in the slug, At/on at~r (Evans & Jones, 1962), arm in a bivalve, Veneru~ phiIippinarum (Horiuchi, 1963). The greatest carbohydrase activity was found to be an amylase in Tegu/a and in Vowrup/s. On the other hand, a cellulase activity w u highest in Helix.
CARBOHYDRASH8 OF 8 T R O M B U S G I G A S I.,INNI~
| 195
In S. g/gas, as well, amylases are the most active carbohydrases. These enzymes occur in the crystallinestyle and in the hepatopancreas. The style amylase shows two pH optima; pH 5.8 and 6.8 (Fig. I). Two pH optima were also described for the style a-amylase of Crassostrea virginica (Black & Pengelley, 1964). These authors suggest that the style of the adult may contain a second amylase that is not present in the larva. During development of the oyster, Black & Pengelley (1964) indicated that amylase reached a high level of activity before the larva begins to feed. The crystallinestyle of the gastropod Cr@idula is the most acid region of the gut at pH 5.8. Stomach contents ranged between pH 5"8 and 6.2. The pH of the gut increased through mid and hind gut to pH 8.3 (Yonge, 1925). The pH of the gut contents of S. gigas varied between pH 5-25 and 6.65. Changes in the absorption spectrum of the iodine--polysaccharide complex during amylolytic hydrolysis reveals the mode of action of the amylases. A rapid color change with iodine occurs during hydrolysis of starch (Hanes & Cattle, 1938), and amylose (Swanson, 1948), with different amylases from a-malt, .4spergillus, saliva and pancreas. On the other hand, this color change never occurs during hydrolysis by fl-malt-amylase, the position of maximum absorption remaining almost constant. Marked changes in the absorption spectrum during hydrolysis strongly suggest that both the crystalline style and the hepatopancreas of S. gigas produce a-amylases. Both the optimum temperature and the thermostability of the style amylase present marked differences from the style cellulase. The latter activity was undiminished up to 45°C (Horiuchi & Lane, 1965). The greatest activity and maximum thermostability of style amylase were found at approximately 28°C. This may be an adaptive response to the average temperature of the littoral environment in the Miami area. In this connection Barrington's comment (1962) that optimum temperatures for enzyme activity in poikilotherms need not always be 37°C should be recalled S. gigas is shown to digest such structural (i.e. cell-wall) polysaccharides as cellulose, carrageenan and sodium alginate. Hashimoto & Onoma (1949) examined the activity of cellulase, alginase, xylanase and amylase in the gastric teeth of the stomach of the gastropod, Dolabella, and found activity similar to that in the crystalline style. Franssen & Jeuniaux (1965) have also described the distribution of alginolytic activity in the style of a bivalve, Tapes, and in 13 different species of gastropods. An alginase seems to be a common constituent of the enzymatic equipment of gastropods. It occurs not only in those phytophagous marine species feeding on brown algae containing alginic acid, but also in marine carnivores, and in phytophagous terrestrial and fresh-water species. It is of interest to compare the digestive activity of the style with that of the hepatopancreas (Table 1). Polysaccharides are reduced to low molecular weight fragments by enzymes liberated in the stomach when the crystalline style dissolves. This digestive process is probably accelerated by the mechanical mixing resulting from style rotation. Further breakdown of partially digested food materials and
1196
SHIRO HORIUCHI AND CHARLES E. LANE
disaccharides occurs in the hepatopancreas. Morton (1951) described a strong amylolytic activity in the style of the gastropod, Struthiolaria. The style of this form hydrolyzed neither disaccharides nor cellulose. Morton ascribes equal importance to the mechanical and enzymatic functions of the style of Struthiolaria. Morton (1952) concluded that the original function of the crystalline style was essentially mechanical trituration. He considered that it developed during evolution from a rotating faecal rod, a so-called "protostyle" devoid of special enzymatic activity. With evolution and dietary specialization the crystalline style acquired its present enzyme activity. Graham (1939), on the contrary, in his comprehensive account of the style-bearing gastropods, described the style as a device to provide digestive enzymes found in a number of rather specialized prosobranchs. Coupin (1900) demonstrated the enzymatic nature of the style in Cardium edule. Yonge (1932b), and Graham (1939), described the crystalline style in microphagous herbivorous gastropods. Mansour (1946) claimed that in the lamellibranchs enzymes are liberated into the stomach by the fragmentation of holocrine digestive cells of the hepatopancreatic diverticula. However, Morton (1951) emphasized that in Struthiolaria such cellular fragments consist principally of excretion products and pass directly to the intestine. Traces of enzymes are probably added to stomach contents by this process. However, this snail generally conforms entirely to Yonge's rule that stylebearers digest intracellularly. In S. gigas, the hepatopancreas seems to be involved in gastric digestion through enzymes produced by ceils of the diverticula, even though the primitive function of these cells was phagocytosis.
REFERENCES BARRINGTONE. J. W. (1962) Digestive enzymes. In Advances in Comparative Physiology and Biochemistry (Edited by LOWENSTEINO.) Vol. 1, pp. 1-65. Academic Press, New York. BLACK R. E. & PENGELLEYE. T. (1964) Alpha amylase development in embryos of Crassostrea virginica. Biol. Bull., Woods Hole 126, 199-204. CouPIN H. (1900) Sur les fonetions de la tige eristalline des Ac~phales. C.r. Acad. Sci., Paris 130, 1214-1216. EVANSW. A. L. & JONESE. G. (1962) Carbohydrases in the alimentary tract of the slug, Arion alter L. Comp. Biochem. Physiol. 5, 149-160. FR~'qSSENJ. & JmrNXAOXCH. (1965) Digestion de l'aeid alginique chez les inverttbrts. Cah. Biol. mar. 6, 1-21. GALLXD. R. & GImE A. C. (1959) Carbohydrate digestion in a herbivorous snail, Tegula funebralis, fl. expl Zool. 140, 415--440. GRAHAMA. (1939) On the structure of the alimentary canal of style-bearing prosobranehs. Proc. zool. Soc. Lond. B 109, 75-112. HANm C. S. & CATTLE M. (1938) Starch-iodine coloration as an index of differential degradation by the amylases. Proc. R. Soc. B 125, 387-414. HASHIMOTOY. & ONOMA K. (1949) On the digestion of higher carbohydrates by moUusca (Do/abella scapu/a and Teredo sp.). Bull. flap. Soc. scient. Fish. 15, 253-258. HoamcHx S. (1963) On the nature of carbohydrases of digestive diverticula in the marine lamellibranch, Venerupis philippinarura. Sci. Pep. Tokyo Kyoiku Da/g., B 11, 133-152.
CARBOHYDRASES
OF S T R O M B U S
G I G A S LINNI~
1197
HORIUCHI S. • LANE C. E. (1965) Digestive enzymes of the crystalline style of Strombus gigas Linn6 I. Cellulase and some other carbohydrases. Biol. Bull., Woods Hole 129, 273-281. LOWRY O. H., ROSENBROUGHN. J., FAm~A. L. &RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent, ft. biol. Chem. 193, 265-275. MANsotm K. (1946) Food and digestive organs of lamellibranchs. Nature, Land. 158, 378. M~USE B. J. D. & FLtmGEL W. (1958) Carbohydrate-digesting enzymes in the sugar-gland juice of Cryptochitan stelleri Middendorff (Polyplacophora, Mollusca). Arch* neerl. Zool. 13, Suppl. 1,301-313. MORTON J. E. (1951) The ecology and digestive system of the Struthiolariidae (Gastropoda). Quart. Jl microsc. Sci. 92, 1-25. MORTON J. E. (1952) The role of the crystalline style. Proc. malac. Soc. Lond. 29, 85-92. MYERS F. L. & NORTHCOT~D. H. (1958) A survey of the enzymes from the gastro-intestinal tract of Helix pomatia. J. exp. Biol. 35, 639-648. NELSON N. (1944) A photometric adaptation of the Somogyi method for the determination of glucose. J. biol. Chem. 153, 375-380. PRoss~a C. L. & BROWN F. A. (1961) In Comparative Animal Physiology (2nd ed.), pp. i 106-134. Saunders Philadelphia, I~NDALr. J. E. (1964) Contributions to the biology of the queen conch, Strombus gigas. Bull. mar. Sci, Gulf Caribb. 14(2), 246-294. ROBERTSON R. (1961) The feeding of Strombus and related herbivorous marine gastropods. Notul. Nat. 343, 1-9. SOMOGYI M. (1928) The distribution of sugar in normal human blood, y. biol. Chem. 78, 117-127. SOMOGYI M. (1952) Notes on sugar determination. J. biol. Chem. 195, 19-23. SWANSON M. A. (1948) Studies on the structure of polysaccharidesMIV. Relation of the iodine color to the structure, ft. biol. Chem. 172, 825-837. YONGE C. M. (1925) The hydrogen ion concentration in the gut of certain lamellibranchs and gastropods, ft. mar. Biol. Ass. U.K. 13, 938-952. YONGE C. M. (1932a) On the size attained by the crystalline style in Tridacna and Strombus. Proc. malac. Soc. Land. 20(1), AA A,5" YONGE C. M. (1932b) Notes on feeding and digestion in Pterocera and Vermetus, with a discussion on the occurrence of the crystalline style in the gastropods. Sci. Rep. Brit. Mus. (Nat. Hist.) (Gt. Barrier Reef Exped.) 1, 259-281.