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Evidence for the origin of the crystalline style enzymes of Telescopium telescopium (Linn.) (Gastropoda)
Comp. Biochem. Physiol., Vol, 65B. pp. 717 to 721
0305-0491/80/0401-0717502.00/0
© Pergamon Press Ltd 1980. Printed in Great Britain
EVIDENCE FOR THE ORIGIN OF THE CRYSTALLINE STYLE ENZYMES OF TELESCOPIUM TELESCOPtUM
(LINN.) (GASTROPODA) DAVID YELLOWLEES Department of Chemistry and Biochemistry, James Cook University of North Queensland, Townsville, Australia (Received 7 August 1979) Abstract--1. The laminarinase and fl-glucosidase activities previously detected in the style are also present in the style sac tissue. 2. The stomach wall also produces laminarinase and fl-glucosidase activities in addition to other polysaccharide degrading enzymes that are found in the style. 3. Evidence is presented that indicates that the laminarinase and fl-glucosidase activities present in the style are the same proteins as occur in the style sac and stomach. 4. The enzymes present in the style are probably secreted from the style sac into the style matrix and these may be supplemented in the proximal area by diffusion, back along the style, of enzymes produced in the stomach wall.
INTRODUCTION
Almost all bivalves and a few gastropods possess a crystalline style associated with their digestive systems. The crystalline style is a hyaline rod which appears to have several functions including the ability to store or transport enzymes for future use in the extracellular digestion of food materials in the stomach. The site of deposition of the style, which is principally proteinaceous in nature (Bailey & Worboys, 1960; Doyle, 1966), has been studied by various workers. Kristensen (1972) concluded on the basis of Trypan Blue staining that addition of new style material in bivalves occurred along the length of the style that was enclosed in the style sac. The actual site of secretion has been suggested by several workers to be a narrow strip of cells along the typhlosole (Bernard, 1973; Fretter & Graham, 1962; Morton, 1952). Driscoll (1972), however, reported that while style material is secreted by tall, slender glandular cells found along the ventral groove of the style sac of the mesogastropod Cerithidea californica, the major site of secretion is at the distal end. Alexander & Rae (1974) concluded that the distal end of the style sac is the sole region of style secretion in Telescopium telescopium. Morton (1952) stated that "a problem is presented concerning the real nature and origin of style enzymes, so strikingly different in mode of occurrence from any other recorded digestive enzymes". Little if any work has been done on the source of these enzymes in the intervening years. It has been assumed that enzyme secretion is in parallel with the secretion of the structural proteins. A previous study detailed the unusual distribution of laminarinase activity along the length of the style (Alexander et al., 1979). The present report details evidence for the source of the digestive enzymes in the crystalline style of T. telescopium.
MATERIALS AND METHODS
Animals Specimens of Telescopium telescopium were collected from mangrove swamps in the vicinity of Townsville, Queensland. Animals were used immediately following collection. Materials Laminarin, fucoidan, p-nitrophenyl fl-D-glucopyranoside and p-nitrophenyl fl-D-galactopyranoside were obtained from Koch-Light Laboratories, Colnbrook, Bucks, England. CM-Cellulose (high viscosity) was purchased from B.D.H. Chemicals Ltd, Poole, England. Hemicellulose (spear grass) was a gift from Professor Richards of this Department, Laminarin was reduced to decrease the reducing power of the substrate (Alexander et al., 1979). All other substrates were laboratory samples prepared by standard methods. Preparation of extracts Style sacs were dissected from T. telescopium and divided into five equal segments each representing twenty per cent of the total length of the style sac. Corresponding segments were pooled. A ciliated section of the stomach, anterior to the gastric shield but remote from the digestive diverticula portals, was also dissected from the same animals. All fractions were washed with 0.2 M phosphate-citrate buffer pH 6.0 before homogenizing in ice-cold buffer (3-5 ml) using a Potter-Elvehjem homogenizer. The resultant fractions were centrifuged at 27,000 g for 10 min at 4°C. The supernatants were used for all further experiments except in the case of stomach samples which had to be dialysed against buffer for 24 hr before use. Extracts from crystalline style were prepared by the method of Alexander et al. (1979). Analytical measurements Reducing sugar was measured by a modified NelsonSomogyi method on one-half of the scale recommended (Robyt & Whelan, 1968), the absorbance being read at 600nm. Protein was measured by a modified Lowry method (Miller, 1959). 717
718
DAVIDYELLOWLEES
Enzyme assays The activity of enzyme fractions towards p-nitrophenyl glycosides was estimated using a digest (225 ,ul) containing either p-nitrophenyl fl-D-glucopyranoside (10mM) or p-nitrophenyl /~-o-galactopyranoside (20mM) in 0.ZM phosphate-citrate buffer pH 5.25. Enzyme (25#1) was added to start the reaction which was carried out at 37°C. The reaction was stopped by the addition of 2 ml of 1 M Na2CO3. The absorbance was read at 400nm. Column fractions were analysed using a final concentration of 1 mM p-nitrophenyl glycoside• fl-l,3-glucanase activity was measured using a digest (1 ml) containing reduced laminarin (4mg), 0.2 M phosphate citrate buffer pH 5.6 (0.3 ml) and enzyme (0.2 ml). This was incubated at 37'C and aliquots (0.2ml) were removed at suitable intervals for estimation of reducing sugar. The activity of enzyme extracts towards other polysaccharide substrates was measured in a digest (1 ml) containing substrate dissolved in 0.2 M phosphate-citrate buffer pH 5.8 (0.8 ml) and enzyme (0.1 ml). The concentration of substrate solutions were fucoidan, 6mg/ml; amylopectin, 6 mg/ml; CM-cellulose, 3 mg/ml; hemicellulose, 6 mg/ml; and xylan, 8 mg/ml. The digest was incubated at 37°C and aliquots (0.2 ml) were removed after 0, 20, 40 and 60 rain for the estimation of reducing power.
I'0 F
(a)
A
t
J t\\
: (c)
lon-exchanye chromatoyraphy Ion-exchange chromatography was performed on a column (12 × 0.9 cm) of DEAE-Sepharose CL-6B equilibrated with 20 mM sodium phosphate buffer pH 7.1. Following application of the sample to the column, the column was washed with buffer followed by a 150ml gradient of 0-0.5 M NaCI in buffer. Fractions (3.5 ml) were collected and analysed for their activity towards reduced laminarin, p-nitrophenyl fl-D-glucopyranoside and p-nitrophenyl fl-o-galactopyranoside.
Purification oJ"enzymes from crystalline styles Glucanase I and II were prepared from the 0-20% segment of crystalline styles from T. telescopium by the method of Cutler & Yellowlees (1979). Purified fl-glucosidase from the same source was purified by ion-exchange chromatography on DEAE-Sepharose CL-6B, gel filtration on Sephadex G-100 and affinity chromatography using the ligand p-aminophenyl fl-D-thioglucopyranoside covalently linked to Sepharose 4B (unpublished results).
RESULTS Enzyme activities of the style, style sac and stomach fractions All fractions were capable of hydrolysing p-nitrophenyl fl-D-galactopyranoside, p-nitrophenyl fl-D-glucopyranoside and reduced laminarin. Results obtained for the hydrolysis of the latter two substrates by the style and the style sac are illustrated in Table 1. The ability of the stomach extract to hydrolyse the polysaccharides laminarin, fucoidan, amylopectin, xylan, hemicellulose and CM-cellulose, and the glycosides p-nitrophenyl fl-o-glucopyranoside and p-nitrophenyl fl-o-galactopyranoside are shown in Table 2.
Ion-exchange chromatoyraphy Extracts of stomach (1 ml), (~20% fraction of the style sac (1 ml) and a mixture of purified glucanase I, glucanase II (Cutler and Yellowlees, 1979) and purified fl-glucosidase (unpublished results) from T. telescopium were fractionated separately on a DEAE-
?2
•
,
A
20
,{L2)
~0.2
!o,
_
lib "~~,-~,
10
£ ~ _ 30 FRACTION NUMBER
• I 50
Fig. 1. Fractionation of a mixture of purified style glucanase I and II and fl-glucosidase Ca), and extracts from style sac (b) and stomach wall (c) on DEAE-Sepharose CL-6B; O O, laminarinase (glucanase) activity; ~-----O, fl-glucosidase activity.
Sepharose CL-6B column. The elution patterns for each of these experiments, which were run in triplicate, are illustrated in Fig. 1. The fraction of each fl-glucosidase/fl-galactosidase peak showing maximum activity was used to measure the ratio of these two activities at saturating substrate concentrations. The ratios are detalied in Fig. 1.
DISCUSSION The crystalline style of both bivalves and gastropods is composed entirely of a protein or glycoprotein get and does not possess any cellular structure. The hydrolytic enzymes present in the style must therefore be synthesised and secreted by cells in associated tissues. Previous investigations have not studied this problem but have been restricted to the occurrence and level of enzyme activity in the style itself. Results in this communication show that in 7". telescopium, fl-glucosidase, fl-galactosidase and laminarinase activities are present in both the style and style sac. Table 1 shows that these enzymes occur
719
Evidence for the origin of the crystalline style enzymes Table 1. Percentages of the total fl-glucosidase and laminarinase activity along the style sac from the stomach (0-20) to the distal end (80-100) Laminarinase
8-Glucosidase SEGMENT
0 - 20
Style
Style
sac
Style
Style
sac
65
38
81
48
20
-
40
16
14
8
17
40
- 60
i0
20
2
17
60
-
4
14
3
7
80
- 100
4
14
6
ii
80
along the whole length of the two tissues. The level of activity in the style sac is in fact much greater than in the style as evidenced by the activities of laminarinase and fl-glucosidase presented in Table 2, and their proportional levels along the style and style sac as detailed in Table 1. This would appear to confirm that the enzymes in the style are derived at least in part from the adjacent style sac. This was suggested by Alexander & Rae (1974) who found secretory centres along the typhlosole and ventral groove of the style sac of T. telescopium that were not involved in the deposition of the structural components of the style. Alexander et al. (1979) have previously reported the unusual distribution of laminarinase along the length of the style. This experiment was repeated, but the
respective style sac segments were pooled in addition to the corresponding ones from the style. Table 1 shows that these two enzymes are distributed along the whole length of the style and style sac. The distribution occurring in the style is also evident in the style sac, however the activity in the 0-20~o segment in the sac is not as elevated in relation to the other segments as it is in the style. All style sac segments possess more activity than the corresponding style segments indicating that there may be sufficient amounts of enzyme in the style sac to account for the enzyme levels in the style. Previous results have shown that proteins can diffuse laterally into the style (Alexander et al., 1979). The possibility that the high level of activity in the style segment adjacent to the stomach could in part
Table 2. Activity of stomach, style and style sac (0-20~ segment) extracts towards polysaccharide and glycoside substrates ACTIVITY (lag glucose equivalents/hr/mg
SUBSTRATE
STOMACH
STYLE
protein)
STYLE (0-20%
SAC
Segment)
Laminarin
683
65
425
Fucoidan
105
85
-
CM-Cellulose
21
26
-
Amylopectin
23
45
-
Xylan
45
23
-
Hemicellulose
46
25
-
0-Nitrophenyl 8-Dglucopyranoside
547
-
189
p-Nitrophenyl 8-Dgalactopyranoside
432
-
160
720
DAVID YELLOWLEES
be derived from diffusion of enzymes from the stomach contents was investigated. Measurement of activity towards both laminarin and p-nitrophenyl fl-D-glucopyranoside revealed that the specific activity of these two enzymes in the stomach wall was greater than in either the style or style sac (Table 2). The activity of the stomach wall extract towards other polysaccharide substrates, that were hydrolysed by T. telescopium style preparations (Alexander et al., 1979), was also checked. While the activity was not as great as that towards either laminarin or p-nitrophenyl fl-Dglucopyranoside, significant levels of activity were detected in all cases. Since the stomach wall preparation was not contaminated by either style sac or digestive gland tissue these enzymes must be synthesized in the stomach wall itself. On present evidence no conclusion as to whether they are involved in intracellular or extracellular digestion can be drawn. However, even if their role is intracellular rather than extracellular, leakage would still occur leading to elevated levels in the stomach fluid which could contribute to the high levels of laminarinase and fl-glucosidase occurring in the proximal end of the style. The tissue used for this experiment was taken from an area of the stomach wall anterior to the gastric shield and style sac and so enzymes produced in this region would come into contact with the style tip in their passage from the stomach into the midgut. There are few reports of digestive enzymes in the stomach walls of molluscs although detailed histological studies have been made. Laminarinase among other carbohydrases has however been detected in the stomach wall of the crustacean Balanus nubilis (Harnden, 1968) and Hashimoto & Onoma (1949) reported the existence of several polysaccharide degrading enzymes in the gastric teeth of the gastropod Dolabella scapula that were also present in the organism's crystalline style. Morton & Stone (1958) also found evidence for various polysaccharide and glycoside hydrolysing enzymes in the stomach walls of molluscs. Much evidence is still required as to the extent of the occurrence of digestive enzymes in the stomach and their role in digestion. The occurrence of laminarinase and fl-glucosidase activities in the stomach wall, style sac and style does not necessarily indicate that they are the same proteins. Sufficient quantities of pure enzymes were not available for the preparation of specific antibodies to test this proposition, however other evidence indicated that this might be the case. Chromatography of the various extracts on DEAE-Sepharose CL-6B using a 04).5 M NaCI gradient showed that in each case the laminarinase activity was separated into two distinct peaks of activity (Fig. 1). These two peaks had identical mobilities to those of the purified style enzymes, glucanase I and II from 7". telescopium (Cutler & Yellowlees, 1979). In addition both the style sac and stomach wall extracts possessed a fl-glucosidase (A) activity that had the same mobility as that of the purified fl-glucosidase/fl-galactosidase from the style of T. telescopium. In each case a fl-galactosidase activity migrated at the same rate as the fl-glucosidase peak. The stomach wall extract also possessed another fl-glucosidase peak (B) that was not evident in either style or style sac extracts. The enzymes present in the style and style sac appear therefore to be
similar at least in charge to those of the stomach wall. Further evidence for the identity of the enzymes in the various tissues is provided from the ratio of the fl-glucosidase/fl-galactosidase activities in the fl-glucosidase peak (A). The purified fl-glucosidase from the style of T. telescopium can hydrolyse both p-nitrophenyl //-Dglucopyranoside and p-nitrophenyl fl-o-galactopyranoside. The ratio of the two activities is 1.2 both during purification and in the purified enzyme itself (unpublished results). The ratio of the activities in the fractions showing maximal activity following ionexchange chromatography (Fig. 1) was 1.2 in each extract. Although the purified enzyme was prepared from the 0-20~o segment a comparison of the ratios of the fl-glucosidase/fl-galactosidase along the length of the style gave a value between 1.1 and 1.2 in all cases. The fl-glucosidase peak (B) in the stomach wall extract does not exactly co-migrate with the fl-galactosidase activity. A ratio of 1.9 for the tube with the maximum fl-glucosidase activity was obtained. Unlike the other fl-glucosidase/fl-galactosidase activity it is probable that these two fl-glycosidase activities are not the same protein. In conclusion it would appear that secretion of enzymes from the style sac could account for the laminarinase and fl-glucosidase activity found in the style. It is probable, however, that the high activity of these enzymes found in the 0-20~o segment of the style is contributed to by diffusion of enzymes back into the style from the stomach fluid. Acknowledoements The author is grateful to Dr Chris Alexander for the careful dissections of the ,~tyle sac and stomach and to Mr Robert Cutler for helpful discussions. REFERENCES
ALEXANDERC. G., CUTLER R. L. & YELLOWLEESD. (1979) Studies on the composition and enzyme content of the crystalline style of Telescopium telescopium (Linn.) (Gastropoda). Comp. Biochem. Physiol. 64B, 83-89. ALEXANDERC. G. & RAE J. C. (1974) The structure and formation of the crystalline style of Telescopium telescopium (Linn.) (Gastropoda: Prosobranchia). Veliger 17, 56--60. BAILEY K. & WORBOYS B. D. (1960) The lamellibranch crystalline style. Biochem. J. 76, 487~,91. BERNARDF. R. (1973) Crystalline style formation and function in the oyster Crassostrea gioas (Thunberg, 1795). Ophelia 12, 159-170. CUTLER R. L. & YELLOWLEESD. (1979) Purification and characterisation of two endo-(1 ~3)-fl-D-glucanases from Telescopium telescopium. Carbohydr. Res. 75, 221-229. DOYLEJ. (1966) Studies on the chemical nature of the crystalline style. In Some Contemporary Studies in Marine Science (Edited by BARNES H.) pp. 253-263. Allen & Unwin, London. DRISCOLL A. L. (1972) Structure and function of the alimentary tract of Batillaria zonalis and Cerithidea californica, style-bearing mesogastropods. Velioer 14, 375-386. FRETTER V. & GRAHAMA. (1962) In British Prosobranch Molluscs. The Ray Society, London. HARNDEND. G. (1968) Digestive carbohydrases of Balanus nubilis. Comp. Biochem. Physiol. 25, 303-309. HASHIMOTO Y. & ONOMA K. (1949) On the digestion of higher carbohydrates by mollusca (Dolabella scapula and Teredo sp.). Bull. Jap. Soc. scient. Fish. 15, 253-258. KRISTENSENJ. H. (1972) Structure and function of crystalline styles of bivalves. Ophelia 10, 91-108.
Evidence for the origin of the crystalline style enzymes MILLER G. L. (1959) Protein determination for large numbers of samples. Analyt. Chem. 31, 964. MORTON J. E. (1952) The role of the crystalline style. Proc. malac. Soc. Lond. 29, 85-92. MORTON J. E. & STONE B. A. (1958) The distribution of
721
cellulases and related enzymes in mollusca. Proc. malac. Soc. Lond. 33, 127-141. ROBYT J. F. & WnELAN W. J. (1968) The or-amylases. In Starch and its Derivatives 4th edn (Edited by RADLEY J. A.) pp. 431-433. Chapman & Hall, London.
0305-0491/80/0401-0717502.00/0
© Pergamon Press Ltd 1980. Printed in Great Britain
EVIDENCE FOR THE ORIGIN OF THE CRYSTALLINE STYLE ENZYMES OF TELESCOPIUM TELESCOPtUM
(LINN.) (GASTROPODA) DAVID YELLOWLEES Department of Chemistry and Biochemistry, James Cook University of North Queensland, Townsville, Australia (Received 7 August 1979) Abstract--1. The laminarinase and fl-glucosidase activities previously detected in the style are also present in the style sac tissue. 2. The stomach wall also produces laminarinase and fl-glucosidase activities in addition to other polysaccharide degrading enzymes that are found in the style. 3. Evidence is presented that indicates that the laminarinase and fl-glucosidase activities present in the style are the same proteins as occur in the style sac and stomach. 4. The enzymes present in the style are probably secreted from the style sac into the style matrix and these may be supplemented in the proximal area by diffusion, back along the style, of enzymes produced in the stomach wall.
INTRODUCTION
Almost all bivalves and a few gastropods possess a crystalline style associated with their digestive systems. The crystalline style is a hyaline rod which appears to have several functions including the ability to store or transport enzymes for future use in the extracellular digestion of food materials in the stomach. The site of deposition of the style, which is principally proteinaceous in nature (Bailey & Worboys, 1960; Doyle, 1966), has been studied by various workers. Kristensen (1972) concluded on the basis of Trypan Blue staining that addition of new style material in bivalves occurred along the length of the style that was enclosed in the style sac. The actual site of secretion has been suggested by several workers to be a narrow strip of cells along the typhlosole (Bernard, 1973; Fretter & Graham, 1962; Morton, 1952). Driscoll (1972), however, reported that while style material is secreted by tall, slender glandular cells found along the ventral groove of the style sac of the mesogastropod Cerithidea californica, the major site of secretion is at the distal end. Alexander & Rae (1974) concluded that the distal end of the style sac is the sole region of style secretion in Telescopium telescopium. Morton (1952) stated that "a problem is presented concerning the real nature and origin of style enzymes, so strikingly different in mode of occurrence from any other recorded digestive enzymes". Little if any work has been done on the source of these enzymes in the intervening years. It has been assumed that enzyme secretion is in parallel with the secretion of the structural proteins. A previous study detailed the unusual distribution of laminarinase activity along the length of the style (Alexander et al., 1979). The present report details evidence for the source of the digestive enzymes in the crystalline style of T. telescopium.
MATERIALS AND METHODS
Animals Specimens of Telescopium telescopium were collected from mangrove swamps in the vicinity of Townsville, Queensland. Animals were used immediately following collection. Materials Laminarin, fucoidan, p-nitrophenyl fl-D-glucopyranoside and p-nitrophenyl fl-D-galactopyranoside were obtained from Koch-Light Laboratories, Colnbrook, Bucks, England. CM-Cellulose (high viscosity) was purchased from B.D.H. Chemicals Ltd, Poole, England. Hemicellulose (spear grass) was a gift from Professor Richards of this Department, Laminarin was reduced to decrease the reducing power of the substrate (Alexander et al., 1979). All other substrates were laboratory samples prepared by standard methods. Preparation of extracts Style sacs were dissected from T. telescopium and divided into five equal segments each representing twenty per cent of the total length of the style sac. Corresponding segments were pooled. A ciliated section of the stomach, anterior to the gastric shield but remote from the digestive diverticula portals, was also dissected from the same animals. All fractions were washed with 0.2 M phosphate-citrate buffer pH 6.0 before homogenizing in ice-cold buffer (3-5 ml) using a Potter-Elvehjem homogenizer. The resultant fractions were centrifuged at 27,000 g for 10 min at 4°C. The supernatants were used for all further experiments except in the case of stomach samples which had to be dialysed against buffer for 24 hr before use. Extracts from crystalline style were prepared by the method of Alexander et al. (1979). Analytical measurements Reducing sugar was measured by a modified NelsonSomogyi method on one-half of the scale recommended (Robyt & Whelan, 1968), the absorbance being read at 600nm. Protein was measured by a modified Lowry method (Miller, 1959). 717
718
DAVIDYELLOWLEES
Enzyme assays The activity of enzyme fractions towards p-nitrophenyl glycosides was estimated using a digest (225 ,ul) containing either p-nitrophenyl fl-D-glucopyranoside (10mM) or p-nitrophenyl /~-o-galactopyranoside (20mM) in 0.ZM phosphate-citrate buffer pH 5.25. Enzyme (25#1) was added to start the reaction which was carried out at 37°C. The reaction was stopped by the addition of 2 ml of 1 M Na2CO3. The absorbance was read at 400nm. Column fractions were analysed using a final concentration of 1 mM p-nitrophenyl glycoside• fl-l,3-glucanase activity was measured using a digest (1 ml) containing reduced laminarin (4mg), 0.2 M phosphate citrate buffer pH 5.6 (0.3 ml) and enzyme (0.2 ml). This was incubated at 37'C and aliquots (0.2ml) were removed at suitable intervals for estimation of reducing sugar. The activity of enzyme extracts towards other polysaccharide substrates was measured in a digest (1 ml) containing substrate dissolved in 0.2 M phosphate-citrate buffer pH 5.8 (0.8 ml) and enzyme (0.1 ml). The concentration of substrate solutions were fucoidan, 6mg/ml; amylopectin, 6 mg/ml; CM-cellulose, 3 mg/ml; hemicellulose, 6 mg/ml; and xylan, 8 mg/ml. The digest was incubated at 37°C and aliquots (0.2 ml) were removed after 0, 20, 40 and 60 rain for the estimation of reducing power.
I'0 F
(a)
A
t
J t\\
: (c)
lon-exchanye chromatoyraphy Ion-exchange chromatography was performed on a column (12 × 0.9 cm) of DEAE-Sepharose CL-6B equilibrated with 20 mM sodium phosphate buffer pH 7.1. Following application of the sample to the column, the column was washed with buffer followed by a 150ml gradient of 0-0.5 M NaCI in buffer. Fractions (3.5 ml) were collected and analysed for their activity towards reduced laminarin, p-nitrophenyl fl-D-glucopyranoside and p-nitrophenyl fl-o-galactopyranoside.
Purification oJ"enzymes from crystalline styles Glucanase I and II were prepared from the 0-20% segment of crystalline styles from T. telescopium by the method of Cutler & Yellowlees (1979). Purified fl-glucosidase from the same source was purified by ion-exchange chromatography on DEAE-Sepharose CL-6B, gel filtration on Sephadex G-100 and affinity chromatography using the ligand p-aminophenyl fl-D-thioglucopyranoside covalently linked to Sepharose 4B (unpublished results).
RESULTS Enzyme activities of the style, style sac and stomach fractions All fractions were capable of hydrolysing p-nitrophenyl fl-D-galactopyranoside, p-nitrophenyl fl-D-glucopyranoside and reduced laminarin. Results obtained for the hydrolysis of the latter two substrates by the style and the style sac are illustrated in Table 1. The ability of the stomach extract to hydrolyse the polysaccharides laminarin, fucoidan, amylopectin, xylan, hemicellulose and CM-cellulose, and the glycosides p-nitrophenyl fl-o-glucopyranoside and p-nitrophenyl fl-o-galactopyranoside are shown in Table 2.
Ion-exchange chromatoyraphy Extracts of stomach (1 ml), (~20% fraction of the style sac (1 ml) and a mixture of purified glucanase I, glucanase II (Cutler and Yellowlees, 1979) and purified fl-glucosidase (unpublished results) from T. telescopium were fractionated separately on a DEAE-
?2
•
,
A
20
,{L2)
~0.2
!o,
_
lib "~~,-~,
10
£ ~ _ 30 FRACTION NUMBER
• I 50
Fig. 1. Fractionation of a mixture of purified style glucanase I and II and fl-glucosidase Ca), and extracts from style sac (b) and stomach wall (c) on DEAE-Sepharose CL-6B; O O, laminarinase (glucanase) activity; ~-----O, fl-glucosidase activity.
Sepharose CL-6B column. The elution patterns for each of these experiments, which were run in triplicate, are illustrated in Fig. 1. The fraction of each fl-glucosidase/fl-galactosidase peak showing maximum activity was used to measure the ratio of these two activities at saturating substrate concentrations. The ratios are detalied in Fig. 1.
DISCUSSION The crystalline style of both bivalves and gastropods is composed entirely of a protein or glycoprotein get and does not possess any cellular structure. The hydrolytic enzymes present in the style must therefore be synthesised and secreted by cells in associated tissues. Previous investigations have not studied this problem but have been restricted to the occurrence and level of enzyme activity in the style itself. Results in this communication show that in 7". telescopium, fl-glucosidase, fl-galactosidase and laminarinase activities are present in both the style and style sac. Table 1 shows that these enzymes occur
719
Evidence for the origin of the crystalline style enzymes Table 1. Percentages of the total fl-glucosidase and laminarinase activity along the style sac from the stomach (0-20) to the distal end (80-100) Laminarinase
8-Glucosidase SEGMENT
0 - 20
Style
Style
sac
Style
Style
sac
65
38
81
48
20
-
40
16
14
8
17
40
- 60
i0
20
2
17
60
-
4
14
3
7
80
- 100
4
14
6
ii
80
along the whole length of the two tissues. The level of activity in the style sac is in fact much greater than in the style as evidenced by the activities of laminarinase and fl-glucosidase presented in Table 2, and their proportional levels along the style and style sac as detailed in Table 1. This would appear to confirm that the enzymes in the style are derived at least in part from the adjacent style sac. This was suggested by Alexander & Rae (1974) who found secretory centres along the typhlosole and ventral groove of the style sac of T. telescopium that were not involved in the deposition of the structural components of the style. Alexander et al. (1979) have previously reported the unusual distribution of laminarinase along the length of the style. This experiment was repeated, but the
respective style sac segments were pooled in addition to the corresponding ones from the style. Table 1 shows that these two enzymes are distributed along the whole length of the style and style sac. The distribution occurring in the style is also evident in the style sac, however the activity in the 0-20~o segment in the sac is not as elevated in relation to the other segments as it is in the style. All style sac segments possess more activity than the corresponding style segments indicating that there may be sufficient amounts of enzyme in the style sac to account for the enzyme levels in the style. Previous results have shown that proteins can diffuse laterally into the style (Alexander et al., 1979). The possibility that the high level of activity in the style segment adjacent to the stomach could in part
Table 2. Activity of stomach, style and style sac (0-20~ segment) extracts towards polysaccharide and glycoside substrates ACTIVITY (lag glucose equivalents/hr/mg
SUBSTRATE
STOMACH
STYLE
protein)
STYLE (0-20%
SAC
Segment)
Laminarin
683
65
425
Fucoidan
105
85
-
CM-Cellulose
21
26
-
Amylopectin
23
45
-
Xylan
45
23
-
Hemicellulose
46
25
-
0-Nitrophenyl 8-Dglucopyranoside
547
-
189
p-Nitrophenyl 8-Dgalactopyranoside
432
-
160
720
DAVID YELLOWLEES
be derived from diffusion of enzymes from the stomach contents was investigated. Measurement of activity towards both laminarin and p-nitrophenyl fl-D-glucopyranoside revealed that the specific activity of these two enzymes in the stomach wall was greater than in either the style or style sac (Table 2). The activity of the stomach wall extract towards other polysaccharide substrates, that were hydrolysed by T. telescopium style preparations (Alexander et al., 1979), was also checked. While the activity was not as great as that towards either laminarin or p-nitrophenyl fl-Dglucopyranoside, significant levels of activity were detected in all cases. Since the stomach wall preparation was not contaminated by either style sac or digestive gland tissue these enzymes must be synthesized in the stomach wall itself. On present evidence no conclusion as to whether they are involved in intracellular or extracellular digestion can be drawn. However, even if their role is intracellular rather than extracellular, leakage would still occur leading to elevated levels in the stomach fluid which could contribute to the high levels of laminarinase and fl-glucosidase occurring in the proximal end of the style. The tissue used for this experiment was taken from an area of the stomach wall anterior to the gastric shield and style sac and so enzymes produced in this region would come into contact with the style tip in their passage from the stomach into the midgut. There are few reports of digestive enzymes in the stomach walls of molluscs although detailed histological studies have been made. Laminarinase among other carbohydrases has however been detected in the stomach wall of the crustacean Balanus nubilis (Harnden, 1968) and Hashimoto & Onoma (1949) reported the existence of several polysaccharide degrading enzymes in the gastric teeth of the gastropod Dolabella scapula that were also present in the organism's crystalline style. Morton & Stone (1958) also found evidence for various polysaccharide and glycoside hydrolysing enzymes in the stomach walls of molluscs. Much evidence is still required as to the extent of the occurrence of digestive enzymes in the stomach and their role in digestion. The occurrence of laminarinase and fl-glucosidase activities in the stomach wall, style sac and style does not necessarily indicate that they are the same proteins. Sufficient quantities of pure enzymes were not available for the preparation of specific antibodies to test this proposition, however other evidence indicated that this might be the case. Chromatography of the various extracts on DEAE-Sepharose CL-6B using a 04).5 M NaCI gradient showed that in each case the laminarinase activity was separated into two distinct peaks of activity (Fig. 1). These two peaks had identical mobilities to those of the purified style enzymes, glucanase I and II from 7". telescopium (Cutler & Yellowlees, 1979). In addition both the style sac and stomach wall extracts possessed a fl-glucosidase (A) activity that had the same mobility as that of the purified fl-glucosidase/fl-galactosidase from the style of T. telescopium. In each case a fl-galactosidase activity migrated at the same rate as the fl-glucosidase peak. The stomach wall extract also possessed another fl-glucosidase peak (B) that was not evident in either style or style sac extracts. The enzymes present in the style and style sac appear therefore to be
similar at least in charge to those of the stomach wall. Further evidence for the identity of the enzymes in the various tissues is provided from the ratio of the fl-glucosidase/fl-galactosidase activities in the fl-glucosidase peak (A). The purified fl-glucosidase from the style of T. telescopium can hydrolyse both p-nitrophenyl //-Dglucopyranoside and p-nitrophenyl fl-o-galactopyranoside. The ratio of the two activities is 1.2 both during purification and in the purified enzyme itself (unpublished results). The ratio of the activities in the fractions showing maximal activity following ionexchange chromatography (Fig. 1) was 1.2 in each extract. Although the purified enzyme was prepared from the 0-20~o segment a comparison of the ratios of the fl-glucosidase/fl-galactosidase along the length of the style gave a value between 1.1 and 1.2 in all cases. The fl-glucosidase peak (B) in the stomach wall extract does not exactly co-migrate with the fl-galactosidase activity. A ratio of 1.9 for the tube with the maximum fl-glucosidase activity was obtained. Unlike the other fl-glucosidase/fl-galactosidase activity it is probable that these two fl-glycosidase activities are not the same protein. In conclusion it would appear that secretion of enzymes from the style sac could account for the laminarinase and fl-glucosidase activity found in the style. It is probable, however, that the high activity of these enzymes found in the 0-20~o segment of the style is contributed to by diffusion of enzymes back into the style from the stomach fluid. Acknowledoements The author is grateful to Dr Chris Alexander for the careful dissections of the ,~tyle sac and stomach and to Mr Robert Cutler for helpful discussions. REFERENCES
ALEXANDERC. G., CUTLER R. L. & YELLOWLEESD. (1979) Studies on the composition and enzyme content of the crystalline style of Telescopium telescopium (Linn.) (Gastropoda). Comp. Biochem. Physiol. 64B, 83-89. ALEXANDERC. G. & RAE J. C. (1974) The structure and formation of the crystalline style of Telescopium telescopium (Linn.) (Gastropoda: Prosobranchia). Veliger 17, 56--60. BAILEY K. & WORBOYS B. D. (1960) The lamellibranch crystalline style. Biochem. J. 76, 487~,91. BERNARDF. R. (1973) Crystalline style formation and function in the oyster Crassostrea gioas (Thunberg, 1795). Ophelia 12, 159-170. CUTLER R. L. & YELLOWLEESD. (1979) Purification and characterisation of two endo-(1 ~3)-fl-D-glucanases from Telescopium telescopium. Carbohydr. Res. 75, 221-229. DOYLEJ. (1966) Studies on the chemical nature of the crystalline style. In Some Contemporary Studies in Marine Science (Edited by BARNES H.) pp. 253-263. Allen & Unwin, London. DRISCOLL A. L. (1972) Structure and function of the alimentary tract of Batillaria zonalis and Cerithidea californica, style-bearing mesogastropods. Velioer 14, 375-386. FRETTER V. & GRAHAMA. (1962) In British Prosobranch Molluscs. The Ray Society, London. HARNDEND. G. (1968) Digestive carbohydrases of Balanus nubilis. Comp. Biochem. Physiol. 25, 303-309. HASHIMOTO Y. & ONOMA K. (1949) On the digestion of higher carbohydrates by mollusca (Dolabella scapula and Teredo sp.). Bull. Jap. Soc. scient. Fish. 15, 253-258. KRISTENSENJ. H. (1972) Structure and function of crystalline styles of bivalves. Ophelia 10, 91-108.
Evidence for the origin of the crystalline style enzymes MILLER G. L. (1959) Protein determination for large numbers of samples. Analyt. Chem. 31, 964. MORTON J. E. (1952) The role of the crystalline style. Proc. malac. Soc. Lond. 29, 85-92. MORTON J. E. & STONE B. A. (1958) The distribution of
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cellulases and related enzymes in mollusca. Proc. malac. Soc. Lond. 33, 127-141. ROBYT J. F. & WnELAN W. J. (1968) The or-amylases. In Starch and its Derivatives 4th edn (Edited by RADLEY J. A.) pp. 431-433. Chapman & Hall, London.