Corap.Biochem.Physiol., 1978,VoL 59B,pp. 81 to 85. PergamonPress.

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A NEW PROTEOLYTIC ENZYME ISOLATED FROM M Y T I L US GALLOPRO V I N C I A L I S : MYTILIDASE. PURIFICATION, CRYSTALLIZATION AND PARTIAL CHARACTERIZATI ON
IOAN FLOREA DUMITRU, DANA IORDACHESCU* AND STELIAN NICULESCU*

Faculty of Biology, Bucharest, Romania

(Received 2 March 1977)

Abstract--1. A procedure for the advanced purification of acid protease(s) obtained from the hepatopancreas of the sea mussel, Mytilus galloprovincialis, is described. 2. Proteolytic activities at pH 1.6 and 2.6 could not be separated, although presenting different optimal action parameters. 3. The molecular weight of mytilidase, determined by gel filtration on a Sephade'x" G-200 column, was 36,000 + 600.

INTRODUCTION

The enzymes present in different tissues and morphological formations of the Mytilidae family--Mytilus edulis and Mytilus galloprovincialis--have been the subject of study of numerous investigations carried out by Skardiv (1957), Minafra (1968), Goromsova (1971), Goromsova & Shapiro (1970), De Zwann (1972), De Zwann & Van Merrewijk (1973), Strusi (1964) and Reid (1966). Isolation and study of the proteolytic enzymes of invertebrates and especially of mussels have revealed the presence of enzymes at prevalently neutral or alkaline pH. Thus, Bundy & Gustafson (1973) purified a protease-like trypsin from the starfish, Kozlovskaya & Vaskovsky (1970) carried out a comparative study on some extracts with a proteolytic activity obtained from 50 species of marine invertebrates, Pignero & Rocca (1969) found evidence of marked peptidase and esterase activities in the hepatopancreas of Loligo vulgaris, and Reid & Rauchert (1970), who studied proteolytic enzymes in the bivalve mollusc Chlamys hericius, demonstrated the presence of an activity similar to that of trypsin, chymotrypsin, carboxi- and aminopeptidase in mammals. Reid (1966) showed that in the digestive tube of Mya arenaria, proteolysis takes place at an acid pH and necessitates the activation of enzyme with an endopeptidase activity, and Strusi (1964) mentions that the digestibility of Mytilus galloprovincialis reaches maximum values in the summer months. In an earlier work, we reported on the presence of two proteolytic activities at acid pH in the marine 1975). Study of the distribution of these two proteolytic activities in five morphological formations of the mollusc showed that the hepatopancreas is characterized by accentuated proteolytic activity. Two hypotheses were discussed: the existence of two distinct acid proteases, and the existence of a single protease with two optimal pH values. The former assumption is sus*Laboratory of Enzymology, Institute of Biological Sciences, Bucharest, Romania. 81 c.a.P. 59/IB--F

tained by certain experimental data: at both optimal pH values different specificities with regard to natural substrates, different requirements for saturation with hemoglobin, different optimal temperatures for the reaction, etc. have been recorded. The behaviour of the two proteolytic activities sharply differ from that of pepsin isolated from the stomach of swine, which does not attack peptones. The present paper describes a procedure for the advanced purification of acid protease(s), without succeeding, however, in their chromatographic separation; it likewise deals with certain physicoehemical properties of the purified enzyme, the conditions in which it crystallizes and the shape of the crystals.
MATERIAL AND METHODS

All of the reagents presented a high degree of purity. Hemoglobin, the Folin-Cioc~.lteu reagent, absolute ethylic alcohol and ammonium sulfate were Merck products; polyacrylamide, myoglobin, cytrochrome c were from B.D.H.; Sephadex G-100, SE-Sephadex C-25 Dextran Blue from Upsala Pharmacia and Bio-Gel P-100 from Bio-Rad. The molluscs were collected on the Romanian shore of the Black Sea, selecting shellfish 345 cm long. The hepatopancreases were separated, stored at - 15C, weighed accurately, then triturated in a Potter homogenizer (1 g wet tissue/20 ml water). The tissue homogenate kept for extraction at 4C for 1 hr was centrifuged at 10,000 rev/min for 15min, the supernatant representing the total proteic extract. Proteolytic activity was determined according to the spectrophotometric method described by Anson & Mirsky (1933); the reaction mixture in a final volume of 1 ml contained 10 mg hemoglobin and 0.2-10~g protein and presented two different pH values--l.6 and 2.6. The determinations were performed at the two optimal activity pH's, the value of which varied within restricted limits in terms of the month in which the molluscs were collected. Protein concentrations were measured by the method of Lowry et al. (1951). Electrophoresis in polyacrylamide gel was carried out according to the method described by Davies (1964), in a buffer glycocol Tris-HC1 solution, 1 10-~ M, pH8.6,

82

IOAN FLOREA DUMITRU, DANA IORDACHESCUAND STELIAN NICULESCU

Protein : = : Enzyme at pH 16 -o-o-o- Enzyme at ~ pH 2 6 .~ ~.

~30 I10

tD

0.81 i 07 c 06 0.5

c 90 ~ so ~ 70
5o ~ ~o ~ 50

~. 03 ~ 2' ~3. 0.4:Z OI i i~i ~ 1

iO i5

i
I,,,Jl,,,~l,,,,I
20 25 Tube number

30

35

0 ~ 0

Fig. 1. Elution curve of the enzymatic preparation with acid pH proteolytic activity from a Sephadex G-100 medium column (2.8 25 cm), equilibrated in 0.3% NaC1. The 3 ml fractions from the eluate obtained with the same saline solution were analyzed from the viewpoint of protein concentration and proteolytic activity at pH 1.6 and 2.6. in 8 x 0.4 cm tubes. Electrophoretic separation was obtained at 5 mA/tube and 350 V. After 90 min the electrophoregrams were stained with Amido-Schwartz 10 B, prepared in a methylic alcohol-acetic acid-water mixture (5:1:5), and then deeoloured electrophoretically using a 7% acetic acid solution.

RESULTS

Purification of the enzyme

The total proteic extract was acidulated by treatment with 0.5 N HzSO4 to pH 3. Precipitation was perfected in the course of 1 hr at 4C; the inert catalytic proteins were then removed by centrifugation at 10,000 rev/min for 15 min. The proteolytic enzyme(s) in the supernatant obtained was separated by fractional precipitation with ammonium sulfate. Most of the proteolytic activity was found in the fraction precipitating at 0.34).7 saturation in (NH4)2SO4. The enzymatically active fraction was subjected to gel filtration on a Sephadex G-100 column (2.8 x 25 cm), equilibrated in a 0.3% NaC1 solution. Elution of the proteins was obtained with the same saline solution, at an elution rate of 20 ml/hr, collecting samples of 3 ml each. Figure 1 gives the elution of protein and proteolytic activity. Eluates 22 34 (peak I) exhibiting a remarkable enzymatic activity, were pooled and subjected again to fractional precipitation with ammonium sulfate. Within the 04).4 saturation range formed a proteic precipitate that was removed by centrifugation at 10,000 rev/min. To the supernatant obtained, (NH4)2SO4 was again added up to a saturation of 0.4 and 0.7, when the protein(s) with the proteolytic acivity studied precipitated. The precipitate was then dissolved in a corresponding volume of 0.005 M Na2SO4 and adjusted to pH 3 with 0.5 N H2SO4. The enzymatic preparation, resulting from the prior stage, by precipitation at a saturation of 0.4-0.7 in ammonium sulfate, was separated by chromatography on a Bio-Gel P-100 column (2.8 x 15 cm), previously

equilibrated with 0.005 M Na2SO,, pH 3, collecting 3 m samples. As may be seen from Fig. 2, this chromatographic separation gave three protein peaks, noted in continuation II, III and IV, most of the enzymatic activity being found, however, in peak lII. Eluates 14--20 were pooled and used for the next purification stage. The partly purified enzymatic preparation was again separated by chromatography on a strongly acid ion-exchange column--SE-Sephadex C-25 (1.8 x 23 cm). The ion exchanger was equilibrated in a 0.05M Na2SO4 solution, pH 3; 3ml samples were taken up. Elution was done stepwise, increasing the ionic strength of the saline solution, as indicated in Fig. 3. Four protein peaks were obtained, V-VIII, peaks VI and VII presenting a particularly marked enzymatic activity. Following this purification procedure, the enzymatic samples presented an acid-proteolytic activity approximately 60 times that of the total proteic extract initially used. Table 1 summarizes the results obtained in 20 experiments concerning specific activity at two pH values and the purification factors recorded in the different stages of purification, The data in Figs 1-3 and Table 1 demonstrate that the elution curves of the two acid pH proteolytic activities in the Sephadex G-100, Bio-Gel P-100 and SE-Sephadex C-25 columns have similar profiles and that the purification factors present in the various stages of the process have close but not identical values. Finally, peak VI obtained by separation on the SESephadex C-25 medium column, with a 50-61-fold enriched proteolytic activity, was crystallized in an
Protein - - o - - o - - - E n z y m e 0 t pH 1 6 : -- E n z y m e ot pH 2 6 ] I l

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/ =
/ i
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~ ~

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5 I0 Tube 15 number 20 25

Fig. 2. Chromatography on Bio-Gel P-100 column (2.8 x 15 cm) equilibrated in 0.005 M Na2SO4, pH 3, of a partially purified enzymatic preparation from the hepatopancreas of a marine mussel, by precipitation at pH 3, within 0.34).7 saturation range in ammonium sulfate, chromatography on Sephadex G-100 and renewed precipitation with ammonium sulfate (0.4-0.7 saturation). FJution was obtained with the same saline solution, collecting 3 ml fractions which were correspondingly analyzed.

A new proteolytic enzyme isolated from Mytilus galloprovincialis t0-Protein Enzyme ot oH 1.61 --*-*--*-Enzyme of pH 2.6]6o .~ .ZM / ,% ]~ ~ 50 o. ~'
0.4M c:

83

50--

the hepatopancreas of the marine mussel. The elution curves of the two activities---at pH 1.6 and pH 2.6-obtained for determination of the molecular weight, were superposable, indicating similar or almost similar molecular weights (Fig. 6).
DISCUSSION

40--

40

~3c
"~ 2 ~-

~ l ~

(~

i~
0

Determination of the optimal action parameters of the purified proteolytic enzyme(s) furnished data very

IO Tube

15 number

20

25

30

Fig. 3. Elution curve on a SE-Sephadex C-25 chromatographic column (1.8 x 23 cm) of the enzymatic preparation

obtained by separation on Bio-Gel P-100. Elution was performed stepwise by increasing the concentration of the Na2SO4 solution, pH 3, as shown; 3 ml fractions were collected and analyzed.
(a)

absolute ethylic alcohol solution. Two types of crystals were obtained (Fig. 4a and b), a needle-like and a prismatic type, lending support to the hypothesis of two different enzymes. The crystals separated by centrifugation and taken up in a small volume of distilled water, adjusted to pH 3 with a 1 N HCI solution, exhibited the same specific activity as the purified preparation used for crystallization of the enzyme. The purification stages were accompanied by an electrophoretic study (Fig. 5) that indicated a single protein band for peaks VI and VII. The appearance of several protein peaks with a catalytic activity in the course of purification might be explained by autodigestion and the appearance of catalytically active intermediary products, as reported by Bohak (1969) for pepsin isolated from the chicken stomach mucosa. The molecular weight, determined by Whitaker's method (1963) using a Sephadex G-200 column (2.8 23 cm) was 36,400 ___600 for the sample presenting proteolytic activity at acid pH, purifed from

(b) Fig. 4. The two types of crystals obtained by crystallization in absolute ethyl alcohol, at pH 3 and pH 1.5 (a and b, respectively) of a purified enzymatic preparation from the hepatopancreas of the marine mussel ( x 960).

Table 1. The purification of mytilidase from the hepatopancreas of Mytilus 9alloprovincialis

Purification stage Total extract Supernatant after precipitation at pH 3 Precipitate at 0.3-0.7 saturation of ammonium sulfate Fraction I, Sephadex G-100 Precipitate at 0.4-0.7 saturation in ammonium sulfate Fraction III. Bio-Gel P-100 Fraction VI, SE-Sephadex C-25 Fraction VII, SE-Sephadex C-25 Volume (ml) 140 Protein (mg/ml) (mg) 2.24 313.6 Enzymatic activity 62 pH 1.6 Specific activity 28 Purification factor 1 Enzymatic activity 41 pH 2.6 Specific activity 18.3 Purification factor 1

135

1.18

159.3

79

66.1

2.36

44

37.3

2.04

50 35

1.02 0.62

51.0 21.7

99 126

97 203.2

3.4 7.3

76 87

74 140

3.5 7.6

20 20 6 6

0.48 0.20 0.05 0.058

9.6 4.0 0.3 0.348

128 140 70 45

266 700 1400 778

9.5 25 50 28

79 122 55 50

164.5 610 1100 863

9.0 33.3 61 50

84

lOAN FLOREA DUMITRU, DANA IORDACHESCU AND STELIAN NICULESCU

Fig. 5. Disc electrophoresis in polyacrylamide gel of samples collected at different stages of the purification process. (a) Total proteic extract; (b) supernatant pH 3; (c) fraction precipitated in ammonium sulfate at 0.3~0.7 saturation; (d) fraction I; (e) fraction precipitated in ammonium sulfate at 0.4-43.7 saturation; (f) fraction III; (g) fraction VI; (h) fraction VII. close to those of the non-purified preparation (Dumitru et al., 1975), save for the optimal pH values which, with the purified sample, were slightly shifted towards more acid values. This might be accounted for by the presence in the non-purified extracts of catalytically inert proteins. The enzyme of pH 1.6 has an optimal action temperature of 37C, presents a reaction rate which increases proportionally only within the first 5 rain and the maximum rate was recorded in the presence of 10mg hemoglobin. The enzyme of pH 2.6 has an optimal action temperature of 50C, a proportional increase in the reaction rate during the first 15 min, and is saturated in the substrate in the presence of 14 mg hemoglobin. Differences in the enzymatic reaction in terms of pH were likewise observed in the presence of copper ions which stimulate their catalytic activity (unpublished results) and with regard to different natural substrates which they hydrolyze: bovine serum albumin, ovalbumin, gelatin, caseine, peptone of different origin, etc. Additional proof that there are two pH acid proteolytic enzymes are the two types of crystals obtained by crystallization of the purified preparation

in the presence of absolute ethylic alcohol. Preliminary studies, to be published, showed that these acid proteases (protease) are to be found in the cell in the form of zymogen, and that at pH values smaller than 3 they are transformed into a catalytically active enzyme. The activation process appears to take place extremely slowly, gradually evolving at 4C for one week and being catalyzed both by the medium protons and by the newly formed active enzyme. By the purification procedure described it was not possible to separate the two proteolytic activities, the purification factors presenting close but not identical values in the various stages of the process. If in the purified enzymatic preparation there are two different proteolytic enzymes, then their behaviour on Sephadex columns, and in the case of electrophoresis in polyacrylamide gel, might be attributed to their identical or extremely similar molecular weights. A proteolytic enzyme like trypsin, in the form of zymogen and with a molecular weight equal to 26,300, with physicochemical properties similar to those of the enzyme(s) studied by us, was isolated by Bundy & Gustafson (1973) from the starfish. The molecular weight of the proteolytic enzyme(s) isolated from the hepatopancreas of the marine mussel is very close to that of pepsin isolated from mammals by Bovey & Yanary (1960). Supplementary data regarding the rate of the enzymatic reaction in the case of the two enzymatic activities, in the presence of anions and cations, confirms the existence of two proteolytic enzymes in the purified enzymatic preparation (unpublished data). REFERENCES ANSON M. L. & MIRSKY A. E. (1933) The estimation of pepsin with hemoglobin. J. 9en. Physiol. 16, 59-63. BOHAK Z. (1969) Purification and characterization of chicken pepsinogen and chicken pepsin. J. biol. Chem. 244, 4638~,648. BOVEY F. A, & YANARY S. S. (1960) Pepsin. In The Enzymes (Edited by BOVERP. D., MVRBXCKK. & LARDY H.) Vol. 4, pp. 63-92. Academic Press, New York. BUNDYH. F. & GUSTAFSON J. (1973) Purification and characterization comparative biochemistry of a protease from the starfish Pisaster 9iganteus 44, 241 251. Comp. Biochem. Physiol. 44B, 241 251. DAVIESJ. B. (1964) Disk electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121, 40~427. DE ZWANNA. (1972) Pyruvate kinase in muscle extracts

6C 5C Cythocrom I1~11 c.

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-tal I0

jVI i og iob i n

~ " . . . ~ , ~ . ~ p r otease 56.400 ~ ' 1 1 Hoemoglobin ~ B l u e

41

dexfran
l,
55

[
42

I
45

I
44

I
45

I
46

I
47

I
48

I
49

I
50

I
51

t
52

I
54

log

moleculor

weight

Fig. 6. Determination of the molecular weight of the proteolytic enzyme(s) in acid pH, purified from the hepatopancreas of Mytilus galloprovincialis, using Sephadex G- 100 column and Whitaker's method (1963).

A new proteolytic enzyme isolated from Mytilus galloprovincialis of the sea mussel Mytilus edulis L. Comp. Biochem. Physiol. 42, 7-14. DE ZWANN A. & VAN MARREWlJK W. J. A. (1973) Intracellular localization of pyruvate carboxylase, phosphoenolpyruvate carboxikinase and "malic enzyme" and the absence of glyoxylate cycle enzymes in the sea mussel Mytilus edulis. Comp. Biochem. Physiol. 44, 1057-1066. DUMITRU I. F., NICULESCU S., IORDACHESCU D. & GHITESCU E. (1975) Evidencing some acid proteases in the sea mussel Mytilus galloprovincialis. Rev. roum. biochim. 12, 159-165. GOROblOSOVA S. A. (1971) Etude comparative de la phosphorylase musculaire chez quelques invertebrates. Zh. evol. Biokhim. Fiziol. Suppl. 3-5, 1971. GOROMOSOVA S. A. & SHAPIRO A. Z. (1970) Activit6 de ramylase dans l'h6patopancreas et les muscles de Mytilus. Biol. Moria 18, 89-98. KOZLOVSKAYAE. P. t~ VASKOVSKYV. E. (1970) A comparative study of marine invertebrates. Comp. Biochem. Physiol. 34, 137-142. LOWRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALL

85

R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MINAFRA S. (1968) La fosfomonoesterasi acida in alcune specie di molluschi marini. Biologica. lat, 21, 223-234. PIGNEgO A. & ROCCA A. (1969) Proteolytic and peptidasic activities of a particulate fraction of Loli#o vulgaris hepatopancreas, Comp. Biochem. Physiol. 29, 1271-1275. REID R. G. B. (1966) Digestive tract enzymes in the bivalve Lima hians Gmelin and Mya arenaria L. Comp. Biochem. Physiol. 17, 417-433. REID R. G. B. & RAUCHERT K. (1970) Proteolytic enzymes in the bivalve mollusc Chlamys hericius Gould. Comp. Biochem. Physiol. 35, 689-695. SKARDIV M. E. (1957) Sull'attivita tiaminasica nel Mytilus galloprovincialis. Distribuzione dell attivita enzimatica nei vari organi. Boll. Soc. ital. biol. sperim. 33, 1089-1090. STRUSI A. (1964) Some chemical characteristics of mussels (Mytilus galloprovincialis) grown in Mor Piccolo and Mar Grande. Boll. Pescd. Piscic. Idrobiol. 19, 199-218. WHITAKER J. (1963) Determination of molecular weights of proteins by gel filtration of Sephadex. Analyt. Chem. 35, 1950-1957.