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Biotechnology Letters 26: 393–397, 2004. ? 2004 Kluwer Academic Publishers. Printed in the Netherlands.

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Puri?cation of a ?brinolytic enzyme (myulchikinase) from pickled anchovy and its cytotoxicity to the tumor cell lines
Yong-Kee Jeong1,? , Woong Suk Yang1 , Kwang Hyuk Kim3 , Kyung Tae Chung1 , Woo Hong Joo4 , Jae Hyun Kim5, Dong-Eun Kim2 & Jeong Uck Park6
Departments of 1 Life Science and Biotechnology and 2 Biotechnology and Bioengineering, Dong-Eu University, Busan 614-714, Korea 3 Department of Microbiology, Kosin Medical College, Busan 602-702, Korea 4 Department of Biology, Changwon National University, Changwon 641-773, Korea 5 School of Life Sciences, Chungbuk National University, Cheongju, Chungbuk 361-763, Korea 6 Department of Microbiology, Gyeongsang National University, College of Medicine, Chinju 660-758, Korea ? Author for correspondence (Fax: +82-51-894-0840; E-mail: ykjeong@dongeui.ac.kr)
Received 27 July 2003; Revisions requested 18 August 2003/20 November 2003; Revisions received 18 November 2003/23 December 2003; Accepted 24 December 2003

Key words: amidolytic activity, cytotoxicity, ?brinolytic enzyme, myulchikinase Abstract A ?brinolytic enzyme, myulchikinase, from a Korean seasoning ingredient, myul-chi-jeot-gal, has been puri?ed to electrophoretic homogeneity. The molecular mass of the myulchikinase was estimated to about 28 kDa by SDSPAGE and gel ?ltration. Amino acid sequence of the NH2 -terminal of myulchikinase showed signi?cant homology with other ?brinolytic enzymes including trypsin from star?sh, katsuwokinase, and rat pancreatic elastase II. The puri?ed myulchikinase hydrolyzed various synthetic substrates with different substrate speci?city and cytotoxic to the tumor cell lines. Introduction Fibrin is a major protein component of blood clots which are normally formed from ?brinogen by the action of thrombin. The accumulation of ?brin in the blood vessels usually increases thrombosis, leading to myocardial infarction and other cardiovascular diseases. The blood clot, ?brin, is lysed by plasmin which is formed by activation of the proenzyme, plasminogen, by either plasmin activators or tissue-type plasminogen activators (tPA). These activators, such as urokinase (Toki et al. 1985), are widely used for the therapeutic purpose by intravenous infusion. They are of human origin and generally safe but are very costly to produce. Oral administration of the ?brinolytic enzyme, nattokinase, can enhance ?brinolytic activity in plasma and the production of tPA (Sumi et al. 1990). If a ?brinolytic enzyme is provided by fermented foods, it can be directly used as a potent natural agent for oral thrombolytic therapy to prevent thrombosis and the related diseases. In this respect, some other ?brinolytic enzymes from different fermented foods such as shiokara (Sumi et al. 1995) and chung-kook-jang (Kim et al. 1996) have been isolated. Similarly, a ?brinolytic enzyme was also puri?ed from earthworm (Park et al. 1998). Myul-chi-jeot-gal is a picked anchovy product popular in Korea and is used primarily as seasoning ingredient. In this paper, we report the puri?cation of a ?brinolytic enzyme from myul-chi-jeot-gal and its cytotoxicity to tumor cell lines. Materials and methods Chemicals Chromogenic substrates, such as S-2251 (DLvalyl-leucyl-lysine-p-nitroanilide, a synthetic substrate for plasmin), S-2238 (DL-phenyl-Pip-arginine

394 p-nitroanilide, a synthetic substrate for thrombin), S-2266 (DL-valyl-leucyl-arginine-p-nitroanilide, a synthetic substrate for kallikrein), S-2444 (pyroglutamyl-glycyl-arginine p-nitroanilide, a synthetic substrate for urokinase), and S-2288 (DL-isoleucylprolyl-argininep-nitroanilide, a synthetic substrate for serine protease) were purchased from Chromogenix (Milan, Italy). Cell culture supplies, including fetal bovine serum, were obtained from Invitrogen (Carlsbad, USA). All other chemicals were purchased from Sigma-Aldrich. Puri?cation of a ?brinolytic enzyme Cleared supernatant of myul-chi-geot-gal were prepared by centrifugation at 12 000 × g for 20 min. The cleared supernatant of myul-chi-geot-gal then dialyzed against water to remove NaCl and other condiments. The dialyzed myul-chi-geot-gal was precipitated with ammonium sulfate (85% saturation) and dialyzed against 20 mM Tris/HCl (pH 7.5) buffer containing 0.2 M NaCl and then load onto DEAESephadex A50 column. The proteins were eluted with a linear gradient from 0.1 M to 0.5 M NaCl in the 20 mM Tris/HCl (pH 7.5) buffer. Fractions with ?brinolytic activity were eluted between 0.25 M and 0.35 M NaCl. These fractions were pooled and precipitated with ammonium sulfate (85% saturation) and dialyzed against 20 mM Tris/HCl (pH 8) buffer containing 0.2 M NaCl and then load onto a Sephadex G50 gel ?ltration column previously equilibrated in 20 m M Tris/HCl (pH 7.5) buffer containing 0.2 M NaCl. All puri?cation steps were carried out at room temperature except for centrifugation, which was conducted at 4 ? C. Enzyme assays Fibrinolytic activity was determined by both the plasminogen-free ?brin plate method and the plasminogen-rich ?brin plate method (Astrup & Müllertz 1952). Plasminogen-free ?brin plate was made up of the ?brinogen solution (2.5 ml of 1.2% human ?brinogen in 0.1 M sodium phosphate buffer, pH 7.4), 10 U thrombin solution and 1% agarose. Plasminogen-rich ?brin plate was made up 2 ml 1.5% ?brinogen and 5 units plasminogen. The sterilized paper disc (5 mm diam.) was overlaid on the ?brin plate. To observe the ?brinolytic activity of the enzymes, 100 ?l puri?ed recombinant protein solution was carefully dropped on to the disc and incubated at 37 ? C for 18 h. After measuring the dimension of the clear zone on the ?brin plate, the number of units was determined according to standard curve by using plasmin. Amidolytic activity was measured spectrophotometrically using chromogenic substrates for proteases. The reaction mixture (1 ml) contained 20 ?l enzyme solution, 0.5 mM chromogenic substrate, and 0.1 M sodium phosphate buffer (pH 7.4). After incubation for 5 min at 37 ? C, the amount of p-nitroaniline that was liberated was determined from the absorbancy at 405 nm. One unit of amidolytic activity was de?ned as nmol substrate hydrolyzed per min per ml enzyme. Amino acid sequencing of the puri?ed ?brinolytic enzyme The puri?ed ?brinolytic enzyme on a SDS-PAGE gel was electroblotted to a polyvinylidene di?uoride membrane (Bio-Rad, USA) and stained with Coomassie Brilliant Blue R-250. The stained protein portion was excised and the NH2 -terminal amino acid sequence of the puri?ed ?brinolytic enzyme was determined by the automated Edman method using a gas-phase protein sequencer (Model 476A, Applied Biosystems, USA). Cell culture K562 (human erythroleukemia) and YAC-1 (murine lymphoma) cells were maintained in RPMI1640 containing 10% (v/v) fetal bovine serum, while MCF 10A (human breast non-tumorigenic epithelial) cells were grown in Dulbecco’s modi?ed Eagle’s medium containing with 10% (v/v) fetal bovine serum supplemented with penicillin (100 IU ml?1 ) and streptomycin (100 ?g ml?1 ). The cells were grown in 5% (v/v) CO2 at 37 ? C. For the viability assays, cells were plated at 3 × 105 /well in 24-well plates. During the viability studies, the myulchikinase was added to the cells 24 h after plating. MTT assay MCF 10A, K562, and YAC-1 cells response to myulchikinase were measured using the methyl-thiazolyl tetrazolium (MTT) assay according to the modi?ed method of Espevik & Nissen-Meyer (1986). The myulchikinase was incubated with the cells for 24 h, after which time MTT was added to give 0.5 mg ml?1 . Cells were incubated with the MTT for 4 h in a CO2 incubator. The cells then lysed to dissolve the formed formazan crystals by the addition of a lysis solution (50% dimethylformamide, 20%, SDS, pH 7.4) and

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Table 1. Puri?cation steps of the myulchikinase from myul-chi-geot-gal. Step Total protein (mg) 7 600 242 18 2 Total activitya (U) 16 000 8 000 3 744 2 128 Speci?c activity (U mg?1 ) 2 33 208 1 064 Fold puri?cation Yield (%)

Myulchi-geot-gal (NH4 )2 SO4 precipitation DEAE-Sephadex A50 Sephadex G50

1 16 99 507

100 50 23 10

a Fibrinolytic activity was determined by both the plasminogen-free ?brin plate method and the

plasminogen-rich ?brin plate method. Table 2. Amidolytic activity of myulchikinase from myul-chi-jeot-gal to different chromogenic substrates compared with other ?brinolytic enzymes. Synthetic substrate S-2251b S-2238c S-2266d S-2444e S-2288f Enzyme activitya (U) Myulchikanse Nattokinase 7 900 (100) 9 300 (118) 5 300 7 000 (89) 8 700 (110) 69 (100) 14 (20) 14 (20) 0 (0) ND

Subtilsin 12 000 (100) 6 800 (6) 3 400 (3) 0 (0) ND

Carlsberg 46 (100) 50 (118) 26 (66) 20 (89) ND

CK 420 000 (100) 22 000 (5) 17 000 (66) 0 (0) ND

ND indicates ‘not determined’. a Amidolytic activity was measured spectrophotometrically as described in Materials and methods. The values in parentheses are percentages calculated on the basis of enzyme activity to S-2251. Each value is the mean of three determinations. b Synthetic substrate for plasmin. c Synthetic substrate for thrombin. d Synthetic substrate for kallikrein. e Synthetic substrate for urokinase. f Synthetic substrate for serine protease.

were incubated 18 h in a humidi?ed CO2 incubator. The degree of MTT reduction in each sample was subsequently assessed by measuring absorbancy at 540 nm at 37 ? C. Background absorbancy values, as assessed from cell-free wells, were subtracted from the absorbancy values of each test sample.
Fig. 1. Comparison of myulchikinase with other proteases for NH2 -terminal amino acid sequence. Boxed areas represent amino acids of myulchikinase which are identical to those of trypsin 1 from star?sh (Estell et al. 1980), katsuwokinase (Sumi et al. 1995) and elastase II (Szilagyi et al. 1995). Numbers denote the position of amino acid of each protein.

Results and discussion Puri?cation of the myulchikinase The ?brinolytic enzyme from myul-chi-geot-gal was puri?ed to electrophoretic homogeneity by the steps listed in Table 1. The molecular size of the puri?ed ?brinolytic enzyme was about 28 kDa by SDS-PAGE and the mobility on gel ?ltration implied that the puri?ed protein is monomeric (data not shown). We have named this ?brinolytic enzyme from myul-chi-geotgal as myulchikinase.

We next examined the amidolytic activity of myulchikinase to different chromogenic substrates. As shown in Table 2, myulchikinase showed signi?cant amidolytic activity on S-2238 (synthetic substrate for thrombin), S-2251 (synthetic substrate for plasmin) and lesser effect on S-2266 (synthetic substrate for kallikrein). Of note, myulchikinase could hydrolyze

396

Fig. 2. The effect of myulchikinase on tumor cells. Cell viability was indirectly measured using MTT assay. Line graphs show viability of cells, as measured by MMT reduction. Open circles show the cell viability in the presence of myulchikinase, and closed circles show the viability in the absence of myulchikinase. The means ± S.D. of 8 determinations are presented.

S-2444 (synthetic substrate for urokinase) and S-2288 (synthetic substrate for serine proteases), which is a unique feature comparing with other ?brinolytic enzymes. In addition, the amidolytic activity of myulchikinase on S-2238, S-2666 and S-2444 was higher, compared to nattokinase (Sumi et al. 1987), subtilisin (Vasantha et al. 1984), Carlsberg (Jacobs et al. 1985) and CK (Kim et al. 1996). The NH2 -terminal amino acids sequencing of myulchikinase revealed that the ?rst 12 amino acid residues of NH2 -terminus of myulchikinase protein were I-V-G-G-E-E-G-T-A-N-S-T. Amino acid sequences of the myulchikinase were used to search data bases for other related sequences with BLAST program (Altschul et al. 1997). As shown in Figure 1, the alignment with the ?rst 12 amino acid sequences of NH2 -terminus of myulchikinase with other known ?brinolytic enzymes showed that the myulchikinase has signi?cant homology with trypsin 1 from star?sh (Estell et al. 1980), katsuwokinase from a Japanese traditional fermented food (Sumi et al. 1995) and rat pancreatic elastase II (Szilagyi et al. 1995). In this respect, this high level of sequence homology may re?ect that the puri?ed myulchikinase from myulchi-geot-gal have a horizontal relationship with other ?brinolytic enzymes. Cytotoxicity of myulchikinase on tumor cells

whereas in cancer spread, the controlling mechanisms appear to be lost (Liotta et al. 1991). In the light of this notion, we further evaluated the cytotoxic effect on the tumor cells by the puri?ed myulchikinase. To measure cell viability, we used an MTT assay. (The yellow MTT is reduced in metabolicably active cells to form insoluble purple formazan crystals which are solubilized by a detergent. The color is then quanti?ed spectrophotometrically.) As shown in Figure 2, MTT reduction in K562 and YAC-1 cells after a 24 h exposure to 6 ?g myulchikinase ml?1 decreased by approx. 35% and 70%, respectively. As a control, MTT reduction in MCF 10A cells treated with myulchikinase was assessed and, under all conditions, it did not signi?cantly alter MCF 10A cell MTT reduction relative to the untreated cells with myulchikinase. These results may suggest that myulchikinase is toxic to tumor cells, whereas human breast non-tumorigenic epithelial cells (MCF 10A) had no effect to their viability. In view of these reports, it can be suggested that myulchikinase may provide important insight into investigation of oral ?brinolytic therapy. Further work should focus on the precise mechanism for myulchikinase to potentate ?brinolysis and whether or not brings side effects. References

The proteolytic enzymes are involved in physiological destruction and tissue remodeling processes which partially resemble cancer invasion and metastasis. The main difference appears to be that in the physiological events, proteolysis is controlled and self-limiting,

Altschul SF, Madden TL, Scaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl. Acids Res. 25: 3389–3402. Astrup T, Müllertz S (1952) The ?brin plate method for estimating ?brinolytic activity. Arch. Biochem. Biophys. 40: 346–351.

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Espevik T, Nissen-Meyer J (1986) A highly sensitive cell line, WEHI164 clone 13, for measuring cytotoxic factor/tumor necrosis factor from human monocytes. J. Immunol. Methods 95: 99–105. Estell DA, Laskowski Jr. M (1980) Dermasterias imbricata trypsin 1: an enzyme which rapidly hydrolyzes the reactive-site peptide bonds of protein trypsin inhibitors. Biochemistry 19: 124–131. Jacobs M, Eliasson M, Uhlen M, Flock JI (1985) Cloning, sequencing and expression of subtilisin Carlsberg from Bacillus licheniformis. Nucl. Acids Res. 13: 8913–8926. Kim WK, Choi K, Kim Y, Park H, Choi J, Lee Y, Oh H, Kwon I, Lee S (1996) Puri?cation and characterization of a ?brinolytic enzyme produced from Bacillus strain CK 11-4 screened from Chungkook-Jang. Appl. Environ. Microbiol. 62: 2482–2488. Liotta LA, Steeg PS, Stetler-Stevenson WG (1991) Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation. Cell 64: 327–336. Park YD, Kim LW, Min BG, Seo JW, Jeong JM (1998) Rapid puri?cation and biochemical characteristics of lumbrokinase III from earthworm for use as a ?brinolytic agent. Biotechnol. Lett. 20: 169–172. Sumi H, Hamada H, Nakanishi K, Hiratani H (1990) Enhancement of the ?brinolytic activity in plasma by oral administration of NK. Acta Haematol. 84: 139–143. Sumi H, Hamada H, Tsushima H, Mihara H, Muraki H (1987) A novel ?brinolytic enzyme (Nattokinase) in the vegetable cheese natto: a typical and popular soybean food in the Japanese diet. Experimentia 43: 1110–1111. Sumi H, Nakajima N, Yatagai C (1995) A unique strong ?brinolytic enzyme (katsuwokinase) in skipjack ‘Shiokara’, a Japanese traditional fermented food. Comp. Biochem. Physiol. 112: 543–547. Szilagyi CM, Sarfati P, Pradayrol L, Morisset J (1995) Puri?cation, characterization and substrate speci?city of rat pancreatic elastase II. Biochim. Biophys. Acta. 1251: 55–65. Toki N, Sumi H, Sasaki K, Boreisha I, Robbins KC (1985) Transport of urokinase across the intestinal tract of normal human subjects with stimulation of synthesis and/or release of urokinase-type proteins. J. Clin. Invest. 75: 1212–1222.


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