Vol.

THE JOURNAL OF B~LOGIC~L CHEMISTRY 245, No. 15, Issue of August 10, pp. 3964-3972,

Printed in

1970

U.S.A.

Purification and Properties of Trypsin-like Starfish Dermasterias imbricatae

ZENAIDO CAMACHO,~ JAMES R. BROWN, AND
From the Clayton Texas 7871% Foundation Biochemical G. BARRIE KITTO Department

Proteases
(Received for publication,

from
February

the
9, 1970:

Institute,

of Chemistry,

The University

of Texas, Austirl,

SUMMARY

Isolation and purification of trypsin-like proteases from the pyloric caeca of the starfish Dermasterias imbricata resulted in the separation of two different proteins which possessed tryptic-like activity. Both enzymes were homogeneous as judged by polyacrylamide electrophoresis and both have molecular weights of 25,000 to 26,000 as determined by gel filtration. The enzymes were inhibited by diisopropyl phosphorofluoridate and N-tosyl-L-lysylchloromethane, but not by soybean trypsin inhibitor. Enzyme stability, as affected by temperature and pH, showed marked dzerences between the two enzymes. Hydrolysis of benzoyl-DL-arginine-p-nitroanilide hydrochloride by each enzyme was optimal at pH 8.0 to 8.5. The starfish enzymes hydrolyze this synthetic substrate at rates 3.2- and 1.2-fold faster than bovine pancreatic trypsin does. Proteolytic digestion of glucagon and amino acid analysis of the resulting peptides revealed a cleavage specificity for both starfish proteases similar to that of bovine pancreatic trypsin. The enzymes appear to exist as inactive precursors in the pyloric caeca, with spontaneous activation taking place on incubation at 20. The activation was accelerated by addition of exogenous pancreatic trypsin and calcium. However, neither of the purified starfish enzymes showed dependence on calcium ion concentration for enzymatic activity or stability and were unaffected by ethylenediaminetetraacetate.

fish. DeVillez and Buschlen (6) later showed the presence 01 tryptic digestive enzymes in various species of Crustacea. .4 tryptic-like enzyme was also reported by Bewley and DeVillen in the earthworm Lumbricus terrestris linnaeus (7). Recently, Yang and Davis (8) have found a tryptic-like enzyme in adult simuliids (Diptera) ; Gates and Travis (9) have reported the presence of a tryptic-like enzyme in the shrimp Penaeus setijerus. Lecadet and Dedonder have shown the presence of a tryptic-like enzyme in the larvae of the butterfly Pieris brassicae (10) Studies on starfish proteases were initiated by FrCdBricq in 187E (11) who reported that pyloric caeca extracts were capable of hy drolyzing fibrin under alkaline conditions. Similar results were reported by Chapeaux (12), Stone (13), and Van der Heyde (14) Sawano (15), who found that extracts of pyloric caeca fron Distolasterias nipon were capable of hydrolyzing protein, peptone polypeptides, and dipeptides, established pH optima for these substrates. Recent work has centered on determining the homology ol these proteases to that of the well studied bovine pancreatic trypsin. In an attempt to locate additional homologues of thr serine protease trypsin, the proteolytic enzymes of the starfisl Dermasterias imbricata were investigated. Although enzyme: appearing to have chymotryptic and carboxypeptidase act&it> were found in this particular starfish, the present work was con. centrated on elucidating the characteristics of the tryptic-like enzymes. While these studies were in progress, Winter and Seu. rath (16) published a preliminary report of the purificat.ion and properties of trypsins from the starfish Evasterias troche&.
EXPERIMENTAL PROCEDURE

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Because of the essential role a serine group plays in the catalytic function of a number of proteolytic enzymes, this group of enzymes has become known as the serine proteases (1). Trypsin and chymotrypsin are two serine proteases which have been studied intensively and presently serve as models of this group. Trypsin-like activity in vertebrates and invertebrates has been reported for various species by a number of authors (2-4). Degkwitz (5) has investigated a trypsin-like enzyme in cray* This work was supported in part by Grants AI 07853 and HI102899 from the National Institutes of Health and by grants from the Clayton Foundation for Xesearch in Biochemistry. $ Recipient of Predoctoral Fellowship 5-TOl-GMOO789-08 from the National Institlltes of Health.

JPateriaZsSubstrates and inhibitors were obtained from the following sources: benzoyl-r,L-arginine-p-nitroanilide hydrochloride, Nutritional Biochemical; hippuryl-L-arginine, Mann: diisopropyl phosphorofluoridate, Calbiochem ; and Na-tosyl-Llysylchloromethane and Kunitz soybean trypsin inhibitor, Sigma. Proteins used as standards for molecular weight determination by gel filtration included cytochrome c (horse heart) type III: egg albumin (ovalbumin) grade V, and bovine pancreatic t,rypsin from Sigma and bovine serum albumin (Fraction V) from Nutritional Biochemicals. Dextran blue 2000 from Pharmacia was used to determine the void volume. Porcine glucagon used fol specificity studies was obtained from Mann. L-1-TosylamidoSphenylethyl chloromethyl ketone-treated bovine pancreatic trypsin was obtained from Worthington.

3964

Issue of August

10, 1970

Z. Camacho,

J. R. Brown,

and G. B. Kitto

3965

Spetimens-The starfish (U. imbricata) were obtained live from Pacific Biomarine, Venice, California. Upon arrival they were dissected immediately and homogenates of the pyloric caeca were prepared as described below. Enzyme Aspays-Enzymic activity of trypsin was determined using BAPAI as substrate according to the method of Erlanger, Kokowsky, and Cohen (17). Cleavage of the substrate was measured at room temperature in a Zeiss PM& II spectrophotometer at 410 rnp. Enzyme activity is expressed as O.D.410 per 10 min (A0.D. 1.0 = 1.0 enzyme unit) after correction for enzyme blanks. Specific activity is expressed as enzyme units per mg of protein. All tests were carried out in duplicate and a blank was run with each assay. Carboxypeptidase activity was determined from the initial rates of hydrolysis of hippuryl-L-arginine at 25, following the procedure of Folk et al. (18). An enzyme unit of carboxypeptidase activity is equivalent to 1 pmole of substrate hydrolyzed per min under these conditions. Protein DeterminationProtein was determined according to the method of Lowry et al. (19). Crystalline bovine albumin, Fraction V from bovine plasma, was used as a standard. Determination of Molecular Weight---The molecular weight of each protease was estimated by the method of Andrews (20) using a calibrated Sephadex G-100 column of inner dimensions 2 cm x 50 cm. All experiments were conducted at 4. Each of the standard proteins was dissolved in 1 ml of buffer for application to the column. Sucrose was added to each sample and the higher density protein charge carefully layered above the Eel. Collection of the column effluent in 1.5-ml fractions was begun with the addition of the sample to the top of the gel bed. The flow rate of the column was maintained at a constant rate (45 ml per hour) by variation in hydrostatic pressure obtained by adjustment of the position of the buffer reservoir. Fractions were collected in an LKB fraction collector and the absorbance at 280 rnp was monitored with the use of an LKB XJvicord II and recorder. Each protein sample was run individually. The exact height of the gel ked in the column was maintained constant throughout the experiment. Values used for the rrolecular weight of reference proteins nere as follows: cytochrome c, 12,400; ovalbumin, 45,000; bovine pancreatic trypsin, 23,425; bovine serum albumin, 66,500; and soybean trypsin inhibitor, 21,200. Each Dermasterias protease was also applied to the column individually. The protein concentration and tryptic activity of the fractions was determined spectrophotometrically. Specijicity Studies-Porcine glucagon was selected for the determination of specificity of each protease because its amino acid sequence is known (21) and it is a relatively small protein. Cleavage specificity on this protein was determined for the starfish proteases and for TPCK-treated bovine trypsin. Tryptic peptides were isolated by high voltage electrophoresis using a procedure similar to that described by Brown and Hartley (22). Amino acid analyses of eluted peptides were performed on a Beckman-Spinco model 120 C amino acid analyzer (23). ilnalytical Polyacrylamide BlectrophoresisThe method of Ornstein (24) and Davis (25) was followed. Samples containing 50 to 125 pg of protein were mixed with the sample gel (0.2 ml) and placed in a small tube (inner dimensions 6 x 120 mm) which had been covered with Parafilm at one open end. After 1 The abbreviations used arc: BAPA, benzoyl-DL-argininep-nit,roanilide hydrochloride; TPCK, L-l-tosylamido-2-phenylethyl chloromethyl ketone; TLCK, - N*-tosyl-L-lysylchloro_. . . methane; JIPP, diisopropyl phosphorofiuorldate.

the sample gel had polymerized, a spacer gel (0.15 ml) was placed on top of the sample gel. The remainder of the tube was then filled with separating gel (approximately 5 ml). The concentration of acrylamide in the small pore separator gel was 7.5%. Runs were made at room temperature with 0.05 M Tris-glycine buffer at pH 8.3. The voltage was adjusted to approximately 2 to 5 ma per gel with migration from the cathode to the anode. The electrophoresis was stopped when a tracer dye (bromophenol blue) had traveled at least 6 cm into the gel. The gels were stained for 1 hour with a solution of 1 y0 Amido black dissolved in 7 y0 acetic acid. Destaining was accomplished electrophoretitally with 7% acetic acid placed in the buffer reservoirs. Preparative Polyacrylamide Electrophoresis-The apparatus used for preparative polyacrylamide electrophoresis was obtained from Canal Industrial Corporation. A sample gel containing 7 mg of protein (0.2 ml), a spacer gel (0.15 ml), and a separator gel (approximately 3 ml) were placed in the upper column of the apparatus. The gels were prepared according to the method of Davis (25). The concentration of acrylamide in the small pore separator gel was 7.5%. The height of the gel in the column was approximately 2 cm. Tris-glycine buffer (0.05 M, pH 8.3) was the electrolyte solution at the cathode and anode. Current for the electrophoresis was set at 5 ma. Tris-N,N, N,N-tetramethylethylenediamine buffer (0.37 M, pH 8.9) was used for elution at a rate of 0.5 ml per min; 2-ml fractions were collected. The column containing the gel was cooled throughout the electrophoretic run with circulating cold water (4). Time of the run was 3 hours. Temperature Stability-The temperature stability of the two proteases was determined by incubating each enzyme at different temperatures for a 20-min period, removing the enzyme on completion of the incubation period, placing it immediately into ice for 1 min, and using an aliquot of the enzyme in a regular assay at room temperature. A more sensitive indication of temperature stability was obtained by incubating each enzyme at 53 and measuring loss of enzymatic activity with time. Aliquots were withdrawn at different times, placed immediately into ice for 1 min, and an aliquot was removed and assayed for enzymatic activity at room temperature. pH Optima and Stability Studies-To determine the pH optima of the two proteolytic enzymes, the substrate (BAPA) was prepared in various buffer solutions ranging from pH 2.0 to 10.0. Assays with a constant amount of enzyme were carried out at various pH values, always using as a blank the substrate at the appropriate pH. To study the stabilities of the two proteolytic enzymes with respect to pH, equivalent amounts of enzyme were incubated in buffers of varying pH at room temperature for 1 hour. Aliquots were then removed and placed in the standard assay system using BAPA as substrate. The 0.01 M buffer solutions used were as follows: HCl-KC1 buffer, pH 2.0; acetate buffer, pH 4.0; phosphate buffer, pH 6.0; Tris-HCI buffer, pH 8.0; phosphate buffer, pH 8.0; glycine-NaOH buffer, pH 10.0. Tissue Localization of Starfish Trypsin-like Proteases-To determine the specific tissue location of the proteases, a D. imlrricata starfish was dissected and the gonads, pyloric caeca, and the pyloric and cardiac regions of the stomach were removed. Each tissue was weighed and homogenized in cold 0.05 M Tris buffer, pH 8.2, and the enzymatic activity per g, wet weight, in each tissue was determined.

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3966

Starfish

Trypsin-like

Proteases

Vol. 245, No.

15

Inhibitor Studies-The effects of a number of trypsin inhibitors on the two proteases were examined by incubating the enzyme with each test substance for different periods of time and determining residual enzyme activity. Concentrations of soybean trypsin inhibitor ranging from 1 to 60 pg were incubated with the enzyme for 5 min, at which time the mixture was added to the substrate BAPA and the enzyme activity determined. TLCK (0.9. x low6 M) incubation with the enzymes was carried out for a period of 10 min. DFP concentrations used for inhibitor studies were 1.1 x 10B3 M and 4.3 x 10es M. Incubation with the enzyme was for a 5-min period. Controls without inhibitor were run in each case. Ultracentrifugal Analysis-A Spinco model E analytical ultracentrifuge equipped with phase plate schlieren optics and RTIC automatic control was used for the determination of the protease B enzyme sedimentation coefficient. Sedimentation velocity experiments were done at 60,000 rpm in an An-D rotor containing 2 double sector cells with quartz windows. The maximum ordinate on schlieren photographs determined from measurements made with a microcomparator (Gaertner Scientific Corporation) was used to obtain the sedimentation coefficient. Enzyme Activation and Calcium Requirement Studies-To determine whether the tryptic-like enzymes exist in the pyloric caeca in an inactive form, with subsequent activation, a starfish was sacrificed, the pyloric caeca were rapidly removed, placed in 0.05 M Tris buffer, pH 8.2, and immediately homogenized for 2 min in a blender. Two-milliliter aliquots of the homogenate were then mixed with an equal volume of the same buffer containing the following additions: (a) none, (b) 0.02 M calcium chloride, (c) 0.02 M calcium chloride plus 55 ~g of bovine pancreatic trypsin, or (d) 55 pg of bovine pancreatic trypsin. The samples were then incubated at 20 and assayed for tryptic activity, with BAPA as substrate, as a function of time. An additional sample, with no additions, was incubated at 0. The effect of calcium on the proteolytic activity of the fully activated enzymes was determined by incubating the enzyme obtained in an homogenate fraction, activated in the absence of calcium, with different concentrations of CaClz (0.001 M and 0.02 M) for 15- and 30-min periods. Aliquots were removed at these times and assayed for tryptic activity using BAPA as subEDTA effects on enzymatic activity were determined in strate. the same manner using lo+ M and 1e3 M EDTA. Controls in the absence of CaClz or EDTA were run simultaneously. Deionized distilled water was used to prepare all reagents. To determine whether the stability of the starfish proteases was affected by the presence of calcium, each enzyme was incubated at 53 with and without 0.02 M CaC12. Aliquots were removed at various times, chilled immediately, and assayed for residual activity at room temperature by the standard procedure with BAPA as substrate. Enzyme Isolation and Purificatiolz-Initial attempts to extract the starfish enzymes with 0.25 N H&Sod, as described by Northrup, Kunitz, and Herriot (26) for the purification of vertebrate trypsins, proved unsuccessful as no enzyme activity was detected in the homogenate. Enzymatic activity was observed, however, when 0.05 M Tris-HCl buffer, pH 8.2, was used in the extraction procedure. When the pH of the homogenate was lowered to pH 1.7 (pH of acid extraction), complete enzymatic denaturation was noted within 10 min. Therefore, live D. imbricata starfish were dissected and the pyloric caeca removed and placed in cold 0.05 M Tris-HCl buffer, pH 8.2. Unless otherwise noted, all

further purification steps were carried out at 4. The mixture (200 mg of tissue per ml of buffer) was homogenized in a TenBroeck tissue homogenizer for 5 min and the homogenate centrifuged at 3000 x g for 10 min in a Sorvall (RC 2B) superspeed centrifuge to remove large insoluble tissue fragments. Polyoxyethylene lauryl ether was added to the viscous homogenate to give a 0.2% solution and the mixture allowed to sit at 4 for 24 hours. In early experiments it was found that there was an increase in enzymatic activity when homogenates were left standing for several hours or when they were frozen and thawed, suggesting that the enzymes were slowly being solubilized, or activated, from a particulate fraction. Further experiments showed that solubilization of the proteases was greatly facilitated by the detergent treatment described above. The homogenate was then centrifuged at 16,000 x g for 30 min. The supernatant fraction was differentially precipitated with solid (NH&SOI and that portion precipitating between 40 and 60% saturation was collected by centrifugation at 20,000 x g for 20 min. The sediment was suspended in cold 0.05 M Tris buffer, pH 8.2, and subsequently dialyzed against the same buffer overnight, the buffer being replaced periodically. An acetone powder was then prepared from the dialyzed solution by the method of Morton (27). This was done by precipitating the protein with cold ( - 15) acetone and drying the precipitate with cold peroxide-free diethyl ether at 4. The powder was suspended in ice-cold 0.05 M Tris buffer, pH 7.5, mechanically stirred at 4 for 30 min to permit complete solubilization of the enzyme, and centrifuged at 20,000 g for 45 min to remove enzymatically inactive insoluble material. The supernatant fraction was pumped directly onto a large Pharmacia model KlOO/ 100 column (inner dimensions 100 mm x 1,000 mm) packed with Sephadex G-100, equilibrated, and eluted with 0.1 M phosphate buffer, pH 7.5. Eluted fractions containing tryptic activity were concentrated by pressure dialysis by using a UM-10 membrane in a Diaflo model 410 ultrafiltration cell. The concentrated enzyme was then dialyzed against the same buffer and placed on a DEAE-cellulose column. DEAE-cellulose (DE-52, microgranular) was purchased from Whatman and treated according to the manufacturers instructions. The slurry was poured into a column with inner dimensions of 5.5 X 65 cm. The enzyme was eluted at 4 using a linear gradient from 0.1 to 1.0 M potassium phosphate buffer, pH 7.5. The flow rate was 1.5 ml per min and &O-ml fractions were collected. Two cleanly separated peaks of tryptic activity were observed. These were termed proteases A and B in the order of their elution from the column. However, protease B was found to be contaminated with carboxypeptidase activity. Attempts to separate the carboxypeptidase activity from the protease B enzyme using a smaller DEAE-cellulose column (inner dimensions 1.5 x 30 cm) and eluting with a slightly shallower gradient than the previous column (0.1 to 1.0 M phosphate buffer, pH 7.5) proved unsuccessful. Additional attempts to separate these two enzymatic activities on a Sephadex G-100 superfine column were also unsuccessful. Successful separation of the protease B enzyme and carboxypeptidase was achieved on a smaller DEAE-cellulose column (Pharmacia) of inner dimensions 1.5 X 30 cm. The enzyme eluted from the Sephadex G-100 superfine column was concentrated as described above and dialyzed against 0.01 br phosphate buffer, pH 7.5. The protein charge was then placed on a DEAE-cellulose column equilibrated with 0.01 M phosphate

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Issue of August

10, 1970

Z. Camacho, J. R. Brown, and G. B. Kitto

3967

Isolation

TABLE I and purification of tryptic-like enzymes from Dermasterias imbricata starfish

Protein

Total activity

Specific activity

Yield

-fw %

Purification procedure Homogenate.................. 40 to 60% (NHI)~SO~ precipitate. Acetone powder. . Sephadex G-100.. DEAE-cellulose Protease A.. Protease B. Further purification of protease Ab Sephadex G-100 _, DEAE-cellulose. Preparative acrylamide electrophoresis Further purification of protease Bb DEAE-cellulose
0.1-l.OM................... 0.01-0.1 M..................

33,000 13,500 13,200 1,170 180 44 49 11 2

3,825 3,780 1,705 1,256 376 716 275 180 46

0.12 0.28 0.13 1.1 2.2 16.3 5.6 16.3 23.0

100 98.8 45.1 32.0 9.8 18.7 7.2 4.7 1.2

0
10 20

I
30

I
40

I
50 60

FRACTION

NUMBER

FIG. 1. Sephadex G-100 gel filtration of the acetone powder preparation of Dermasterias proteases using a lOO- X 1000~mm column. Each fraction contained approximately 100 ml. Elution was with 0.1 M phosphate buffer, pH 7.5. Fractions 20 to 55 were pooled and used for further purification.

24 4

688 212

28.7 53.0

18.0 5.6

a Proteases A and B were separated by this initial DEAEcellulose step. Their further purification is described separately. b Yield calculated from the total initial activity. buffer, pH 7.5, and eluted with a shallow gradient of 0.01 M to 0.1 M phosphate buffer, pH 7.5. The flow rate was adjusted to 1.0 ml per min and 2-ml fractions were collected. Fractions containing tryptic activity were combined and concentrated as described above. A single band of protein was noted for the purified protease B following analytical acrylamide electrophoresis. Protease A was further purified on a Sephadex G-100 column (2.5 x 30 cm). The enzyme was slowly eluted with 0.1 M phosphate buffer, pH 7.5; 2-ml fractions were collected. Fractions containing tryptic activity were pooled, concentrated as described above, and dialyzed against 0.01 M phosphate buffer, pH 7.5. Further purification was achieved on a DEAE-cellulose column (1.5 x 30 cm). The enzyme was eluted with a linear gradient, of 0.01 M to 0.10 M phosphate buffer, pH 7.5. Fractions containing trypsin activity were pooled and concentrated as above. The presence of three protein bands were observed on analytical acrylamide gels at this stage of the purification. The concentrated enzyme was then subjected to preparative acrylamide electrophoresis. Two-milliliter fractions were collected and the absorbance at 280 rnp was monitored with an LKB Uvicord and recorder assembly. The criterion for the purity of the enzyme was again the appearance of a single protein band following analytical acrylamide electrophoresis.
RESULTS

Tissue Localization of Dermasterias Tryptic-like Proteasea-A major fraction of the tryptic-like activity in Dermasterias tissues was found to be located in the pyloric caeca (9.24 units per g, wet, weight). Lesser amounts were found in the cardiac and pyloric regions of the stomach (3.3 and 3.2 units per g, wet weight, re-

spectively). No activity was noted in other tissues. For this reason, pyloric caeca were utilized as a source of the tryptic-like enzymes for further purification. Enzyme Isolation and Pur$ication-The purification of the starfish trypsins is summarized in Table I. Differential (NH&SO, precipitation was successful in purifying the enzymes slightly with little loss of enzymatic activity. In this particular experiment,, the acetone powder preparation of the protein resulted in a large loss of enzymatic activity with no detectable purification. This is in contrast to earlier preparations of the enzymes where this particular step resulted in both high yield and purification. As shown in Fig. 1, a single peak of tryptic-like activity was obtained after Sephadex G-100 chromatography. A 9.2-fold purification was achieved with this step. The two tryptic-like enzymes were clearly separated in the initial DEAE-cellulose column (Fig. 2) and designated as proteases A and B. The figure shows contamination of the protease B with carboxypeptidase activity. Attempts to separate carboxypeptidase and protease B tryptic activity on a smaller DEAE-cellulose column by eluting with the same gradient (0.1 M to 1.0 M phosphate buffer, pH 7.5) at, a very slow rate of ionic change resulted in considerable purification of both enzymes but no separation of the two enzymatic activities. Separation of these two enzymatic activities was accomplished on a second DEAE-cellulose column eluted with a shallower gradient, as shown in Fig. 3. This step resulted in a large loss of enzymatic activity. The final product, however, appeared homogeneous, as judged by the presence of a single band of protein on analytical polyacrylamide electrophoresis. The purified protease B had a specific activity 3.2 times greater than that of crystalline bovine trypsin using BAPA as a substrate and the enzyme was purified 465-fold from the crude extract. The enzyme sedimented as a single symmetrical peak in the analytical ultracentrifuge with a .SPO,~ 2.5 S at a concentration of 2.0 mg of per ml, in 0.1 M potassium phosphate buffer, pH 7.5. Protease A, from the initial DEAE-cellulose column was purified slightly in a second Sephadex G-100 column step. Further purification was achieved in a DEAE-cellulose column eluted with a shallow ionic gradient. Final purification of this enzyme was accomplished by preparative polyacrylamide electrophoresis (Fig. 4). BAPA hydrolysis by the purified protease A indicated

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3968
+ 0.8,

StarJish Trypsin :-l&e Proteases

,0.2

Vol.

245, No. 15

40

80

120

160

200

FRACTION

NUMBER
0 20 40 60 80 100

FIG. 2. DEAE-cellulose chromatography of the Dermasterias proteases following the gel filtration step. Gradient elution was carried out with 0.1 to 1.0 M phosphate buffer, pH 7.5. Fractions of 8 ml were collected. The size of the column was 5.5 X 65 cm. Fractions 40 to 92 were pooled and designated as Protease A; Fractions 100 to 148, Prot,ease B. Trypsin activity, 0 ; carboxypeptidase activity, A; protease A, A ; protease B, B.

FRACTION
FIG.

NUMBER preparative Procedure polyacrylfor de-

amide tails.

4. Purification electrophoresis.

of protease A by See Experimental

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b OT+0442 i Ii I
i 0
d 03 z go.2 5 z > 0.1 N cl

40

80

120

160

200

FRACTION

NUMBER ELUTION
FIG. 5. Estimation of tryptic-like proteases by column (2 X 50 cm) was sium phosphate buffer, with dextran blue 2000, ovalbumin, n ; protease trypsin, A; cytochrome

FIG. 3. Separation of carboxypeptidase and protease B activity by DEAE-cellulose chromatography. The column (1.5 X 30 cm) was equilibrated with 0.01 M phosphate buffer, pH 7.5, and eluted with a linear gradient elution from 0.01 to 0.10 M phosphate buffer, pH 7.5; 2-ml fract,ions were collected. Protease B activity, 0; carboxypeptidase activity, A.

VOLUME

(ml)

a specific activity 1.3 times higher than that of crystalline bovine trypsin and represented a 202-fold purification. The enzyme moved as a single band on analytical polyacrylamide electrophoresis at several protein concentrations. Insufficient material was available for ultracentrifugal analysis. Determination of Molecular Weight-The approximate molecular weight of each protease was determined by means of a Sephadex G-100 column according to the method of Andrews (20) as detailed under Experimental Procedure. The results are presented in Fig. 5. By this method, the molecular weight of both starfish proteases is 25,000 to 26,000. This figure differs only slightly from the molecular weight of bovine pancreatic trypsin (23,425). In this particular experiment, a molecular weight of 24,000 for the bovine enzyme was obtained by gel filtration. Inhibitor Studies-The effects of DFP and TLCK on the tryptic activity of both starfish proteases and on bovine pancreatic trypsin, run as a control, are shown in Ta.ble II. Both

the molecular weights of Dermasterias gel filtration on Sephadex G-100. The equilibrated and eluted with 0.1 M potaspH 7.5. The void volume, determined was 51 ml. Bovine serum albumin, 0; A, 0; protease B, 0 ; bovine pancreatic c, a; soybean trypsin inhibitor, V.
TABLE

II
by specijic
DFP

Inhibition

of proteolytic

activity

inhibitors
I

I
pmole

I
70 inhibilion

Bovine Protease Protease

trypsin. A. . B. .

. .

0.0017 0.0020 0.0018

88 84 89

39 27 23 I I

62 48 57

starfish enzymes were found to be inhibited by DFP and TLCK, to extents similar to that observed with the bovine enzyme. It was found, however, that soybean trypsin inhibitor had no effects Stoichiometric inhibition of on either of the starfish proteases.

Issue of August 10, 1970

Z. Camacho, J. R. Brown,

and G. B. Kitto

3969

g f z 5 [r: s

50-

25-

20

I 30

40

50

60

70

TEMPERATURE,

C
I 4 I 6 I 8 I 10

FIG. 6. Thermal inactivation of the two proteases from Dermasterias imbricata. Each enzyme was incubated at the indicated temperatures for 20 min and an aliquot was removed and assayed for enzyme activity in a regular assay at room temperature. Protease A, 0 ; protease B, 0.

PH

B,

8. Effect of pH on stability of D. imbricata proteases A and B. Each enzyme was incubated at the indicated pH values for 1 hour at room temperature and the residual enzymic activity was determined in the standard assay. ProteaseA, 0 ; protease
FIG. l

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PH

9. Effect of pH on the activity of proteases A and B from D. imbricata. Protease A, 0 ; protease B, 0.

FIG.

ing temperature (Fig. 6). After heating at 60 for 20 min, no enzymatic activity wasdetectablein the proteaseB fraction and only a slight amount of the original activity was still presentin the proteaseA preparation. When eachenzyme wasincubated at 53 and aliquots withdrawn at different times, a marked difference in the temperature stability of the two enzymes was I I I I I noted (Fig. 7). IO 20 30 40 50 The effect of pH on the stability of the Dermasteri~ proteases INCUBATION TIME (min) is illustrated in Fig. 8. While purified proteaseA is relatively FIG. 7. Effect of calcium ion on enzyme stability of proteases A stableat pH 2, retaining 58% of the initial activity after an hour and B. Each protease was incubated at 53 in the presence and at this pH, the proteaseB is completely inactivated by this proabsence of calcium and equivalent units of enzyme removed at different times and the residual enzyme activity determined at cedure and showssomedenaturation even at pH 6. The proteaseA retained full activity over the pH range 6 to 10 under room temperature using BAPA as substrate. Protease A + theseconditions. Cal&, 0 ; protease A - CaCla, A; protease B + and - CaCla, 0. Metal Ion Studies-Initial experiments indicated that the extracts remained unchanged bovine pancreatic trypsin by soybeantrypsin inhibitor was ob- tryptic-like activity of Dermasterias for long periodsof time, in the absence addedcalcium,suggestof served in a concurrent control experiment. Stability Measuremenfc-Temperaturestability measurements ing the lack of a calcium requirementfor protein stability or acshowa drop in enzymatic activity for eachprotease with increas- tivity. Further studies showedthat the starfish enzymeshad

3970

StarJish Trypsin-like

Proteases -4 - Try +

Vol. 245, No. 15 j. - Leu - Asp - Ser - Arg + +

His

- Ser - Gln - Gly - Thr

- Phe - Ser - Asp - Tyr

- Ser - Lys +

+ + - Arg - Ala
FIG.

- Gln - Asp - Phe - Val - Gln - Trp - Leu - Met - Asn - Thr 10. Cleavage specificity of Dermasterias proteases A and B on glucagon. See text for details.
bovine
I I

treated

pancreatic
I I

trypsin
I

gave

cleavage
I

products
I I

identical

with

those

obtainedwith

protease

A control experiment with B. Protease A, T ; protease

TPCKB, 1 .

0 t t

2 w

0.2-

I IO

I 20

I 30

I 40

I 60

I 80

I
100

I 120

140

TfME

(min)

FIG. 11. Activation of tryptic-like proteases in a crude homogenate of Dermasterias pyloric caeca. Live starfish were dissected and the pyloric caeca homogenized with 0.05 M Tris buffer, pH 8.2, for 2 min in a Waring Blendor. Separate 2-ml aliquots were mixed with equal volumes of (a) 0.05 M Tris buffer (0) ; (b) 0.02 M calcium chloride in Tris buffer (0); (c) 55 rg of bovine pancreatic trypsin in Tris buffer (A); (d) 0.02 M calcium chloride + 55 pg of bovine pancreatic trypsin in Tris buffer (A) and incubated at 20. A further sample (e) in Tris buffer was incubated at O(m). Aliquots were removed at the indicated times and assayed for BAPA activity under the standard assay conditions.

served with the Dermasterias proteaseA except that additional cleavagewasnoted at the Ser-Lys bond (positions11and 12)and at the Ser-Arg bond (positions16 andl7). The resultsare summarized in Fig. 10. Excellent yields of all peptides were obtained, enabling an unambiguous interpretation of cleavage points. The additional hydrolysis observedwith the protease A may have resulted from minor contamination with carboxypeptidase B-like activity, although no suchactivity was observable usinga short term regular assaywith hippuryl-L-arginine assubstrate. In peptide mapsof the starfish protease-treatedglucagon, several weakly staining peptides were observedin addition to the major products. Quantitative aminoacid analysisof the eluted minor peptidesshowed that thesepeptideswerepresentin yields less than lo%, and in mostcases than 5a/ of the major less hydrolytic fragments. Activation of Tryptic-lilce Proteaaes in TissueHomogenates--In order to investigate the possibility that the Dermasterias proteases exist in the pyloric caecain zymogenform, fresh tissueextracts were incubated at 20 in Tris buffer alone and with the addition of calcium chloride and bovine pancreatic trypsin and aliquots were assayed various times for BAPA activity. The at resultsof suchan experiment are illustrated in Fig. 11. On incubation at 20, spontaneous activation wasobserved,the rate of activation being considerablyenhancedby the presence calof cium and exogenous trypsin. No activation was observedwith a samplekept at 0.
DISCUSSION

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identical activities, with BAPA as substrate, in the presence or absenceof CaClz or EDTA. Neither does calcium appear to exert any marked stabilizing effect on the Dermasterias trypticlike proteases. When the temperaturestabilities of the enzymes were examinedat 53 in the presence absence CaClz (Fig. and of 7), no effect of the metal ion was seenwith the proteaseB and only a very slight protection, almost within the range of experimental error, was observedwith proteaseA. pH Optima-The effect of pH on the activity of the Derma-sterias proteases shownin Fig. 9. Both enzymesshowmaximal is activity in the pH range8 to 8.5 with BAPA assubstrate. Speci$city of Dermasterias Tryptic-like Proteases-The hydrolytic specificity of the starfish proteases wasexaminedusing glucagon as a model substrate. A parallel control study was performed using TPCK-treated bovine pancreatic trypsin. After separationand characterization of the peptidesfrom hydrolysatesof glucagon,asdescribed under Experimental Procedure, it was apparent that the starfish proteaseB rapidly cleavedthe bonds on the COOH-terminal side of lysine (position 12) and arginine (positions17 and 18). An identical pattern of cleavage wasobservedwith bovine trypsin. A similar specificity wasob-

Studieson the tissuedistribution of tryptic-like enzymesin the starfish D. imbricata revealedthat a major part of this enzymatic activity is located in the pyloric caeca,a finding which correlates well with the physiology of theseorgans,which servemany of the functions of an exocrine pancreas. Pairs of pyloric caecalie alongeacharm of the starfishand are connected largeductsto by the pyloric stomach. Histological examination by Anderson (28) has shownthe presence numeroussecretory cells in the of saclikeglandular pouches the pyloric caeca. Extensive granuof lar inclusions thesecellswere presumedto be zymogen granin ules. Evidence for the importance of the pyloric caecain the digestive process beenprovided by Anderson (29) who found has that starfish with the pyloric caecaremovedwere able to ingest food normally but were unable to digest it. It is possible that the smaller amounts of tryptic activity observedwith starfish stomachextracts may have originated in the pyloric caecaand remained attached to the stomach epithelium, as the stomach wall lacksthe zymogen-likecellssoapparentin the pylorio cseca. The presentresults on the activation of the trypsin-like proteasesin Dermasterias consistentwith theseenzymesoccurare

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ring in an inactive zymogen form in the pyloric caeca. Whether activation is normally accomplished by a simple autocatalytic process or whether a specific enterokinase is involved, as is the case with vertebrate trypsins, awaits further study. The finding of two tryptic-like enzymes in Dermasterias parallels the preliminary report of two tryptic enzymes being present in the pyloric caeca of Evasterias troche& (16). Whether the two tryptic enzymes in starfish represent separate gene products, or whether they are modifications of the same protein must await detailed structural examination. The latter possibility is suggested by the recent studies of Schroeder and Shaw (30), who demonstrated the formation of several active forms of bovine trypsin upon autocatalytic activation of the zymogen. The two Dermasterias proteases differ most markedly in their sensitivity to denaturation, both by heat and extreme pH. The protease A shows considerably greater thermostability than does protease B and retains partial activity after 1 hour at pH 2, a feature more similar to the vertebrate trypsins. It is of interest that both starfish enzymes show significantly more activity with the synthetic substrate benzoyl-nn-arginine-p-nitroanilide than does bovine pancreatic trypsin, the starfish proteases A and B having 1.3 and 3.2 times, respectively, the activity of the bovine enzyme. Both Dermasterias tryptic-like proteases have molecular weights of approximately 25,000 which is similar to that of the vertebrate trypsins. The few studies available on other invertebrate tryptic-like enzymes indicate similar molecular weights for these enzymes. Winter and Neurath (16) found one of the tryptic enzymes from Evasterias to have a molecular weight, by gel filtration, of 25,000. A molecular weight of 24,700 was observed for the trypsin for the crayfish Orconectes, and Bewley and DeVillea (7) have estimated a molecular weight of approximately 26,000 for a trypsin isolated from the earthworm Lumbicus terrestris. The observed pH optima of the Dermasterias proteases is consistent with their action in the slightly alkaline medium of the starfish stomach (14,31) and is similar to the pH-activity profiles of vertebrate trypsins (17, 32). Similar pH optima were reported for trypsins from Crustacea (6) and from the earthworm Lumbricus (7). The DwmasteriQs enzymes are virtually unaffected in stability by the presence of calcium and are not inhibited by EDTA. In contrast, the vertebrate trypsins are markedly stabilized by calcium (33,34) and activated by this ion (35). Zwilling et al. (36) found no calcium effects with a crayfish trypsin and Winter and Neurath (16) reported that EDTA slightly activated a trypsin from the starfish Evasterias. In the initial studies on the Dermasterias proteases the two enzymes isolated were referred to as tryptic-like on the basis of their ability to hydrolyze the synthetic substrate benzoyl-narginine-p-nitroanilide. That the invertebrate enzymes are at least functionally analogous to vertebrate trypsins is suggested by the results of experiments with specific inhibitors, and on the analysis of the cleavage products of protease-treated glucagon. Both starfish proteases were inhibited by DFP and by TLCK suggesting an essential role for a serine (37) and a histidine residue (38) respectively, in the catalytic function of these enzymes. Unlike many vertebrate trypsins (23) the Dermasterias enzymes were unaffected by even high concentrations of soybean trypsin inhibitor. Recently, Travis and Roberts (39) reported that human trypsin is not inhibited by soybean trypsin inhibitor and suggested that this might be related to the reduced number of

disulfide bonds in the human enzyme compared with other vertebrate trypsins. The hydrolysis products from the starfish protease digestion of glucagon provided further evidence for the similarity of the Dermasterias enzymes and vertebrate trypsins. The Dermasterias protease B gave as major hydrolytic products peptides identical with those obtained with bovine pancreatic trypsin, with the expected cleavage on the carboxyl side of lysine and arginine residues. The starfish protease A enzyme gave similar products except for additional cleavages at Ser-Lys bond at positions 11 and 12 and a Ser-Arg bond at positions 16 and 17. The additional splits observed with the protease A could result either from a broader specificity of the starfish enzyme compared with bovine trypsin, or from a slight contamination with carboxypeptidase activity. Although no carboxypeptidase activity was detectable in the enzyme in a regular assay, it is possible that amounts sufficient to produce hydrolysis of the glucagon peptides over the long incubation time employed would be undetected by this procedure. The present results clearly indicate close similarities between the two tryptic-like proteases of D. imbricata and vertebrate trypsins. In particular, the results obtained with the specific inhibitors DFP and TLCK and the cleavage specificity of the starfish enzymes are suggestive of homology with vertebrate trypsins, but definitive evidence must await detailed structural analysis. Purification of the Dermasterias enzymes, in sufficient quantities to permit such studies, is in progress. AcklzowledgmentsWe thank Glenda Anderson, Katherine Yndo, Sharyn Marshall, Vivian Sellers, and James Lemburg for their assistance in various aspects of this work. REFERENCES 1. HARTLEY, B. S., Annu. Rev. Biochem., 29, 45 (1960). 2. DESNUELLE, P., in P. D. BOYER, H. LARDY, AND K. MYRB~~CK (Editors), The enzymes, Vol. Q, Academic Press, New York, 1960, p. 119. 3. ZENDZIAN, E. N., AND BARNARD, E. A., Arch. Biochem. Biophys., 122, 699 (1967). 4. BARRINGTON, E. J. W., Advan. Comp. Physiol. Biochem., 1, 1 (1962). 5. DEGKWITZ, E., Veroeff. Inst. Meeresforsch. Bremerhaven, 6, 1 (1957). 6. DEVILLEZ, E. J., AND BUSCHLEN, K., Comp. Biochem. Physiol., 21, 541 (1967). 7. BEWLEY. G. C.. AND DEVILLEZ, E. J., Comp. Biochem. Physiol., 26, 1061 (1967). 8. YANG, Y. J., AND DAVIS, D. M., J. Insect Physiol., 14, 205 (1968). 9. GATEs,B. J., AND TRAVIS, J., Fed. Proc., 27, 770 (1968). 10. LECADET, M., AND DEDONDER, R., Bull. Sot. Chim. Biol., 48, 661 (1966). 11. FR~D&RICQ, L., Arch. Zool. Exp. Gen., 7, 391 (1878). 12. CHAPEAUX, M., Bull. Acad. Belg., Ser. S,26, 227 (1893). 13. STONE, E., Amer. Natural., 31, 1035 (1897). 14. VAN DER HEYI~E, H. C., Dissertation, Amsterdam, 1922. 15. SAWANO, E., Sci. Rep. Tokyo Bunrika Daigaku, Sec. B, 2, 26 (1936). 16. WINTER, W. P., AND NEURATH, H., Fed. Proc., 28, 492 (1969). 17. ERLANGER, B. F., KOKOWSKY, N., AND COHEN, W., Arch. Biochem. Biophys., 96, 271 (1961). 18. FOLK, J. E., PIEZ, K. A., CARROLL, W. R., AND GLADNIER, J. A., J. Biol. Chem., 236, 2272 (1960). 19. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem., 193, 265 (1951). 20. ANDREWS, P., Biochem. J., 91, 222 (1964).

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Proteases

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21. BROMER, W. W., STAUB, A., DILLER, E. R., BIRD, H. L., SINN, L. G.. AND BEHRENS. 0. K.. J. Amer. Chem. Sot.. , 79.. 2794

(1957 j.

22. BROWN, J. R., AND HARTLEY, B. S., Biochem. J., 101, 214 (1966). 23. SPACKMAN, D. II., STEIN, W. H., AND MOORE, S., Anal. Chem., 30, 1190 (1958). 24. ORNSTEIN, L., Ann. N. Y. Acad. Sci., 121, 321 (1964). 25. DAVIS, B. J., Ann. N. Y. Acad. Sci., 121, 494 (1964). 26. NORTHROP, J. H., KUNITZ, M., AND HERRIOT, R. M., Crystalline enzymes, Columbia University Press, New York, 1955, p. 262. 27. MORTON, R. K., in S. P. COLOWICK AND N. 0. KAPLAN (Editors), Methods in enzymology, Vol. 1, Academic Press, New York, 1955, p. 34. 28. ANDERSON, J. M., Biol. Bull., 106,47 (1953); in R. A. BOOLOOTIAN (Editor), Physiology of echinodermata, Interscience Publishers, New York, 1966, p. 329.

29. ANDERSON, J.M., Biol. Bull., 119,371 (1960). 30. SCHROEDER, D. D., AND SHAW, E., J. Biol. Chem., 248, 2948 (1968). 31. IRVING, L., J. Gen. Physiol., 10,345 (1926). 32. BUCK. F. F.. VITHAYATHIL. A. J.. BIER. M.. AND NORD. F. F.. Awh. Biochem. Biophys.,97,4i7 (1962). 33. NORD, F. F., AND BIER, M., Biochim. Biophys. Acta, 12, 56 (1953). GREEN, N. M., J. Biol. Chem., 206, 535 (1953). 2 SIPOS, T., AND MERKEL, J. R., Biochem. Biophys. Res. Commm., 81, 522 (1968). 36. ZWILLINQ. R., PSLEIDERER, G., SONNEBORN, H.-H., KRAFT. V., AND.STU~KY, I., Camp: Biochem. Physiol:, 98.1275 (1969): 37. DIXON. G. H.. Go. S.. AND NEURATH. , H.. , Biochim. Bkhus. 1 _ Acta; 19, 193 (19.&I). 38. SHAW, E., MARES-GUIA, M., AND COHEN, W., Biochemistry, 4, 2219 (1965). 39. TRAVIS, J., AND ROBERTS, R. C., Biochemistry, 8, 2884 (1969).

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