Comp. Biochem. Physiol. Voi. 88B, No. 2, pp. 503-506, 1987 0305-0491/87 $3.00 + 0.00 Printed in Great Britain © 1987 Pergamon Journals Ltd

DIGESTIVE PROTEASES IN THE MIDGUT GLAND OF THE ATLANTIC BLUE CRAB,

CALLINECTES SAPIDUS*

JAMES E. D ~ I N O E R Department of Biology, James Madison University, Harrisonburg, VA 22807, USA (Tel.: 703-568-6225)

(Received9December 1986)

Abstract--l. Digestive proteases from the midgut gland of male Atlantic blue crabs, Callinectes sapidus, were investigated. Tentative identities of proteolytic enzymes were determined with synthetic substrates and inhibitors.

2. Trypsin, chymotrypsin, carboxypeptidase A and B and lencine aminopeptidase activities were found and quantified.

3. Activity against Succinyl-(Ala)s-nitroanalide was also found. This as yet unidentified enzyme has a tool. wt of about 26,000 and has elastolytic activity.

INTRODUCTION

There have been numerous reports on the digestive proteases found in the midgut gland or stomach fluid of various Crustacea. These enzymes have been, for the most part, similar to vertebrate digestive pro- teases and include both endo- and exopeptidases. Most reports of specific enzymatic activity have been based on the cleavage of synthetic substrates and inhibition by specific inhibitors.

Trypsin activity has been identified in crayfish (DeVillez, 1965; Pfleiderer et al., 1967), several crabs (Linke et al., 1969; Herbold et al., 1971), shrimp (Galgani et al., 1984; Tsai et al., 1986) and prawn (Lee et al., 1980).

Chymotrypsin activity is equivocal in Crustacea. Several investigators have attempted to demonstrate chymotryptic activity without success (DeVillez, 1965; Pfleiderer et aL, 1967). More recently there has been reported low activity in shrimp (Galgani et al., 1984) and prawn (Lee et al., 1980). This low activity may have been due to 8uboptimum substrates (Tsai et al., 1986).

An endopeptidase of low tool. wt around 11,000 has been detected in several crustaceans including shrimp (Galgani et aL, 1984) and lobster (Brockerhoff et al., 1970) and has been purified from crayfish and partially sequenced (Zwilling et al., 1981).

In addition to these endopeptidases, collagenase has been identified in shrimp (Galgani et al., 1984) and purified from fiddler crab (Eisen et al., 1973). Elastase has not been identified, to our knowledge, in any crustacean (Galgani et al., 1984).

The exopeptidases identified in Crustacea include carboxypeptidase and leucine aminopeptidase. Car- boxypeptidase A and B activities have been found in prawn (Lee et al., 1980), crayfish (DeVillez, 1975;

*This work was supported by a grant from the James Madison University Program of Grants for Faculty Research.

Zwilling et ai., 1979) and shrimp (Galgani et al., 1984; Gates and Travis, 1973). Leucine aminopeptidase activity has been reported in crayfish (DeVillez, 1975), shrimp (Galgani et al., 1984) and prawn (Lee et al., 1980).

The purpose of this study was to determine which digestive proteases are active in the midgut gland of the Atlantic blue crab, Callinectes sapidus.

MATERIALS AND METHODS

Animals and tissues

Adult male blue crabs were obtained from a commercial crab house on the Rappahannock River of Virginia during June. Each midgut gland was removed and frozen for transport to the laboratory. Frozen samples were homogen- ized for 5 rain in a Waring blender at 4°C in water. The homogenate was centrifuged at 10,000g for 30 min at 4°C and the supernate fluid decanted through cheesecloth to retain the thick lipid layer. This supemate was brought to 85% saturation with ammonium sulfate, stirred for 30 rain at room temperature and then centrifuged at 10,000g for 30 min at 4°C. The supernate was discarded and the protein pellet was redissolved in I0 mM Tris-HCl at pH 7.4.

Chemicals The following chemicals were purchased from Sigma

Chemical Company: bovine pancreatic trypsin, chymotryp- sin, and carboxypeptidase A, porcine pancreatic carboxy- peptidase B, porcine kidney leucine aminopeptidase, porcine pancreatic elastase, p-toluene-sulfonyl-L-arglnine methyl- ester (TAME), benzoyl-L-tyrosine ethyl ester (BTEE), hippuryl-L-phenylalanine (HLPA), hippuryl-L-arginine (HLA), succinyl-L-alanine3-p-nitroanilide (SANA), azo- albumin, leucine amide (LA), tosyl-L-lysine chloromethyl ketone (TLCK), tosyl-L-phenylalanine chloromethyl ketone (TPCK), phenyhnethylsulfonyl fluoride (PMSF), potato carboxypeptidase inhibitor and elastin congo red. Other chemicals were analytical grade.

pH Optimum

The pH range over which there was general proteolytic activity against azoalbumin was determined by mixing 10 mM Tris buffer, azoalbumin and Sephadex G-25 filtrate of the crab midgut gland in a series of test tubes. Each sample was adjusted to a particular pH with 0.1 mi dilute HC1 or NaOH and incubated at 30°C for 1 hr. At the end

503

504 JAMES E. Dm, rDINOWR

of the incubation, each tube was again checked for pH and the reaction stopped with 0.5 mi of 5% trichloroacetic acid.

Assay of enzyme activity

General proteolytic activity was determined spectre- photometrically using azoalbumin as substrate following the method of Tomarelli et al. (1949). The sample (0.5 mi) was mixed with an equal volume of 1 mg/ml azoaibumin in 1 M Tris-HCl at pH 7.4 and incubated at 30°C in a shaking water bath for 1 hr. The reaction was terminated by the addition of 0.5 ml of 5% trichloroacetic acid and then centrifuged in a clinical centrifuge for 10 rain at top speed. The supematant fluid was withdrawn, mixed with an equal volume of 0.5 M NaOH and the optical density measured at 425 nm. Non-digested azoalbumin was used as the standard.

Trypsin activity was determined spectrophotometrically using TAME as substrate (Hummel, 1959) and thyme- trypsin activity was determined with BTEE as substrate (Walsh and Wilcox, 1970). Carboxypeptidase A and B activity were determined using HLPA and HLA as substrate following the methods of Folk and Sehirmer (1963) and Folk et aL (1960), respectively. Leucine amide was used as substrate for leucine aminopeptidase (Binkley and Torres, 1960) and SANA as substrate for low reel. wt protease (Zwilling and Neurath, 1981). Elastase activity was deter- mined by the method of Shotton (1970). All of the enzyme assays were conducted with a Beckman 25 spectropho- tometer with temperature controller at 30°C and recorder. Protein concentration was estimated using absorbance at 280/260 nm (Warburg and Christian, 1942).

Activities are reported as /zmol substrate/min per mg protein except for leucine aminopeptidase where 1 unit is a change of 0.001 in optical density.

Chromatography

The redissolved protein from the ammonium sulfate precipitation was passed through a 2.5 x 35 cm column of Sephadex G-25 equilibrated and eluted with 10raM Tris-HCl at pH 7.4. The protein peak was monitored at 280 nm using an I.S.C.O. UA-5 absorbance monitor and collected in an I.S.C.O. Retriever III fraction collector. Pooled fractions were freeze dried and stored over silica gel at room temperature.

DEAE-Sephacel was equilibrated with 10 mM Tris-HC1 at pH 7.4 and used in a 2.5 x 32 em column. Approximately 290 mg of protein in 10 mi of 100 mM Tris-HC1, pH 7.4, was pumped on to the column. Elution was by a linear NaCI gradient from 0.2 to 1.5 M in the same buffer. Elution was monitored and samples collected as above.

The Sephadex G-75 column (1.8 x 56era) was equili- brated and eluted with 10 mM Tris-HCl at pH 7.4, moni- tored and samples collected as above. All chromatography procedures were carried out at 4°C.

Inhibition studies

Inhibition studies were carried out by mixing the crab sample or commercial enzyme solutions with an equal volume of 2X inhibitor solutions. These were incubated for 1 hr at 30°C in a shaking water bath before the enzyme assays.

RESULTS

The Sephadex G-25 filtrate from crab midgut gland had proteolytic activity against azoalbumin over a broad pH range from 5 to 9 (Fig. 1). Maxi- mum activity was around 6.2 although there was substantial activity up to about pH 7.5.

Activity of the Sephadex G-25 filtrate against various synthetic substrates is shown in Table 1. Highest activity was with azoalbumin as substrate. Activity with T A M E suggests a trypsin-like enzyme

.¢: 275

175-

7 5 -

I I I i

3 5 7 9 II pH

Fig. 1. Effect ofpH on proteolytic activity of Sephadex G-25 filtrate from Callinectes sapidus midgut gland against azo-

albumin at 30°C.

and with BTEE a chymotrypsin-like enzyme. Hydrol- ysis of HLPA and H L A indicates the presence of both carboxypeptidase A and B activities, with the latter having about three times the activity of the former. The activity against SANA is an indication of elastase (Feinstein et al., 1973) or a low mol. wt protease (Zwilling and Neurath, 1981). Leucine aminopeptidase activity is indicated by the hydrolysis of LA.

Once the presumptive identities of proteolytic en- zymes in blue crab midgut gland had been determined using synthetic substrates, the crab Sephadex G-25 filtrate and commercial enzymes were tested against a battery of inhibitors in order to further test the identities of these proteolytic enzymes in blue crab. The results are shown in Table 2. With T A M E as substrate, P M S F and T L C K inhibited trypsin and the crab preparat ion to about the same extent, indi- cating that trypsin is in fact present in blue crab midgut gland. T A M E activity was inhibited in both the crab preparation and commercial enzyme by potato carboxypeptidase inhibitor. Some of the T A M E activity may be carboxypeptidase B which will cleave next to arginine but is not inhibited by PMSF.

The BTEE activity was inhibited by PMSF and T P C K and is therefore probably chymotrypsin. HLPA and HLA activity was inhibited by potato carboxypeptidase inhibitor and lack of inhibit ion by the others confirmed the presence of carboxypepti- dase A and B. The SANA activity was completely inhibited by P M S F which indicated that this is not low mol. wt protease but rather a serine protease.

In order to partially purify the various proteases, the Sephadex G-25 filtrate was chromatographed on

Table 1. Activity of Callinectes sapidus midgut gland Sephadex G-25 filtrate with

various synthetic substrates Substrate /~mol/min per mg protein Azoalbumin 12.81 TAME 1.68 BTEE 50 x 10 -3 HLPA 120 x 10 -3 HLA 319 x 10 -3 SANA 3 X 10 -3

LA 200* Activities were measured at 30°C. *Units/rain per mg protein.

Digestive proteases in Callinectes

Table 2. Percent inhibition of commercial enzyme and CaUinectes sapi~ midgut gland Sephadex G-25 filtrate

Inhibitor PMSF TPCK TLCK Potato

Substrate Enzyme 2 mM 2 mM 2 mM 0.4 mM

% Inhibition TAME Trypsin 53 5 i 00 17

Crab 49 0 100 57 BTEE Chymotrypsin 59 81 0 100

Crab 100 92 2 84 HLPA Carboxypeptidase A 0 0 0 100

Crab 0 0 0 100 HLA Carboxypeptidase B 0 0 0 100

Crab 0 0 0 I00 LA Leucine Aminopeptidase 50 20 0 0

Crab 0 9 0 0 SANA Crab 100 39 7 7

505

DEAE-Sephacel and the major protein peaks as- sayed for enzyme activity. The chromatogram is shown in Fig. 2 and the enzyme activity from the protein peaks in Table 3. Peak I had low proteasc activity which was a combination of chymotrypsin and carboxypeptidase B activity. Peak II had the lowest protease activity and was a mixture of chymo- trypsin, carboxypeptidase A and B, SANA and leu- cine aminopeptidase. Peak III was a combination of all enzymes tested for except carboxypeptidase B. The greatest general protease activity was in Peak IV and appears to be due entirely to trypsin activity.

To try to identify the enzyme responsible for the SANA activity, Sephadex G-25 filtrate from crab midgut gland was passed through a Sephadex G-75 column calibrated for mol. wt. These results are shown in Fig. 3. The greatest activity against SANA eluted at a volume indicating a tool. wt of about 26,000. A protease in this tool. wt range which will degrade SANA and is inhibited by PMSF is elastase.

D~CUSSION

The proteases tentatively identified in Callinectes sapidus midgut gland during the course of this study are, with one exception, consistent with previous investigations. However, the trypsin, chymotrypsin, carboxypeptidase A and B and leucine amino- peptidase activities found in the blue crab have different relative activities to those reported from Macrobranchium rosenbergii (Lee et al., 1980) or Penaeus sp. (Galgani et aL, 1984). Although it is difficult to compare the results of other studies be- cause of the use of different activity units and some different substrates, it appears that Macrobranchium

F '° g

• I I I i I 0 0 2 0 0 3 0 0 4 0 0

E l u t i o n V o l u m e (ml )

Fig. 2. Separation of proteins from Callinectes sapidus midgut gland on DEAE-Sephacel.

Table 3. Activities of protein peaks from DEAE-Sephacel in Fig. 3

Substrate Peak I II III IV

Azoalbumin 2.5 0.5 43.0 48,2 TAME 0 0 0.5 3.2 BTEE 20 x 10 .3 8 x 10 -3 57 x 10 .3 0 HLPA 0 154 x 10 -3 379 x 10 -3 0 HLA 191 x 10 -~ 159 x 10 -3 0 0 SANA 0 10 x 10 -3 10 x 10 -3 0 LA 0 0.88 0.33 0

All activities are/~mol/min per mg protein except for LA which is OD/min per mg protein.

has substantially more chymotrypsin activity than trypsin activity whereas Penaeus has abundant tryp- sin and very little chymotrypsin. The blue crab is more like Penaeus in that there is substantially more trypsin than chyrnotrypsin activity. It also appears t ha t t he re is a b o u t twice the c a r b o x y p e p t i d a s e A act ivi ty as c a r b o x y p e p t i d a s e B in b o t h Macro- branchium a n d Penaeus kerathurus w h e r e a s in P. japonicus the re is m u c h m o r e c a r b o x y p e p t i d a s e B t h a n A activi ty. Resu l t s f r o m the b lue c r ab a re m o r e s imi lar to P. japonicus in t h a t in t he blue c r a b c a r b o x y p e p t i d a s e B act ivi ty is a b o u t two a n d h a l f t imes the act ivi ty o f c a r b o x y p e p t i d a s e A.

A l t h o u g h a low tool. w t p r o t e a s e has been f o u n d in several c rus t aceans ( B r o c k e r h o f f et al., 1970; A r m - s t r ong a n d DeVil lez , 1978; Ga l gan i et aL, 1984), the re

0.8

o.6 o

0 . 4

0.2

SAN Activi

(~)

20C

l lO0

u- 0

I

Io

'A

I 3 0 510

Elution Volume (ml)

- 4 . 7

4.5

_J

4.3'

4.1

Fig. 3. Separation and mol. wt determination of SANA activity from Callinectes sapidus midgut gland on Sephadex G-75. Molecular weight standards: [] -- ovalbumin; • -- chymotrypsin; A = myoglobin; • = ribonuclease A. The large trailing peak is nucleic acid. SANA activity units

are change in absorbance at 410 nm/min per 0.I ml.

506 D~ms E. DesoINO~

is no evidence of this enzyme in the blue crab. SANA activity was found but this activity was inhibited by PMSF, indicating a serine protease and the mol. wt of the enzyme responsible for this activity was about 26,000 rather than the expected 11,000 (Zwilling and Neurath, 1981).

SANA was originally developed as a synthetic substrate for elastase (Feinstein et aL, 1973; Bieth et al., 1974) which is also a serine protease (Shotton, 1970). The rather slow but definite activity of blue crab Sephadex G-25 filtrate against elastin-congo red indicates that elastase activity is present. The additional fact that this SANA activity is inhibited by the specific elastase inhibitor MeOSucc-(AIa)2-Pro- AIa-CH2C1 indicates that Callinectes sapidus does have some form of elastase. An enzyme with elasto- lytic activity has been isolated from a non-crustacean invertebrate, Schistosoma mansoni, but this trema- tode protease more closely resembles vertebrate chymotrypsin than elastase (Landsperger et al., 1982). Work is in progress to elucidate the identity of the blue crab protein with SANA activity.

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