Comp. Biochem. Physiol. Vol. 91B, No. 4, pp. 647-650, 1988 0305-0491/88 $3.00 + 0.00 Printed in Great Britain © 1988 Pergamon Press plc

CHARACTERISTICS OF A PROTEINASE FROM OVARIAN FLUID OF THE LUMPSUCKER

(CYCLOPTERUS LUMPUS L.)

ARNT J. RAAE,* JOHN DAVENPORT'[" and BERNT WALTHER*

*University of Bergen, Dept of Biochemistry, Arstadveien 19, 5009 Bergen, Norway (Tel: 29-17-00) and tUniversity College of North Wales, Marine Science Laboratories, Menai Bridge,

Anglesey LL59 5EH, UK

(Received 23 December 1987)

Abstract--1. A proteinase has been isolated from the ovarian fluid of the lumpsucker (Cyclopterus lumpus ).

2. The enzyme was purified essentially to homogeneity by a one step purification procedure using anion-exchange chromatography.

3. The mol. wt of the denatured enzyme is approximately 20,000 as judged by SDS-polyacrylamide gel electrophoresis.

4. The enzyme is inhibited by serine-proteinase inhibitors and acts in the manner of a trypsin-type proteinase both with respect to specific peptide substrates and enzyme inhibitors.

5. The lumpsucker proteinase exhibits low general proteolytic activity but acts effectively on the specific chromogenic peptide substrates.

INTRODUCTION

The lumpsucker, Cyclopterus lumpus L., is a cottoid teleost which spends most of its life offshore, but moves into shallow water to spawn during spring.

While in the ovarian fluid within the oviduct, the eggs of Cyclopterus are soft and covered with a viscous mucus (Fulton, 1907), but they do not stick to one another. Within seconds of being expelled into sea-water, they become extremely sticky and isolated eggs adhere to one another readily for about the next 30min (Davenport et al., 1983). After ½hr the stickiness of isolated eggs declines rapidly until isolated eggs, or any eggs broken from the egg mass, are unable to re-attach (6-24 hr after release from the female parent). Both stickiness and egg hardening are induced by the high concentration of Ca 2+ and Mg 2+ present in sea-water (Lonning et al., 1984).

The adhesion of lumpsucker eggs to one another to form a semi-rigid lattice appears to result from condensation of the viscous slime into an elec- tron dense, papillate layer on the chorion (L~nning and Hagstrom, 1975; Davenport et al., 1983) which generates thick circular patches at the inter- faces between eggs. The viscous slime, which appear to be of a proteinaceous type, forms transparent non-rigid strands immediately upon exposure to sea-water. The condensation of the viscous slime is accompanied by a release of NH3 (Davenport et al., 1983). In this paper we describe the isolation, characterization and possible function of a tryp- sin type proteinase from the ovarian liquid of Cyclopterus.

Mailing address: Arnt J. Raae, University of Bergen, Dept of Biochemistry, Arstadveien 19, N-5009 Bergen, Norway.

MATERIALS AND METHODS

Materials Benzoyl arginine ethyl ester (BAEE), benzoyl tyrosine

ethyl ester (BTEE), phenylmethyl sulphonyl fluoride (PMSF), soybean trypsin inhibitor (STI), casein and apro- tinin were purchased from Sigma. Low tool. wt protein markers were from Bio-Rad, and chromozym TH, chro- mozym TRY, chromozym PL, tosyl-L-leucyl chloromethyl ketone (TLCK) and tosyl-L-phenylalanine-chloromethyl- ketone (TPCK) were obtained from Boehringer. Succinyl- L-alanyl-L-alanyl-L-alanine-p-nitroanilide (Suc-(Ala)3pNA) was obtained from Protein Research Foundation. Mono Q and Superose 12 were purchased from Pharmacia and all inorganic salts were from Merck and of analytical grade.

Collection of ovarian fluid Adult lumpsuckers were collected by gillnets in Sandnes-

sunder, Troms~ (northern Norway), and ovarian fluid was collected as described by Davenport (1983).

Enzyme assays Enzyme activity was measured by a standard assay con-

sisting of 75 raM, Tris-HC1, pH 7.8, 20 mM CaCI2 and 1.0 mM BAEE (total volume of 1 ml). The enzyme assay was carried out in the cuvette (at room temperature, 22°C) and enzyme activity was recorded on a Perkin Elmer 554 spectrophotometer as the increase in absorbancy at 253 nm. One enzyme unit catalyses an increase in A253 of 0.001 per rain at pH 7.8 and 25°C under the specified conditions.

In assays, using the chromogenic peptide substrates, the procedure was essentially identical except for the concentra- tion of substrate which was 0.1 mM and the reaction was monitored at 405 nm.

General proteolytic activity was measured essentially according to the procedure of Drapeau (1976). The assay mixture (0.5 ml) consisted of 1% casein in 50 mM Tris-PO4, pH 7.8. The reaction was carried out for 30 rain at 22°C and stopped by adding 0.5 ml 10% trichloroacetic acid. After centrifugation at 10,000 rpm for 15 min, the A2s o was deter- mined in the supernatant. One unit is the amount of enzyme

647

648 ARNT J. RAAE et al.

which liberates acid soluble fragments equivalent to 0.001 A280 per min under the defined conditions.

Polyacrylamide gel electrophoresis was carried out essen- tially according to Laemmli (1970). The gels (15% w/v acrylamide and 0.1% SDS), were usually run for 4hr at room temperature at 35 mA per slab. Protein bands were visualized by silver staining after the procedure of Marshall and Latner (1981).

Molecular wt determinations under native conditions were carried out on a Superose 12 (HR10/30) column equibrated with 10 mM Tris-HC1 pH 7.6, 10 mM CaC12 and varying concentrations of NaCl.

Enzyme purification Enzyme purification was carried out on a Pharmacia

FPLC (fast protein liquid chromatography) system equipped with a single path UV-1 monitor and LCC 500 control unit.

Ovarian fluid, obtained after mechanical separation from most of the viscous strands, was centrifuged at I0,000 g for 10 min and the supernatant was applied to a Mono Q anion exchanger equilibrated with 10mM Tris-HCl, pH7.8, 20 mM CaC12. The column was washed with the buffer and proteins were eluted with a linear gradient from 0 to 1 M NaC1. The fractions containing enzyme activity were pooled and dialysed against buffer containing 50% glycerol. The enzyme could subsequently be stored at -20°C for several months without any detectable loss of activity.

Protein determination Protein was determined according to Lowry et al. (1951).

RESULTS

Enzyme purification

The enzyme activity in the raw material was very low and hardly detectable by the BAEE standard enzyme assay, as shown in Table 1. The enzyme content in the ovarian fluid corresponded to 8.6 BAEE hydrolysing units per mg protein. After being absorbed onto the anion-exchanger the enzyme activity was released from the column at 0.95 M NaCI. In this step the purification based on protein content is 3.5-fold which is a magnitude of purification normally obtained by ionic exchangers. However, the specific activity of the enzyme fraction is 4000 U/mg which represents a 450-fold increase in specific activity from the crude extract.

Figure 1 presents the protein elution profile from the Mono Q column. The protein profile shows that the crude extract separates into three major protein peaks, one of which does not bind to the exchanger, a second peak which elutes at 0.5 M NaC1 and the third peak, containing the enzyme activity, eluting at 0.95 M NaCI.

Polyacrylamide gel analysis also shows that the protein peak corresponding to the enzyme activity is of high purity. The gel reveals a double protein band with mol. wt of about 20,000-21,000. Gel filtration analysis under native conditions and moderately high salt concentration (0.3 M) resolved this fraction into

three protein peaks. About 30% of the protein was excluded in the void volume whereas the rest eluted in two overlapping protein peaks of tool. wts in the range 10,000-15,000. Of the enzyme activity applied to the column 50% was recovered in the void volume and an additional 24% was found equally distributed in the two low tool. wt protein peaks. Gel filtration at lower salt concentration (0.1 M) did not significantly alter this distribution, whereas at higher salt concentrations, a larger fraction of the enzyme- activity eluted in the void volume.

Enzyme characteristics

In Table 2 the general proteolytic powers of the lumpsucker proteinase are compared with other proteinases such as chymotrypsin, trypsin and thrombin. The table shows that the lumpsucker proteinase hydrolyses casein about 10 times more effectively than thrombin but is over a 100 times less effective than chymotrypsin.

The enzyme was tested against several synthetic chromogenic peptide substrates and the results are summarized in Table 3.

All substrates are cleaved by serine proteinases. Chromozym Try, Chromozym X and BAEE are specialized substrates for Trypsins, and Chromozym PL for Plasmin. BTEE is used to test for chymo- trypsin-type enzymes and Suc(Ala)3 pNA for elastase- like enzymes.

Q

~ ~o

113

.. !i / / , , \ -1,2 / $ J t

I I |

' Y //il -18" I I I | )

- - I ° O

e l ! I ' I

4 8 12 16 Etu/(ion volume (mr)

Ct8

3.4

13.2

Fig. 1. Elution profile of the Mono Q anion-exchanger. Crude extract (0.5ml) was applied to the column and proteins were eluted by applying a gradient of 0-1 M NaC1. Proteins were monitored by the absorbance at 280 nm ( - - ) and enzyme activity was assayed using BAEE as substrate

(O--O).

Table 2. General proteolytic activity

U/mg

Chymotrypsin 3020.0 + 300 Trypsin 1667.0 _+ 200 Thrombin 2.0 + 0.2 C.lumpus proteinase 25.0 4-_ 4

The assay was carried out using casein as substrate as described in Materials and Methods.

Table 1. Purification of proteinase from lumpsucker ovarian liquid

Specific Volume Protein Protein activity

( m l ) (mg/ml) (mg) Units (units/rag)

Crude extract 0.5 0.70 0.35 3 8.6 Mono Q fraction 2.0 0.05 0.10 400 4000

Cyclopterus

Table 3. Activity of the lumpsucker proteinase towards synthetic peptide substrates

Wavelength Substrate (nm)

AA/min #g enzyme C. lumpus Bovine proteinase trypsin

15 50 n.d. n.d. n.d. n.d. 500 1500 500 1200 100 280

BAEE 253 BTEE 256 Suc(ala)3 PNA 405 Chromozym X 405 Chromozym Try 405 Chromozym PL 405

n.d. = not detectable.

The results presented in Table 3 document that the specialized trypsin substrates, chromozyms Try and X, are the best substrates for the Cyclopterus proteinase.

Inhibition studies

The enzyme was tested against several synthetic and natural proteinase inhibitors, and the results are summarized in Table 4.

The serine proteinase inhibitors P M S F and Apro- tinin both accomplish total inhibition of the enzyme, confirming that the enzyme belongs to the serine-type proteinases. The enzyme is also totally inhibited by soybean trypsin inhibitor and furthermore strongly inhibited by the chloromethylketone, TLCK, and less by TPCK. Such a response to the various inhibitors, and especially to the chloromethylketones, classifies the lumpsucker proteinase as a trypsin type enzyme (Keil, 1971). Also crude extract inhibits the enzyme

proteinase 649

Table 4. Effect of inhibitors on the lumpsucker proteinase activity

Inhibitor % Inhibition

None (standard reaction conditions) 0 PMSF 1 nmol 100 Aprotinin 0.5 KIV* 100 STI 5 g g 100 TPCK 10 nmol 0 TLCK 10 nmol 70 Crude extract 15 gg 80 Triton X-100 0.04% 50

Inhibitors and enzyme (2.5#g) in a total volume of 60#1 were preincubated for 20 min before the residual enzyme activity was determined in the standard BAEE hydrolysis enzyme assay.

KIV* = Kallikrein inhibiting units.

to an extent of 80% under the conditions specified in Table 4.

DISCUSSION

This paper describes the isolation and purification of a serine-proteinase from the ovarian fluid of the lumpsucker. The enzyme activity is hardly detectable in the crude extract, both after limited trypsinolysis and addition of high salt concentrations (not shown), but increases about 450-fold after being released from the anion-exchange column. This increase in specific activity is too large to be accounted for by protein purification alone. Several explanations may acocunt for this observation. First, the increase in enzyme activity is of a magnitude which would be expected from an inactive zymogen to an active enzyme. Such

I 2 3 4 5

Fig. 2. SDS-polyacrylamide gel analysis of the protein peaks from the anion-exchange. Lane 1--5 pl crude ovarian fluid; lane 2--20 pl protein peak A, (Mono Q); lane 3--20 pl protein peak B; lane 4--20 pl of enzyme fraction C. The mol. wt standards in lane 5 were phosphorylase B (92,500), bovine serum albumin (66,200), ovalbumin (45,000), carbonic anhydrase (31,000), soybean trypsin inhibitor (21,500) and

lysozyme (14,400). Experimental conditions were as described in Materials and Methods.

650 ARNT J. RAAE et aL

a transformation is normally catalysed by activation enzymes or by self-activation (Kraut, 1971; Keil, 1971). However, there is little evidence that such an event takes place during the short time (20 min) on the ion-exchange column. Also incubation of the purified enzyme with crude extract resulted in 80% inhibition of enzyme activity.

A second explanation is therefore that the enzyme exists complexed to an inhibitor in the crude extract and that the enzyme-inhibitor complex subsequently separates during the ion exchange chromatography step. However, attempts to reconstitute an enzyme- inhibitor complex by combining purified enzyme with the various column fractions failed, even after exten- sive dialysis of the fractions. Thus one cannot exclude the possible existence of a low tool. wt inhibitor which is unable to act at high salt concentrations and which is lost during dialysis.

A third and equally attractive explanation is that the inhibitory action is caused by a naturally occur- ring substrate in the crude extract. The enzyme- substrate complex separates on the ion-exchanger due to high salt concentration and a different affinity to the column material. The failure of the various column fractions to reconstitute the inhibitory activity could therefore either be due to loss of correct three dimensional structure or limited proteolytic degradation of the active form of the substrate. The low general proteolytic activity found to be associ- ated with the lumpsucker proteinase favours such a theory. The proteinase may be associated with the substrate in such a manner that extensive proteolysis does not take place.

SDS-polyacrylamide gel electrophoresis of the active fraction from the anion-exchanger reveals two protein bands of tool. wt 20,000 and 21,000 respec- tively. Gel filtration analysis under native conditions showed that most of the enzyme activity was excluded in the void volume but minor amounts of enzyme activity were found associated with two overlapping protein peaks with mol. wts of about 10,000-15,000. These protein peaks most probably correspond to the double protein band seen by SDS-PAGE, since proteins from fish species are often retarded on gel filtration media (Raae et al., unpublished results). Such retardation is also associated with hydrophobic proteins, and are often enzymes possessing a multi- subunit structure held together by hydrophobic forces. If so, the lumpsucker proteinase subunits may consist of polypeptide chains with mol. wts of 20,000 and 21,000. Equal amounts of enzyme activity were found to be associated with the subunits but the experimental data is at present not sufficient to indicate whether the subunits are of different origin or whether one is a proteolytically processed product of the other.

The enzyme is a serine type proteinase, as judged by the response to the various proteinase inhibi-

tors. Furthermore the enzyme acts in a similar man- ner to trypsin both with respect to several different ehromogenic peptide substrates and the differential response to the chloromethyl ketones TPCK and TLCK.

The proteolytic power of the Cyclopterus proteinase is comparable to that of thrombin, i.e. high specific activity on chromogenic peptide sub- strates and low general proteolytic activity as judged by the poor ability to hydrolyse casein.

It is tempting to speculate that the physiochemical properties of Cyclopterus proteinase may be corre- lated with the peculiarities of spawning of the Cyclo- pterus species, however, more experiments have to be carried out in order to establish the in vivo function of the proteins.

Acknowledgements--We wish to thank Mrs R. Knudsen and Mr H. Sveier for excellent technical assistance and Mr I. F. Pryme for correcting the manuscript.

REFERENCES

Davenport J. (1983) Oxygen and the developing eggs and larvae of the lumpfish, Cyclopterus lumpus. J. mar. Biol. Ass. UK 63, 633-640.

Davenport J., L~nning S. and Kj~svik E. (1983) Ammonia output by eggs and larvae of the Lumpsucker, Cyclopterus lumpus, the cod, Gadus morhua, and the plaice, Pleuronectes platessa. J. mar. Biol. Ass. UK 63, 713-723.

Drapeau G. R. (1976) In Methods in Enzymology (Edited by Lorand L.), Vol. XLV, p. 471. Academic Press, New York.

Fulton T. W. (1907) On the spawning of the lumpsucker (Cyclopterus lumpus) and the paternal guardianship of the eggs. Rep. Fish. Bd Scot. 24, 169-178.

Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.

Lonning S. and Hagstrom B. E. (1975) Scanning electron microscope studies of the fish egg. Astarte 8, 17 22.

Lonning S., Kjorsvik E. and Davenport J. (1984) The hardening process of the egg chorion of the cod, Gadus morhua L., and the lumpsucker, Cyclopterus lumpus L. J. Fish Biol. 24, 505-522.

Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 193-198.

Keil B. (1971) In The Enzymes. Trypsin, Hydrolysis: Peptide Bonds, Vol. III, 3rd edn (Edited by Boyer P. D.), p. 261 265. Academic Press, New York.

Kraut J. (1971) In The Enzymes Chymotrypsinogen : X-Ray Structure II. The Activation Process, Vol. III, Hydrolysis: Peptide Bonds, 3rd edn (Edited by Boyer P. D.), p. 167-169. Academic Press, New York.

Marshall T. and Latner A. L. (1981) Incorporation of methylamine in ultrasensitive silver stain for detecting protein in thick polyacrylamide gels. Electrophoresis 2, 228-235.