Camacho Et Al 1970. Purification and Properties of Trypsin-Like Proteases From the Starfish Dermasterias Imbricatae

8/3/2019 Camacho Et Al 1970. Purification and Properties of Trypsin-Like Proteases From the Starfish Dermasterias Imbricatae

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THE JOURNAL OF B~LOGIC~L CHEMIST RYVol. 245, No. 15, Issue of Augu st 10, pp. 3964-3972, 1970Printed in U.S.A.

Purification and Properties of Trypsin-like Proteases from theStarfish Dermasterias imbricatae

(Received for publication , February 9, 1970:ZENAIDO CAMACHO,~ JAMES R. BROWN, AND G. BARRIE KITTOFrom the Clayton Foundation Biochemical Institute, Department of Chemistry, The University of Texas, Austirl,Texas 7871%

SUMMARYIsolation and purification of trypsin-like proteases from

the pyloric caeca of the starfish Dermasterias imbricata re-sulted in the separation of two different proteins whichpossessed tryptic-like act ivi ty. Both enzymes were ho-mogeneous as judged by polyacrylamide electrophoresisand both have molecular weights of 25,000 to 26,000 asdetermined by gel filt ration. The enzymes were inhibitedby diisopropyl phosphorofluoridate and N-tosyl-L-lysyl-chloromethane, but not by soybean trypsin inhibitor. En-zyme stabil ity, as affec ted by temperature and pH, showedmarked dzerences between the two enzymes. Hydro lysisof benzoyl-DL-arginine-p-nitroanilide hydrochloride by eachenzyme was optimal at pH 8.0 to 8.5. The starfish en-zymes hydrolyze this synthetic substrate at rates 3.2- and1.2-fold faster than bovine pancreatic trypsin does. Proteo-lyti c digestion of glucagon and amino acid analysis of theresulting peptides revealed a cleavage specif icity for bothstarfish proteases similar to that of bovine pancreatic trypsin.The enzymes appear to exist as inactive precursors in the py-loric caeca, with spontaneous activation taking place onincubation at 20. The activation was accelerated byaddition of exogenous pancreatic trypsin and calcium.However, neither of the purified starfish enzymes showeddependence on calcium ion concentration for enzymatic ac-tiv ity or stabili ty and were unaffected by ethylenediamine-tetraacetate.

Because o f the essential role a serine group plays in the cata-lyti c function of a number of proteolytic enzymes, this group ofenzymes has become known as the serine proteases (1). Tryp-sin and chymotrypsin are two serine proteases which have beenstudied intensively and presently serve as models o f this group.Trypsin-like act ivi ty in vertebrates and invertebrates has beenreported 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 andHI102899 from the National Institutes of Health and by grantsfrom the Clayton Foundation for Xesearch in Biochemistry.

$ Recipient of Predoctoral Fellowship 5-TOl-GMOO789-08 fromthe National Institlltes of Health.

fish. DeVillez and Buschlen (6) later showed the presence 01trypt ic digestive enzymes in various species of Crustacea. .4tryptic-like enzyme was also reported by Bewley and DeVillenin the earthworm Lumbricus terrestris linnaeus (7). Recently,Yang and Davis (8) have found a tryptic-like enzyme in adultsimuli ids (Diptera) ; Gates and Travis (9) have reported the pres-ence of a tryptic-like enzyme in the shrimp Penaeus setijerus.Lecadet and Dedonder have shown the presence of a tryptic-likeenzyme in the larvae of the butte rfly Pieris brassicae (10)Studies on starfish proteases were initiated by FrCdBricq in 187E(11) who reported that pyloric caeca extracts were capable o f hydrolyzing fibrin under alkaline conditions. Similar results werereported by Chapeaux (12), Stone (13), and Van der Heyde (14)Sawano (15), who found that extracts of pyloric caeca fronDistolasterias nipon were capable of hydrolyzing protein, peptonepolypeptides, and dipeptides, established pH optima for thesesubstrates.

Recent work has centered on determining the homology olthese proteases to that of the well studied bovine pancreatictrypsin. In an attempt to locate additional homologues of thrserine protease tryps in, the proteolytic enzymes of the starfislDermasterias imbricata were investigated. Although enzyme:appearing to have chymotrypt ic and carboxypeptidase act&it>were found in this particular starfish, the present work was con.centrated on elucidating the characteristics of the tryptic-likeenzymes. While these studies were in progress, Winter and Seu.rath (16) published a preliminary report of the purificat.ion andproperties of trypsins from the starfish Evasterias troche&.

EXPERIMENTAL PROCEDURE

JPateriaZsSubstrates and inhibitors were obtained from thefollowing sources: benzoyl-r,L-arginine-p-nitroanilide hydro-chloride, Nutritional Biochemical; hippuryl-L-arginine, Mann:diisopropyl phosphorofluoridate, Calbiochem ; and Na-tosyl-L-lysylchloromethane and Kunitz soybean trypsin inhibitor,Sigma.

Proteins used as standards for molecular weight determinationby gel filtration included cytochrome c (horse heart) type III:egg albumin (ovalbumin) grade V, and bovine pancreatic t,rypsinfrom Sigma and bovine serum albumin (Fraction V) from Nutri-tional Biochemicals. Dextran blue 2000 from Pharmacia wasused to determine the void volume. Porcine glucagon used folspec ific ity studies was obtained from Mann. L-1-TosylamidoS-phenylethyl chloromethyl ketone-treated bovine pancreatictrypsin was obtained from Worthington.

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Spetimens-The starfish (U. imbricata) were obtained live the sample gel had polymerized, a spacer gel (0.15 ml) was placedfrom Pacific Biomarine, Venice, California. Upon arrival they on top of the sample gel. The remainder of the tube was thenwere dissected immediately and homogenates of the pyloric caeca filled with separating gel (approximately 5 ml). The concen-were prepared as described below. tration of acrylamide in the small pore separator gel was 7.5%.Enzyme Aspays-Enzymic act ivi ty of trypsin was determined Runs were made at room temperature with 0.05 M Tris-glycineusing BAPAI as substrate according to the method of Erlanger, buffer at pH 8.3. The voltage was adjusted to approximatelyKokowsky, and Cohen (17). Cleavage of the substrate was 2 to 5 ma per gel with migration from the cathode to the anode.measured at room temperature in a Zeiss PM& II spectrophotom- The electrophoresis was stopped when a tracer dye (bromophenoleter at 410 rnp. Enzyme act ivi ty is expressed as O.D.410 per 10 blue) had traveled at least 6 cm into the gel. The gels weremin (A0.D. 1.0 = 1.0 enzyme unit) after correction for enzyme stained for 1 hour with a solution of 1 y0 Amido black dissolved inblanks. Specific activ ity is expressed as enzyme units per mg of 7 y0 acetic acid. Destaining was accomplished electrophoreti-protein. All tests were carried out in duplicate and a blank was tally with 7% acetic acid placed in the buffe r reservoirs.run with each assay. Carboxypeptidase act ivi ty was determined Preparative Polyacrylamide Electrophoresis-The apparatusfrom the initial rates of hydrolysis of hippuryl-L-arginine at 25, used for preparative polyacrylamide electrophoresis was obtainedfollowing the procedure of Folk et al. (18). An enzyme unit of from Canal Industrial Corporation. A sample gel containing 7carboxypeptidase act ivi ty is equivalent to 1 pmole of substrate mg of protein (0.2 ml), a spacer gel (0.15 ml), and a separator gelhydrolyzed per min under these conditions. (approximately 3 ml) were placed in the upper column of the

Protein Determination- Protein was determined according to apparatus. The gels were prepared according to the method ofthe method of Lowry et al. (19). Crystalline bovine albumin, Davis (25). The concentration of acrylamide in the small poreFraction V from bovine plasma, was used as a standard. separator gel was 7.5%. The height of the gel in the column was

Determination of Molecular Weight---The molecular weight of approximately 2 cm. Tris-glycine buffer (0.05 M, pH 8.3) waseach protease was estimated by the method of Andrews (20) the electrolyte solution at the cathode and anode. Current forusing a calibrated Sephadex G-100 column o f inner dimensions the electrophoresis was set at 5 ma. Tris-N,N, N,N-tetra-2 cm x 50 cm. All experiments were conducted at 4. Each of methylethylenediamine buffer (0.37 M, pH 8.9) was used forthe standard proteins was dissolved in 1 ml of buffer for applica- elution at a rate of 0.5 ml per min; 2-ml fractions were collected.tion to the column. Sucrose was added to each sample and the The column containing the gel was cooled throughout the electro-higher density protein charge carefully layered above the Eel. phoretic run with circulating cold water (4). Time of the runCollection of the column effluent in 1.5-ml fractions was begun was 3 hours.with the addition of the sample to the top o f the gel bed. The Temperature Stability-The temperature stabili ty o f the twoflow rate of the column was maintained at a constant rate (45 ml proteases was determined by incubating each enzyme at differentper hour) by variation in hydrostatic pressure obtained by ad- temperatures for a 20-min period, removing the enzyme on com-justment of the position of the buffe r reservoir. Fractions were pletion of the incubation period, placing i t immediately into icecollected in an LKB fraction collector and the absorbance at 280 for 1 min, and using an aliquot of the enzyme in a regular assay atrnp was monitored with the use of an LKB XJvicord II and re- room temperature.corder. Each protein sample was run individually. The exact A more sensitive indication of temperature stabili ty was ob-height of the gel ked in the column was maintained constant tained by incubating each enzyme at 53 and measuring loss ofthroughout the experiment. Values used for the rrolecular enzymatic activi ty with time. Aliquots were withdrawn at dif -weight of reference proteins nere as follows: cytochrome c, ferent times, placed immediately into ice for 1 min, and an ali-12,400; ovalbumin, 45,000; bovine pancreatic trypsin, 23,425; quot was removed and assayed for enzymatic act ivi ty at roombovine serum albumin, 66,500; and soybean trypsin inhibitor, temperature.21,200. Each Dermasterias protease was also applied to the col- pH Optima and Stabili ty Studies-To determine the pH optimaumn individually. The protein concentration and trypt ic ac- of the two proteolytic enzymes, the substrate (BAPA) was pre-tivi ty of the fractions was determined spectrophotometrically. pared in various buffer solutions ranging from pH 2.0 to 10.0.

Specijicity Studies-Porcine glucagon was selected for the de- Assays with a constant amount of enzyme were carried out attermination of spec ific ity of each protease because its amino acid various pH values, always using as a blank the substrate at thesequence is known (21) and it is a rela tively small protein. appropriate pH.Cleavage speci fici ty on this protein was determined for the star- To study the stabilities of the two proteolytic enzymes with re-fish proteases and for TPCK-treated bovine trypsin. Tryptic spect to pH, equivalent amounts of enzyme were incubated inpeptides were isolated by high voltage electrophoresis using a buffers of varying pH at room temperature for 1 hour. Aliquotsprocedure similar to that described by Brown and Hartley (22). were then removed and placed in the standard assay system usingAmino acid analyses of eluted peptides were performed on a BAPA as substrate. The 0.01 M buffer solutions used were asBeckman-Spinco model 120 C amino acid analyzer (23). follows: HCl-KC1 buffer, pH 2.0; acetate buffer, pH 4.0; phos-

ilnalytical Polyacrylamide BlectrophoresisThe method of phate buf fer, pH 6.0; Tris-HCI buffer, pH 8.0; phosphate buffer,Ornstein (24) and Davis (25) was followed. Samples containing pH 8.0; glycine-NaOH buffer, pH 10.0.50 to 125 pg of protein were mixed with the sample gel (0.2 ml) Tissue Localization of Starfish Trypsin-like Proteases-To de-and placed in a small tube (inner dimensions 6 x 120 mm) termine the specifi c tissue location of the proteases, a D. imlrri-which had been covered with Parafi lm at one open end. After cata starfish was dissected and the gonads, pyloric caeca, and theEach1 The abbreviations used arc: BAPA, benzoyl-DL-arginine- pyloric and cardiac regions of the stomach were removed.p-nit,roanilide hydrochloride; TPCK, L-l-tosylamido-2-phenyl- tissue was weighed and homogenized in cold 0.05 M Tris buffer,ethyl chloromethyl ketone; TLCK, N*-tosyl-L-lysylchloro- pH 8.2, and the enzymatic act ivi ty per g, wet weight, in each_. . - .methane; JIPP, diisopropyl phosphorofiuorldate. tissue was determined.

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3966 Starfish Trypsin-like Proteases Vol. 245, No. 15Inhibitor Studies-The eff ect s of a number of trypsin inhibitors

on the two proteases were examined by incubating the enzymewith each test substance for different periods of time and deter-mining residual enzyme activity.Concentrations of soybean trypsin inhibitor ranging from 1 to60 pg were incubated with the enzyme for 5 min, at which timethe mixture was added to the substrate BAPA and the enzymeact ivi ty determined. TLCK (0.9. x low6 M) incubation with theenzymes was carried out for a period of 10 min. DFP concentra-tions used for inhibitor studies were 1.1 x 10B3 M and 4.3 x 10esM. Incubation with the enzyme was for a 5-min period. Con-trols without inhibitor were run in each case.Ultracentrifugal Analysis-A Spinco model E analytical ultra-centrifuge equipped with phase plate schlieren optics and RTICautomatic control was used for the determination of the proteaseB enzyme sedimentation coeff icien t. Sedimentation velocityexperiments were done at 60,000 rpm in an An-D rotor containing2 double sector cells with quartz windows. The maximum ordi-nate on schlieren photographs determined from measurementsmade with a microcomparator (Gaertner Scientif ic Corporation)was used to obtain the sedimentation coefficient.Enzyme Activation and Calcium Requirement Studies-To de-termine whether the tryptic-like enzymes exist in the pyloriccaeca in an inactive form, with subsequent activation, a starfishwas sacrificed, the pyloric caeca were rapidly removed, placed in0.05 M Tris buffer, pH 8.2, and immediately homogenized for 2min in a blender. Two-milliliter aliquots of the homogenatewere then mixed with an equal volume of the same buffer con-taining the following additions: (a) none, (b) 0.02 M calciumchloride, (c) 0.02 M calcium chloride plus 55 ~g of bovine pan-creatic trypsin, or (d) 55 pg of bovine pancreatic trypsin . Thesamples were then incubated at 20 and assayed for trypti c ac-tiv ity , with BAPA as substrate, as a function of time. An addi-tional sample, with no additions, was incubated at 0.The ef fect of calcium on the proteolytic activ ity of the full yactivated enzymes was determined by incubating the enzyme ob-tained in an homogenate fraction, activated in the absence ofcalcium, with different concentrations of CaClz (0.001 M and0.02 M) for 15- and 30-min periods. Aliquots were removed atthese times and assayed for trypt ic act ivi ty using BAPA as sub-strate. EDTA eff ect s on enzymatic act ivi ty were determined inthe same manner using lo+ M and 1e3 M EDTA. Controls inthe absence of CaClz or EDTA were run simultaneously. De-ionized distilled water was used to prepare all reagents.To determine whether the stabili ty of the starfish proteaseswas affected by the presence of calcium, each enzyme was incu-bated at 53 with and without 0.02 M CaC12. Aliquots were re-moved at various times, chilled immediately , and assayed forresidual act ivi ty at room temperature by the standard procedurewith BAPA as substrate.

Enzyme Isolation and Purificatiolz-Initial attempts to extractthe starfish enzymes with 0.25 N H&Sod, as described by North-rup, Kunitz, and Herriot (26) for the purification of vertebratetrypsins, proved unsuccessful as no enzyme activit y was detectedin the homogenate. Enzymatic act ivi ty was observed, however,when 0.05 M Tris-HCl buffer, pH 8.2, was used in the extractionprocedure. When the pH of the homogenate was lowered to pH1.7 (pH of acid extraction), complete enzymatic denaturationwas noted within 10 min. Therefore, live D. imbricata starfishwere dissected and the pyloric caeca removed and placed in cold0.05 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 Ten-Broeck tissue homogenizer for 5 min and the homogenate centri-fuged at 3000 x g for 10 min in a Sorvall (RC 2B) superspeedcentrifuge to remove large insoluble tissue fragments. Poly-oxyethylene lauryl ether was added to the viscous homogenate togive a 0.2% solution and the mixture allowed to sit at 4 for 24hours. In early experiments it was found that there was anincrease in enzymatic act ivi ty when homogenates were lef t stand-ing for several hours or when they were frozen and thawed, sug-gesting that the enzymes were slowly being solubilized, or acti-vated, from a particulate fraction. Further experimentsshowed that solubilization of the proteases was greatly facilitatedby the detergent treatment described above.The homogenate was then centrifuged at 16,000 x g for 30min. The supernatant fraction was differentially precipitatedwith solid (NH&SOI and that portion precipitating between 40and 60% saturation was collected by centrifugation at 20,000 x gfor 20 min. The sediment was suspended in cold 0.05 M Trisbuffer, pH 8.2, and subsequently dialyzed against the same buffe rovernight, the buffer being replaced periodically.An acetone powder was then prepared from the dialyzed solu-tion by the method of Morton (27). This was done by precipi-tating the protein with cold ( - 15) acetone and drying the pre-cipitate with cold peroxide-free diethyl ether at 4. The powderwas suspended in ice-cold 0.05 M Tris buffer, pH 7.5, mechani-cally stirred at 4 for 30 min to permit complete solubilization ofthe enzyme, and centrifuged at 20,000 g for 45 min to remove en-zymatically inactive insoluble material. The supernatant frac-tion was pumped directly onto a large Pharmacia model KlOO/100 column (inner dimensions 100 mm x 1,000 mm) packed withSephadex G-100, equilibrated, and eluted with 0.1 M phosphatebuffer, pH 7.5. Eluted fractions containing trypt ic activity wereconcentrated by pressure dialysis by using a UM-10 membranein a Diaflo model 410 ultrafiltration cell. The concentrated en-zyme was then dialyzed against the same buffe r and placed on aDEAE-cellulose column.DEAE-cellulose (DE-52, microgranular) was purchased fromWhatman and treated according to the manufacturers instruc-tions. The slurry was poured into a column with inner dimen-sions of 5.5 X 65 cm. The enzyme was eluted at 4 using a lineargradient 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 col-lected. Two cleanly separated peaks of trypti c act ivi ty were ob-served. These were termed proteases A and B in the order oftheir elution from the column. However, protease B was foundto be contaminated with carboxypeptidase act ivi ty. Attemptsto separate the carboxypeptidase act ivi ty from the protease Benzyme using a smaller DEAE-cellulose column (inner dimen-sions 1.5 x 30 cm) and eluting with a slightly shallower gra-dient than the previous column (0.1 to 1.0 M phosphate buffer,pH 7.5) proved unsuccessful. Additional attempts to separatethese two enzymatic activi ties on a Sephadex G-100 superfinecolumn were also unsuccessfu l. Successfu l separation of theprotease B enzyme and carboxypeptidase was achieved on asmaller DEAE-cellulose column (Pharmacia) of inner dimensions1.5 X 30 cm.The enzyme eluted from the Sephadex G-100 superfine columnwas concentrated as described above and dialyzed against 0.01 brphosphate buf fer , pH 7.5. The protein charge was then placedon 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 3967TABLE I

Isolation and purification of tryptic-like enzymes fromDermasterias imbricata starfish

Purification procedureHomogenate..................40 to 60% (NHI)~SO~ precipi-tate.Acetone powder. .Sephadex G-100..DEAE-celluloseProtease A..Protease B.Further purification of proteaseAb

33,00013,50013,2001,170

18044

Sephadex G-100 _,DEAE-cellulose.Preparative acrylamide elec-trophoresisFurther purification of proteaseBbDEAE-cellulose

49112

0.1-l .OM.. . . . . . . . . . . . . . . . . . 240.01-0.1 M.................. 4

Protein

fw

---

-

Totalactivity Specificactivity

3,825 0.12 1003,780 0.28 98.81,705 0.13 45.11,256 1.1 32.0

376 2.2 9.8716 16.3 18.7

275 5.6 7.2180 16.3 4.746 23.0 1.2

688 28.7 18.0212 53.0 5.6

Yield

%

a Proteases A and B were separated by this initial DEAE-cellulose step. Their further purification is described separately.b Yield calculated from the total initial act ivi ty.buffer, pH 7.5, and eluted with a shallow gradient of 0.01 M to0.1 M phosphate buffer, pH 7.5. The flow rate was adjusted to1.0 ml per min and 2-ml fractions were collected. Fractionscontaining tryptic act ivi ty were combined and concentrated asdescribed above. A single band of protein was noted for thepurified protease B following analytical acrylamide electro-phoresis.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 phos-phate buffer , pH 7.5; 2-ml fractions were collected. Fractionscontaining tryptic acti vity were pooled, concentrated as de-scribed above, and dialyzed against 0.01 M phosphate buffer, pH7.5. Further purification was achieved on a DEAE-cellulosecolumn (1.5 x 30 cm). The enzyme was eluted with a lineargradient, of 0.01 M to 0.10 M phosphate buffer, pH 7.5. Fractionscontaining trypsin act ivi ty were pooled and concentrated asabove. The presence of three protein bands were observed onanalytical acrylamide gels at this stage of the purification. Theconcentrated enzyme was then subjected to preparative acryl-amide electrophoresis. Two-milliliter fractions were collectedand the absorbance at 280 rnp was monitored with an LKB Uvi -cord and recorder assembly. The criterion for the purity of theenzyme was again the appearance of a single protein band follow-ing analytical acrylamide electrophoresis.

RESULTSTissue Localization of Dermasterias Tryptic-like Proteasea-A

major fraction of the tryptic-like act ivi ty in Dermasterias tissueswas found to be located in the pyloric caeca (9.24 units per g, wet,weight). Lesser amounts were found in the cardiac and pyloricregions of the stomach (3.3 and 3.2 units per g, wet weight, re-

I I I0 10 20 30 40 50 60

FRACTION NUMBERFI G. 1. Sephadex G-100 gel filtration of the acetone powderpreparation of Dermasterias proteases using a lOO- X 1000~mmcolumn. Each fraction contained approximately 100 ml. Elutionwas with 0.1 M phosphate buffer, pH 7.5. Fractions 20 to 55 werepooled and used for further purification.

spectively) . No act ivi ty was noted in other tissues. For thisreason, pyloric caeca were utilized as a source of the tryptic-likeenzymes for further purification.Enzyme Isolation and Pur$ication-The purification of thestarfish trypsins is summarized in Table I. Differential (NH&-SO, precipitation was successful in purifying the enzymes slightly

with little loss of enzymatic act ivi ty. In this particular experi-ment,, the acetone powder preparation of the protein resulted in alarge loss of enzymatic act ivi ty with no detectable purification.This is in contrast to earlier preparations of the enzymes wherethis particular step resulted in both high yield and purification.

As shown in Fig. 1, a single peak of tryptic-like act ivi ty wasobtained after Sephadex G-100 chromatography. A 9.2-foldpurification was achieved with this step. The two tryptic-likeenzymes were clearly separated in the initial DEAE-cellulosecolumn (Fig. 2) and designated as proteases A and B. The figureshows contamination of the protease B with carboxypeptidaseactivity.Attempts to separate carboxypeptidase and protease B trypticacti vity on a smaller DEAE-cellulose column by eluting with thesame gradient (0.1 M to 1.0 M phosphate buffe r, pH 7.5) at, a veryslow rate of ionic change resulted in considerable purification ofboth enzymes but no separation of the two enzymatic activi ties.Separation of these two enzymatic activi ties was accomplishedon a second DEAE-cellulose column eluted with a shallowergradient, as shown in Fig. 3. This step resulted in a large loss ofenzymatic act ivi ty. The final product, however, appearedhomogeneous, as judged by the presence of a single band of pro-tein on analytical polyacrylamide electrophoresis. The purifiedprotease B had a specific act ivi ty 3.2 times greater than that o fcrystalline bovine trypsin using BAPA as a substrate and theenzyme was purified 465-fold from the crude extract. The en-zyme sedimented as a single symmetrical peak in the analyticalultracentrifuge with a .SPO,~ f 2.5 S at a concentration of 2.0 mgper ml, in 0.1 M potassium phosphate buffer, pH 7.5.Protease A, from the initial DEAE-cellulose column was puri-fied slightly in a second Sephadex G-100 column step. Furtherpurification was achieved in a DEAE-cellulose column elutedwith a shallow ionic gradient. Final purification of this enzymewas accomplished by preparative polyacrylamide electrophoresis(Fig. 4). BAPA hydrolysis by the purified protease A indicated

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3968 StarJish Trypsin :-l&e Proteases Vol. 245, No. 15+ 0.8, ,0.2

0 40 80 120 160 200

a

FRACTION NUMBERFIG. 2. DEAE -cellulose chromatography of the Dermasteriasproteases following the gel filtration step. Gradient elution was

carried out with 0.1 to 1.0 M phospha te buffer, pH 7.5. Fractionsof 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. Tryps in activity, 0 ; carboxy-peptidase activity, A; protease A, A ; protease B, B.

b OT+0442i Ii I A Ii0- 03dzgo.25z> 0.1Ncl

0 40 80 120 160 200FRACTION NUMBER

FIG. 3. Separation of carboxypeptidase and protease B activityby DEAE-c ellulose chromatography. The column (1.5 X 30 cm)was equilibrated with 0.01 M phosphate buffer, pH 7.5, and elutedwith 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.

a specifi c act ivi ty 1.3 times higher than that of crystalline bovinetrypsin and represented a 202-fold purification. The enzymemoved as a single band on analytical polyacrylamide electro-phoresis at several protein concentrations. Insuf ficient materialwas available for ultracentrifugal analysis.Determination of Molecular Weight-The approximate molecu-lar weight of each protease was determined by means of a Sepha-dex G-100 column according to the method of Andrews (20) asdetailed under Experimental Procedure. The results arepresented in Fig. 5. By this method, the molecular weight ofboth starfish proteases is 25,000 to 26,000. This figure differsonly slightly from the molecular weight of bovine pancreatictrypsin (23,425). In this particular experiment, a molecularweight of 24,000 for the bovine enzyme was obtained by gel filtra-tion.

Inhibitor Studies-The effect s of DFP and TLCK on thetryptic act ivi ty of both starfish proteases and on bovine pan-creatic trypsin, run as a control, are shown in Ta.ble II. Both

0 20 40 60 80 100FRACTION NUMBER

FIG. 4. Purification of protease A by preparative polyacryl-amide electrophores is. See Experimental Procedure for de-tails.

ELUTION VOLUME (ml)FIG. 5. Estimation of the molecular weights of Dermasterias

tryptic-like proteases by gel filtration on Sephadex G-100. Thecolumn (2 X 50 cm) was equilibrated and eluted with 0.1 M potas-sium phospha te buffer, pH 7.5. The void volume, determinedwith dextran blue 2000, was 51 ml. Bovine serum a lbumin, 0;ovalbumin, n protease A, 0; protease B, 0 ; bovine pancrea tictrypsin, A; cytochrome c, a; soybean trypsin inhibitor, V.

TABLE IIInhibition of proteolytic activity by spe cijic inhibitors

I I DFP I

Bovine trypsin. .Protease A. . .Protease B. .

pmole0.00170.00200.0018

I I70 inhibilion

88 3984 2789 I I

6248

23 57

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


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Issue of August 10, 1970 Z. Camacho, J. R. Brown, and G. B. Kitto 3969

g 50-fz5 25-[r:s I

20 30 40 50 60 70TEMPERATURE, CFIG. 6. Thermal inactivation of the two proteases from Derma-sterias imbricata. Each enzyme was incubated at the indicatedtemperatures for 20 min and an aliquot was removed and assayedfor enzyme activity in a regular assay at room temperature.Protease A, 0 ; protease B, 0.

I I I I IIO 20 30 40 50INCUBATION TIME (min)

-

FIG. 7. Eff ect of calcium ion on enzyme stability of proteases Aand B. Each protease was incubated at 53 in the presence andabsence of calcium and equivalent units of enzyme removed atdifferent times and the residual enzyme act ivi ty determined atroom temperature using BAPA as substrate. Protease A +Cal&, 0 ; protease A - CaCla, A; protease B + and - CaCla, 0.bovine pancreatic trypsin by soybean rypsin inhibitor was ob-served in a concurrent control experiment.Stability Measuremenfc-Temperaturestability measurementsshowa drop in enzymatic activity for eachproteasewith increas-

I I I I2 4 6 8 10

PHFIG. 8. Effect o f pH on stability of D. imbricata proteasesAand B. Each enzyme was incubated at the indicated pH valuesfor 1 hour at room temperature and the residual enzymic act ivi tywas determined in the standard assay. ProteaseA, 0 ; proteaseB, l .

PHFIG. 9. Effect of pH on the activ ity of proteases A and B fromD. imbricata. Protease A, 0 ; protease B, 0.

ing temperature (Fig. 6). After heating at 60 for 20 min, noenzymatic activity wasdetectable n the proteaseB fraction andonly a slight amount of the original activity was sti ll present nthe proteaseA preparation. When eachenzyme was ncubatedat 53 and aliquots withdrawn at different times, a marked dif-ference in the temperature stability of the two enzymes wasnoted (Fig. 7).The effect of pH on the stability of the Dermasteri~ proteasesis illustrated in Fig. 8. While purified proteaseA is relativelystableat pH 2, retaining 58% of the initial activity after an hourat this pH, the proteaseB is completely nactivated by this pro-cedure and showssomedenaturation even at pH 6. The pro-teaseA retained full activity over the pH range 6 to 10 undertheseconditions.Metal Ion Studies-Initial experiments indicated that thetryptic-like activity of Dermasteriasxtracts remained nchangedfor long periodsof time, in the absence f addedcalcium,suggest-ing the lack of a calcium requirement or protein stability or ac-tivity. Further studies showed hat the starfish enzymeshad


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3970 StarJish Trypsin-like Proteases Vol. 245, No. 15-4 j.His - Ser - Gln - Gly - Thr - Phe - Ser - Asp - Tyr - Ser - Lys - Try - Leu - Asp - Ser - Arg -

+ + + +

+ +- Arg - Ala - Gln - Asp - Phe - Val - Gln - Trp - Leu - Met - Asn - Th rFIG. 10. Cleavage specificity of Dermasterias proteases A and B on glucagon . See text for details. A control experiment with TPCK-treated bovine pancrea tic trypsin gave cleavage products identica l w ith those obtainedwith protease B. Protease A, T ; protease B, 1 .

I I I I I I I I

0t t

2 0.2-w

I I I I I I I I0 IO 20 30 40 60 80 100 120 140TfME (min)

FIG. 11. Activation of tryptic-like proteases in a crude homog-enate of Dermasterias pyloric caeca . Live starfish were dissec tedand the pyloric caeca homogenized with 0.05 M Tris buffer, pH 8.2,for 2 min in a Waring Blendor. Separate 2-ml aliquots weremixed with equal volumes of (a) 0.05 M Tris buffer (0) ; (b) 0.02 Mcalciu m chloride in Tris buffer (0); (c) 55 rg of bovine pancrea tictrypsin in Tris buffer (A); (d) 0.02 M calcium chloride + 55 pgof bovine pancrea tic trypsin in Tris buffer (A) and incubated at20. A further samp le (e) in Tris buffer was incubated at O(m).Aliquots were removed at the indicated times and assayed forBAPA activity under the standard assay condition s.

identical activities, with BAPA as substrate, n the presence rabsenceof CaClz or EDTA. Neither does calcium appear toexert any marked stabilizing effect on the Dermasterias tryptic-like proteases. When the temperaturestabilities of the enzymeswere examinedat 53 in the presence nd absence f CaClz (Fig.7), no effect of the metal ion was seenwith the proteaseB andonly a very slight protection, almost within the range of experi-mental error, was observedwith proteaseA.pH Optima-The effect of pH on the activity of the Derma-ste-rias proteasess shown n Fig. 9. Both enzymesshowmaximalactivity in the pH range8 to 8.5 with BAPA assubstrate.Speci$city of Dermasterias Tryptic-like Proteases-The hy-drolytic specificity of the starfish proteaseswasexaminedusingglucagon as a model substrate. A parallel control study wasperformed using TPCK-treated bovine pancreatic trypsin. Af-ter separationand characterization of the peptides rom hydroly-satesof glucagon,asdescribed nder Experimental Procedure,it was apparent that the starfish proteaseB rapidly cleaved hebonds on the COOH-terminal side of lysine (position 12) andarginine (positions17 and 18). An identical pattern of cleavagewasobservedwith bovine trypsin. A similar specificity wasob-

served with the Dermasterias roteaseA except that additionalcleavagewasnoted at the Ser-Lys bond (positions11and 12)andat the Ser-Arg bond (positions16 andl7). The resultsare sum-marized in Fig. 10. Excellent yields of all peptides were ob-tained, enabling an unambiguous nterpretation of cleavagepoints. The additional hydrolysis observedwith the proteaseAmay have resulted from minor contamination with carboxypep-tidase B-like activity, although no suchactivity was observableusinga short term regular assaywith hippuryl-L-arginine assub-strate. In peptide mapsof the starfish protease-treatedgluca-gon, several weakly staining peptides were observed n additionto the major products. Quantitative aminoacid analysisof theeluted minor peptidesshowed hat thesepeptideswerepresent nyields ess han lo%, and n mostcasesess han 5a/ of the majorhydrolytic fragments.Activation of Tryptic-lilce Proteaaes in TissueHomogenates--Inorder to investigate the possibility that the Dermasterias pro-teases xist in the pyloric caeca n zymogen orm, fresh tissueex-tracts were incubated at 20 in Tris buffer alone and with theaddition of calcium chloride and bovine pancreatic trypsin andaliquots were assayed t various times for BAPA activity. Theresultsof suchan experiment are llustrated in Fig. 11. On incu-bation at 20, spontaneous ctivation wasobserved, he rate ofactivation being considerablyenhancedby the presence f cal-cium and exogenous rypsin. No activation was observedwitha samplekept at 0.DISCUSSION

Studieson the tissuedistribution of tryptic-like enzymes n thestarfishD. imbricata revealed hat a major part of this enzymaticactivity is ocated n the pyloric caeca,a finding which correlateswell with the physiology of theseorgans,which servemany of thefunctions of an exocrine pancreas. Pairs of pyloric caeca iealongeacharm of the starfishand are connected y largeducts othe pyloric stomach. Histological examination by Anderson(28) has shown he presence f numeroussecretory cells n thesaclikeglandular pouches f the pyloric caeca. Extensive granu-lar inclusionsn thesecellswere presumed o be zymogen gran-ules. Evidence for the importance of the pyloric caeca n thedigestive process asbeenprovided by Anderson (29) who foundthat starfish with the pyloric caeca emovedwere able to ingestfood normally but were unable to digest t. It is possiblehatthe smaller amounts of tryptic activity observedwith starfishstomachextracts may have originated in the pyloric caecaandremained attached to the stomach epithelium, as the stomachwall lacks he zymogen-likecellssoapparent n the pylorio cseca.The present esults on the activation of the trypsin-like pro-teases n Dermasterias re consistentwith theseenzymesoccur-


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Issue of August 10, 1970 Z. Camacho, J. R. Brown, and G. B. Kitto 3971ring in an inactive zymogen form in the pyloric caeca. Whetheractivation is normally accomplished by a simple autocatalyticprocess or whether a specif ic enterokinase is involved, as is thecase with vertebrate trypsins, awaits further study.

The finding of two tryptic-like enzymes in Dermasterias paral-lels the preliminary report of two trypt ic enzymes being presentin the pyloric caeca of Evasterias troche& (16). Whether thetwo tryptic enzymes in starfish represent separate gene products,or whether they are modifications of the same protein must awaitdetailed structural examination. The latter possibility is sug-gested by the recent studies of Schroeder and Shaw (30), whodemonstrated the formation of several active forms of bovinetrypsin upon autocatalytic activation of the zymogen.

The two Dermasterias proteases differ most markedly in theirsens itiv ity to denaturation, both by heat and extreme pH. Theprotease A shows considerably greater thermostability than doesprotease B and retains partial act ivi ty after 1 hour at pH 2, afeature more similar to the vertebrate trypsins. It is of interestthat both starfish enzymes show significantly more act ivi ty withthe synthet ic substrate benzoyl-nn-arginine-p-nitroanilide thandoes bovine pancreatic trypsin, the starfish proteases A and Bhaving 1.3 and 3.2 times, respectively, the act ivi ty of the bovineenzyme.Both Dermasterias tryptic-like proteases have molecularweights of approximately 25,000 which is similar to that of thevertebrate trypsins. The few studies available on other inverte-brate tryptic-like enzymes indicate similar molecular weights forthese enzymes. Winter and Neurath (16) found one of thetryptic enzymes from Evasterias to have a molecular weight, bygel filtration, of 25,000. A molecular weight of 24,700 was ob-served for the trypsin for the cray fish Orconectes, and Bewleyand DeVillea (7) have estimated a molecular weight of approxi-mately 26,000 for a trypsin isolated from the earthworm Lumbi-cus terrestris.The observed pH optima of the Dermasterias proteases is con-sistent with their action in the slightly alkaline medium of thestarfish stomach (14,31) and is similar to the pH-activi ty profilesof vertebrate trypsins (17, 32). Similar pH optima were re-ported for trypsins from Crustacea (6) and from the earthwormLumbricus (7). The DwmasteriQs enzymes are virtua lly unaf-fected in stabili ty by the presence o f calcium and are not in-hibited by EDTA. In contrast, the vertebrate trypsins aremarkedly stabilized by calcium (33,34) and activated by this ion(35). Zwilling et al. (36) found no calcium eff ect s with a crayfishtrypsin and Winter and Neurath (16) reported that EDTAslightly activated a trypsin from the starfish Evasterias.In the initial studies on the Dermasterias proteases the twoenzymes isolated were referred to as tryptic-like on the basisof their ability to hydrolyze the synthetic substrate benzoyl-n-arginine-p-nitroanilide. That the invertebrate enzymes are atleast functionally analogous to vertebrate trypsins is suggestedby the results o f experiments with specifi c inhibitors, and on theanalysis of the cleavage products of protease-treated glucagon.Both starfish proteases were inhibited by DFP and by TLCKsuggesting an essential role for a serine (37) and a histidine resi-due (38) respect ively, in the cataly tic function of these enzymes.Unlike many vertebrate trypsins (23) the Dermasterias enzymeswere unaffected by even high concentrations of soybean trypsininhibitor. Recently, Travis and Roberts (39) reported thathuman trypsin is not inhibited by soybean trypsin inhibitor andsuggested that this might be related to the reduced number of

disulfide bonds in the human enzyme compared with other verte-brate trypsins.The hydrolysis products from the starfish protease digestion of

glucagon provided further evidence for the similarity of theDermasterias enzymes and vertebrate trypsins. The Dermaste-rias protease B gave as major hydrolytic products peptides iden-tical with those obtained with bovine pancreatic trypsin, withthe expected cleavage on the carboxyl side of lysine and arginineresidues. The starfish protease A enzyme gave similar productsexcept for additional cleavages at Ser-Lys bond at positions 11and 12 and a Ser-Arg bond at positions 16 and 17. The addi-tional splits observed with the protease A could result either froma broader spec ific ity of the starfish enzyme compared with bovinetrypsin, or from a slight contamination with carboxypeptidaseact ivi ty. Although no carboxypeptidase act ivi ty was detectablein the enzyme in a regular assay, it is possible that amounts suf fi-cient to produce hydrolysis of the glucagon peptides over the longincubation time employed would be undetected by this proce-dure.

The present results clearly indicate close similarities betweenthe two tryptic-like proteases of D. imbricata and vertebratetrypsins. In particular, the results obtained with the speci ficinhibitors DFP and TLCK and the cleavage specifi city of thestarfish enzymes are suggestive of homology with vertebratetrypsins, but defini tive evidence must await detailed structuralanalysis. Purification of the Dermasterias enzymes, in sufficien tquantities to permit such studies, is in progress.

AcklzowledgmentsWe thank Glenda Anderson, KatherineYndo, Sharyn Marshall, Vivian Sellers, and James Lemburg fortheir assistance in various aspects of this work.

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phys., 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 . Ph ysiol.,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. B iol., 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.

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3972 Starfish Trypsin-like Proteases Vol. 245, No. 1521. 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. , .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., Crystal-line enzymes, Columbia Universi ty Press, New York, 1955,p. 262.27. MORTON, R. K., in S. P. COLOWICK AND N. 0. KAPLAN (Edi-tors), Methods in enzymology, Vol. 1, Academic Press, NewYork, 1955, p. 34.28. ANDERSON, J. M., Biol. Bull., 106,47 (1953); in R. A. BOOLOO-

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Camacho Et Al 1970. Purification and Properties of Trypsin-Like Proteases From the Starfish Dermasterias Imbricatae