A comparative study of the digestive enzymes in the hepatopancreas of Jonah crab (Cancer borealis) and rock crab (Cancer irroratus)

Comp. Biochem. Physiol.. 1976, Vol. 53B, pp. 387 to 391. Pergamon Press. Printed in Great Britain

A COMPARATIVE STUDY OF THE DIGESTIVE ENZYMES IN THE HEPATOPANCREAS OF JONAH CRAB (CANCER BOREALIS) A N D ROCK CRAB (CANCER IRRORATUS) G. L. BRUN AND M. B. WOJTOWICZ Department of the Environment, Fisheries and Marine Service, Halifax Laboratory, P.O. Box 429, Halifax, Nova Scotia, Canada (Received 13 November 1974) A b s t r a c t - - 1 . In general, specific activities of enzymes were greater in the hepatopancreas of Cancer irroratus than in Cancer borealis. Optimum pH activity of each enzyme was similar for both species. 2. Two proteinases similar in optimum pH (3.8 and 8-0) to those in gastric juice of Homarus americanus were found. Molecular weights were estimated at 16,700 and 20,500 for C. borealis and C. irroratus respectively.

3. Among the carbohydrases, chitobiase was most predominant with a specific activity of 0.4 U/mg protein in lyophylised powder extract of C. irroratus. An unusually high level of fl-galactosidase was found compared to other forms of Crustacea. There was also a considerable difference in molecular weights of ~-glucosidase between C. borealis (47,000) and C. irroratus (86,000). 4. In contrast to the American lobster, the hepatopancreas is the apparent source of production of certain esterases in C. irroratus and C. borealis.

INTRODUCTION

Preparation of the material for analysis and enzyme assays

little information on digestive enzymes in the crustaceans Cancer irroratus (Rock crab) and Cancer borealis (Jonah crab) exists in the literature. Telford (1970) compared semi-quantitatively carbohydrase activities in hepatopancreas of these two crabs. Hiltz & Lightle (1970) reported activities of fl-glucuronidase and arylsulphatase in digestive gland of C. irroratus. A more detailed investigation of digestive enzymes of other species of crabs was carried out by Ito & Saito (1963), Nagabhushanam & Sarotini (1968), Sather (1969) and Eisen & Jeffrey (1969). DeVillez & Buschlen (1967) described tryptic digestive enzymes in the hepatopancreas of numerous Crustacea; a caseinolytic and esterolytic activity was found in all amphipod, isopod and decapod species. Another comparative study of proteinases by Kozlovskaya & Vaskovsky (1970) in marine invertebrates showed that many Crustacea and Asteroidea possessed a high activity tested on hemoglobin or casein. Because of the renewed interest in the utilization of crabs in the fishing industry, brought about by mechanized methods of meat separation, it became important to study digestive hydrolases in two closely related animals of potential commercial importance, C. irroratus and C. borealis.

Procedures for preparation of extracts for analysis from fresh hepatopancreas, acetone powder and lyophilized powder, methods for carbohydrases assays and protein determination have been described previously (Wojtowicz, 1972). Summarized methods for activity tests are given in Table 1. The optimal pH values, determined from pH/activity curves (see Fig. 1), were used in particular enzyme testing. Esterases were estimated by automatic potentiometric titrations; pH given in the table were used as their respective end points. Results for various enzymes were expressed in units as follows: amylase, 1 U = 1 #mole maltose liberated per min; carbohydrases and phosphatases tested on p-nitrophenyl substrates, l U = 1 ttmole p-nitrophenol per min; esterases (chymotrypsin, trypsin and tributyrinase), 1 U = 1 m-equiv. NaOH used for titration per min; deoxyribonuclease and ribonuclease, 1 U = increase of OD26 o per min.

VERY

MATERIALS A N D M E T H O D S

Gel filtration

Dextran Sephadex G-200 (Pharmacia, Uppsala) and polyacrylamide Bio-Gel P-100 (Bio-Rad Laboratories, Richmond, California) were used in columns measuring 16 Acifl phosphatase

Amylase

Chltoblase Deccyribonuclease I~-OalactosJdase

~ o[ . . . . . . . E

ct -G)ucosidase

JS-Glucosidase Proteinase {Hb)

Pr~einase (casein)

Rllx)nucleese

The crabs were caught in inshore Nova Scotia waters (Halifax Harbour area) during February and March, and were kept without feeding in tanks of running sea water at the temperature of 9.12°C for 1-2 weeks, until used. The hepatopancreas was excised from 30 live, mature 3 5 7 3 5 7 4 6 4 6 3 5 1 male crabs, pooled, frozen immediately over dry ice and pH used within 30 min. Care was taken not to contaminate Fig. 1. pH activity curves for hepatopancreatic enzymes the sample (15(~200g) with body fluids. in Cancer irroratus. 387

i.!

388

G. L. BRuy AN[) M. B. WOJTOWICZ TABLE 1--ENZYMES AND ASSAY PROCEDURES Classification No.

Enzyme Amylase

3.2.2.1

Substrate

Temperature fmC)

pH

1% soluble starch 0.02 M phosphate buffer

Reference

7.0

25

Bernfeld

(1955)

3.6 5.5 4.7 5.5 5.0

30 30 30 30 30

Harris et al. (1966) Wojtowicz (1972)

0.95 M NacI

Acid phosphatase a-Glucosldase 8-Glucosldase ~-Galactosidase Chitobiase

3.1.3.2 3.2.1.20 3.2.1.21 3.2.1.23 3.2.1.29

Ribonuclease

2.7.7.16

1% ribonucleic acid 0.i M acetate buffer

5.0

37

Kalnitzky

Deoxyribonuclease

3.1.4.5

0.004% highly poly~erized DNA in 0.1 M acetate buffer, 0.005 M MgSO 4

5.0

30

Russell et al. (1964)

37 37

Continuous titration (Prahl & Neurath, 1966 a,b)

(p-nitrophenyl substrates ) *

(1959)

Chymotrypsin Trypsin

3.4.4.5 3.4.4.4

0.01 M ATEE, 0.i M KCI 0.01 M BAEE, 0.1 M KC1 or 0.01 M TAME, 0.01 M CaCI 2

7.8 7.9 8.1

3.7

Trlbutyrinase

3.1.1.3

2% tributyrin in 0.i M CaCI 2

6.5

37

Brockerhoff ef aJ, (1970)

1.5% hemoglobin 0.i M acetate buffer

3.8

35

Wojtowicz & Odense (1970)

8.0

35

3.8

35

8.0

35

Proteinases

or 1.5% hemoglobin 3 M ~ r e a , 0.i M sodium phosphate buffer or 1% casein, 0.2 M citrate buffer or 1% casein, 0.2 M citratephosphate buffer

Laskowskl

(1955)

* Abbreviat/ons used: D~% - deoxyribonucleic acid; ATEE = N-acetyl-L-tyrosine ethyl ester; BAEE = benzoyl-Larglnine ethyl ester hydrochloride; TAM~ = p-tosyl-L-arginlne methyl ester hydrochloride.

× 700 mm. The bed was supported by flow adaptors and upward-flow elution was applied at the speed of 6 or 8 ml/hr. The temperature of the separating system was maintained at 10-1YC. Ammonium acetate 0-1 M was used as the eluant. The columns were calibrated for mol. wt estimation of enzymes with following markers (Pharmacia. Uppsala): cytochrome c --12,400; myoglobin--17,800: chymotrypsinogen 25,000; ovalbumin 45,000; albumin-67,000; ~-globulin 160,000. Dextran-blue (Pharmacia, Uppsala) was used for determining void vol.

Samples consisting of 100 mg lyophilized powder dissolved in 3 ml of buffer were applied to the column. Fractions were collected at intervals of 30 min,

RESULTS The p H of the fresh hepatopancreas varied between 6.60 and 6-80 in both species. Enzyme activities were determined in fresh tissue extract, in acetone powder

TABLE 2--AVERAGE SPECIFIC ACTIVITY OF SOME DIGESTIVE ENZYMES IN HEPATOPANCREAS OF CANCER IRRORATUS AND CANCER BOREALIS

Acetone powder u x 10-3/mg

Enzyme

C. borealis

Acid phosphatase Amylase Chitoblase

C. irroratus

C. borealis

C. irroratus

5

12

17

36

120

240

450

930

90

230

120

400

~aymotrypsin

traces

Deoxyribonucle ase

150

190

8-Galactosldase

6

8-Glucosidase

8

~-Gluoosidase

1

Proteinase

Lyophylised~der U x 10-3/mg

0.30

0.21

0.80

250

330

7

12

30

9

i0

16

1

5

4

(pH 3.8/~)

103

25

40 250

Proteinase

(pS 8.0/~)

300

70

Proteinase

(pH 3.8/casein)

18

5

15

Proteinase

(pH 8.0/casein)

133

30

60

1140

410

3860

Ribonuc leas e

190

Tributyrinase

1.60

0.17

2. ii

Trypsin

(BAEE)

0.88

3.98

i.ii

Trypsin

(TAME)

3.46

25.6

3.25

0.29 7.21 32.9

Digestive enzymes in the hepatopancreas of Jonah and rock crabs 100 ] l"

o[

TABLE 3-"NOIJL'UIJ~ WEZ~aT OF ~

~.:'~ ..........E~erase (BAEE) ~ ~rk I~-Galactosldase

,

,

,

389 ESTIMATED

ON 8EPBADEX G-200

mat~/t~

"'~\

CanQer irroratuJ

30 40 50 60 Temp. °C

Fig. 2. Stability of enzyme extracts (Cancer irroratus), when preheated for 20 min at various temperatures prior to activity assays.

(a.p.) extract and in solutions of lyophylized protein (1.p.). The preliminary results showed that no significant loss of activity occurred during the preparation of powders; consequently 1.p. was used for more detailed assays. Table 2 summarizes the specific activities obtained on a.p. and 1.p. extracts. An amylase ranging in activity between 0.120.93 U/mg was found in both species of crab. Among the enzymes acting on p-nitrophenyl substrates, chitobiase in C. irroratus exhibited the strongest activity, 0.4 U/mg in l.p. The lowest carbohydrase activity found was for ct-glucosidase, 0.001 U/mg in a.p. of C. borealis. Activities for acid phosphatase, fl-galactosidase and fl-glucosidase ranged between 0-005 and 0.036U/rag. No alkaline phosphatase could be detected in hepatopancreas of either species. In most cases the results were higher in dialysed and freeze-dried preparations (1.p.) than in acetone powder, possibly because of the removal of dialysable compounds which may have enhanced enzyme activities in the a.p. Ribonuclease in C. irroratus showed more than a 3-fold increase in activity for l.p. over the a.p. extract. Deoxyribonuclease activity in 1.p. was double that in a.p. for both species. In general, activities were higher for the hepatopancreas of C. irroratus than for the other crab. Tributyrinase was the only hydrolase, for which the results for C. borealis were greater than in C. irroratus. Figure 1 illustrates the pH/activity curves of the various enzymes in C. irroratus hepatopancreas. The

Canoer Ix)realls

C~ttaoilule

110,000

125,000

B-Galact~l£~se

115,000

121t000

~ L d l)hOSphatale

91,000

104,000

;-Gluooll#-~le

86,000

47,000

B-G1~o61~um

51,000

44,000

mmae

38,000

29,000

F4Ome

30,000

22,500

Trypsin (TAm)

20,500

16,700

• z~r~si n (BAR1)

20,500

16,700

a-Ch~trypsln

17,500

optimum pH of enzymes was similar for the two species. The results were checked by preparing the assay mixtures in at least one additional buffer system. Proteinases were active on hemoglobin and casein over a rather wide range of pH, with optima around pH 3"8 (low activity) and around pH 8.0 (high activity). The stability of enzyme extracts upon heating was also investigated (Fig. 2). After preheating the extracts at various temperatures for periods of 20 min it was found that the enzyme, tested on BAEE substrate, was completely destroyed at 60°C. /~-Galactosidase and ribonuclease were inactivated at temperatures of 65 and 68°C respectively, all three enzymes maintaining practically all their activity up to 30°C. We also found that aqueous extracts of these enzymes, prepared from a.p. and 1.p. were stable for at least 48 and 72 hr at room temp and at 5°C respectively. The stability of extracts was confirmed at a slight modification of pH (6.0 and 8.0) in phosphate buffer. Gel filtration

Cancer Irroratus

•-~

5o

E

Cancer Borealis

F.~

0

' '

20

30

' Fraction

, ,

G 20

H\

~ 30

, , 40

no.

Fig. 3. Separation of lyophilized hepatopancreatic proteins on Sephadex G-200. Peak intensities (~ of maximum optical density) are not necessarily relative (in activity) to each other. A = chitobiase; B = ct-glucosidase; C = fl-glucosidase; D = RNase; E = chymotrypsin; F = fl-galactosidase; G = acid phosphatase; H = DNase; I = trypsin (TAME); J = trypsin (BAEE).

Lyophilized samples were separated more or less successfully on columns of Sephadex G-200 (Fig. 3) and Bio-Gel P-100 and mol. wt of the active proteins were estimated according to the method of Andrews (1964) (Table 3). In both species, amylase was the only enzyme which was not recovered after gel filtration. Chitobiase was the most predominant carbohydrase recovered, with an approximate mol. wt of 110,000 and 125,000 in C. irroratus and C. borealis respectively, ct-Glucosidase separated from hepatopancreas of C. irroratus had almost twice the mol. wt of that in C. borealis (86,000 and 47,000). The nucleases were well separated and detected in eluents of two separate runs (peaks D and H on Fig. 3). We found the trypsin, tested on TAME, to correspond exactly in mol. wt with the slightly alkaline proteinase acting on hemoglobin and casein. A sample of trypsin-like proteinase was obtained from a larger column, the fractions of interest pooled, dialysed overnight against water, and then freeze dried. An 8-fold increase of activity against protein substrates has been obtained by that purification step.

390

G.L. Bat~x AND M. B. WoJTowwz

6 5

g2 0

i0

18

26 34 Fradi0n no.

42

50

Fig. 4. Separation of protein on Sephadex G-200 and Bio-

Gel P-100. 100 mg of lyophilized powder was applied, protein in fractions was determined by the Lowry method. Dotted line = Cancer irroratus on G-200; broken line Cancer borealis on Sephadex G-200; solid line = C. irroratus on Bio-Gel P-100. Chymotrypsin was barely detectable after gel filtration of C. irroratus sample (peak E on Fig. 3) but totally lost in C. horealis. Separation on Bio-Gel gave no evidence of retardation of carbohydrases (Wojtowicz, 1972) and the same order of elution and similar recoveries for all proteins was obtained. Figure 4 shows the separation of total protein on both gel beds. In the case of C. irroratus on Sephadex and Bio-Gel, between 72 and 7501; of the protein was recovered after each run: with C. borealis, on Sephadex, about 843~owas obtained. As can be seen from the curves, three peaks were eluted from the Bio-Gel and only two peaks from the Sephadex. D1SCU SSION

The decapods Cancer borealis and Cancer irroratus, belonging to the family Cancridae, are found in tide pools and under littoral rocks in New England, but they are chiefly sublittoral south of Cape Cod (Gosner, 1971). Along the coast of Nova Scotia C. borealis is usually caught in deeper water while C. irroratus favors shallower water (Wilder, 1966); feeding habits of both species seem to be similar (Searratt & Lowe, 1972). The animals attain sizes of l,~16cm carapace width. According to Vonk (1960) and Lockwood (1968), the hepatopancreas of Crustacea is the main or perhaps the only source of digestive enzymes. In Malacostraca the cells of the hepatopancreas produce almost all the digestive juice which is then transported to the stomach. Although experimental data are lacking, it is likely that some forms may have the gastric juice produced in either or both the hepatopancreas and mid-gut. For example, Brockerhoff et al. (1970l have reported the presence of the esterases, trypsin (TAME), chymotrypsin and tributyrinase in the gastric juice of the American lobster (Homarus americanus). However, in subsequent works (unpublished data) we could not detect these enzymes on fresh extract of lobster hepatopancreas. Kleine (1967) also reported a significant difference in the activity of trypsin (tested on TAME) in gastric juice and hepatopancreas from Astacus astacus. Clearly these enzymes, present in the stomach juice of lobster are produced or activated not in hepatopancreas but in some other part of the digestive system. In C. borealis and C.

irroratus, on the other hand, we have found trypsinlike proteinase, chymotrypsin and tributyrinase in the extract of fresh tissue and ill acetone powder and in lyophilized protein of hepatopancreas. We must then conclude that the source of some digestive enzymes (at least three cstcrases) is different in American lobster than in the two Cancer species. The mol. wt of the trypsinqike enzyme on Sephadex G-100 in gastric juice of lobster was 25,000 (Brockerhoff et al., 1970), almost the same (24,000) as that found in shrimp (Gates & Travis, 1969). On the same gel but with G-2(X) instead of G-100 we found tool. wt of 16,7(X) and 20,500 for this enzyme in C. horealis and C. irroratus respectively. Brockerhoff et al. (1970) reported two proteinases pH 4.0 and 8.0 (tested on Azocoll) of tool. wt identical to that of trypsin (TAME). In the present study we also found two proteinases pH 3-8 and 8"0 (acting on both casein and hemoglobin substrates) which were similar in mol. wt to our corresponding trypsin (TAME) esterase. Considerably more detailed work would be needed to characterize these proteinases and trypsinlike proteinases, but hypothetically, because of varying sources, we believe just the same that they play essentially similar roles during digestion in the lobster as in the crabs. In C. borealis and C. irroratus the pH of hepatopancreas was found to be approx 6"6 6.8, comparable to the pH range of 6.4 6.8 found in O. asselus by' Hartenstein (1964). In both crabs amylase was the only enzyme with pH optimum close to this range (pH 7.0). Amylase of Carcim~s maem~s was reported to have an optimum pH of 7.0 by Blandamer & Beethey (1964, 1966) and the corresponding value for Dio,qem's hicristimanus was given as 7-2 by Nagabhushanan & Sarotini (1968). Wojtowicz & Brockerhoff (1972) found an optimum pH of 5.2 for amylase in the American lobster. Our data on C. horealis and C. irroratus compare fairly well with the specific activities obtained by Blandamer & Beechey (1966). Among the glycosidases assayed, chitobiase was the most active. Jeuniaux {1960) detected its presence in the hcpatopancreas of the crab Eriocheir smensis. Brockerhoff et al. (1970) observed a specific activity of 4.7 moles l>nitrophenol/mm per mg protein in gastric .juice of lobster: this value is at least ten times greater than the activity found in crabs (Table 2). A mol. wt of l(t0,000 on Sephadex G-100 obtained by Brockerhoff et al. (1970) is somewhat lower (125,000 and I10,(XX) for crabs) than estimated on G-200, the reason probably being that lhe G-100 gel excludes the molecules of lnol. wt over 10/),0(X). No comparable data for chitobiase in the lobster hepatopancreas were available, but it has been observed that the optima of pH 5.0 was identical for lobster and both Calt(.'~'r c r a b s . The activities of e-glucosidase and/#glucosidase in C. horealis and C. irroratus were similar to the activity found in the gastric juice of lobster (Brockerhoff ez al.. 1970). fi-Galactosidase, however, had an activity twenty to a hundred and fifty times greater in both species of crab than in lobster. This is unusually high wflue compared to other forms of Crustacea (Kooiman, 1964). Kooiman's results obtained on Astacus strongly support the concept of the hepatopancreatic

Digestive enzymes in the hepatopancreas of Jonah and rock crabs origin and secretion of most if not all carbohydrases. A phosphatase with optimum p H 3.6 was found to be twice as active in C. irroratus as in C. borealis. In the gastric juice of American lobster an alkaline phosphatase (pH 9.0) was reported but no acid phosphatase was found. Deoxyribonuclease in the crab hepatopancreas showed p H optimum of 5-0. Similarly Russel et al. (1.964) reported an optimum pH of 5.0 in the clam Mercenaria and in the green sea urchin, and optima at 5.0 and 7.0 in digestive gland of the American lobster. The mol. wt on Sephadex G-200 of RNase in C. borealis (22,500) is similar to that of American lobster (25,000) (Brockerhoff et al., 1970). In this study, no attempts were made to determine other enzymes in the hepatopancreas of the two investigated crabs. More data, not only on these two species but on many other crustaceans, are needed to bring about a more complete understanding of phylogenie differences and similarities in the action of digestive enzymes in Crustacea.

391

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