Comp. Biochem. PhysioL Vol. 106B, No. 3, pp. 763-766, 1993 0305-0491/93 $6.00 + 0.00 Printed in Great Britain © 1993 Pergamon Press Ltd

A VERY STABLE AND POTENT FIBRINOLYTIC ENZYME FOUND IN EARTHWORM LUMBRICUS RUBELLUS

AUTOLYSATE

HIROYUKI SUMI,*'~ NOBUYOSHI NAKAJIMA~" and HISASHI MIHARA* *Department of Physiology, Miyazaki Medical College, Miyazaki 889-16, Japan; and

tDepartment of Nutrition and Food Science, Okayama Prefectural College, Okayama 700, Japan

(Received 25 January 1993; accepted 26 February 1993)

Abstract--l. The autolysate of earthworms was found to exhibit powerful fibrin and thrombin substrate hydrolyzing activity.

2. It also showed a ciot-forming activity in the fibrinogen- or plasma-added system. 3. Zymography revealed that there were three active components with mol. wts of 40,000, 21,000 and

15,000 in the autolysate. 4. The major form with a mol. wt 35,500 (by SDS-PAGE) was further purified. The N-terminal amino

acid sequence of this enzyme (16 residues) was similar to that of the swine pancreatic proelastase.

INTRODUCTION

Earthworms have long been used for antipyretic and diuretic purposes in Chinese medicine, under the name Jiryu, and as a traditional folk remedy in Japan (Tanaka and Nakata, 1974; Jiang 1991). However, the nature of their effective components has remained unclear, with the exception of lumbrifebrine (Tanaka and Nakata, 1974) for antiphyresis and a furan compound of 2-ethyl-5-hexyl-furan-3-sulfonate (Yoneta et al., 1988), which the authors have recently found to inhibit platelet aggregation.

The authors have thus far reported a few fibrinolytic enzymes isolated from earthworm extract (Mihara et al., 1983, 1990, 1991). Since oral adminis- tration of such enzymes induces plasma fibrinolysis in animals and humans, and is clinically effective in treating thrombotic disease (Sumi et al., 1980, 1987, 1990; Toki et al., 1985), capsules containing earthworm powder of high fibrinolytic activity have recently been approved in the Republic of Korea as a health insurance drug for the prevention of adult disease (Daedo, 1990).

The present paper reports some physiochemical properties of a new enzyme, very stable and potent in solution, which was found in earthworm autolysate after very long-term storage.

MATERIALS AND METHODS

The following substances were used: earthworm, Lumbricus rubellus from the Experimental Animal Center, Miyazaki Medical College, Japan; swine pan- creas trypsin (Type I), aprotinin, soybean trypsin inhibitor (SBTI) and diisopropyl fluorophosphate (DFP) from Sigma Chemical Co. (St Louis, MO); E-aminocaproic acid (E-ACA) and trans-4-

aminomethyl-cyclohexanecarboxylic acid (t-AM- CHA) from Daiichi Seiyaku Co. (Japan); human urokinase (high molecular form: mol. wt 53,000 and low molecular form: mol. wt 32,000) from Green Cross Co. (Osaka, Japan); BZ-DL-Arg-pNA and BZ-L-Tyr-pNA from the Protein Research Foun- dation, Osaka University (Osaka, Japan); and pyro--Glu-Gly-Arg-pNA (S-2444), pyro-Glu-Pro-- Val-pNA (S-2428), H-D-Val-Leu-Lys-pNA (S- 2251), H-D-Ile-Pro-Arg-pNA (S-2288), H-D--Phe- Arg-pNA (S-2238) and H-o-Val-Leu-Arg-pNA (S-2266) from Kabi Group, Inc., USA. All other chemicals were obtained from commercial sources and were of the best grades available. For purification of the earthworm enzyme, 50 ml of earthworm au- tolysate (protein: 125 mg) was treated by gel filtration on Cellulofine GCL 2000, equilibrated with 0.01 M phosphate buffer containing 0.15 M NaC1, pH 7.2. The enzyme fraction eluted from the column, with fibrinolytic activity, was further purified by DEAE-Toyopearl and Mono-Q (HPLC) columns (Pharmacia Chemical Co., Uppsala, Sweden), yield- ing a final preparation of 0.16mg with a S.A. 106.3 U/mg protein (each unit corresponding to the amount required to hydrolyze 1 nmole of H-rr-Phe-Pip-Arg-pNA, in pH 7.2, at 37°C/min).

Amidase activity was estimated colorimetrically using several synthetic amido substrates by an end- point method (Claeson et al., 1987): the reaction mixture (1 ml) consisted of the enzyme sample, 2.5 x 10 -4 M substrate and 0.01 M phosphate buffer containing 0.15 M NaCI, pH 7.2. Fibrinolytic activity was determined by the method of Milstone (1941) employing standard fibrin plates (Astrup and Miillertz, 1952). Thromboelastography (TEG) origi- nated by Hartert was performed by TEG apparatus (Hellige) according to the routine procedure with

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764 HmOYUKZ SUMI et al.

(a)

Fig. 1. Strong lysis of artificial thrombus with earthworm autolysate (a) Shows autolysate prepared by adding earthworms to twice their wet weight of water (v/w) and allowing them to stand at room temperature for 7 years. It was a deep black liquid. (b) Shows intensive fibrinolysis after 5, 10, 20 or 30/al of autolysate supernatant was applied to standard fibrin plate, followed by incbation at 37°C for 18 hr. For calculation of the activity, a high molecular form of urokinase (S.A. 67,000 IU/mg protein) was used

for the standard.

human or rat plasma. Protein concentration was estimated by the method of Lowry e t al. (1951) using bovine serum albumin (Armour Pharmaceutical Co.) as the reference protein. The fibrinolytic activity of the protein bands separated on the S D S - P A G E gel was determined by the zymography technique of Tissot e t al. (1982). Isoelectric focussing was carried out according to the method of Vesterberg and Svensson (1966) using ampholytes of pH 3.5-10.5.

RESULTS

Figure 1 shows the state of lysis achieved by applying 5-30/~1 of ear thworm autolysate (a deep black liquid produced after earthworms were added to twice their wet weight of water and allowed to stand in a hermetic container for 7 years) on artificial thrombi (fibrin plate). The fibrinolytic activity of this solution was calculated at 3000 IU/ml or more by the standard fibrin plate method. Moreover, such very high lytic activity was not influenced by the presence of plasminogen in the fibrin plate; the activity was found to be due to a plasmin-like enzyme which exhibited direct fibrinolytic activity.

This enzyme also acted on casein and gelatin (ager-plate method) in the same assay conditions but was weaker on collagen and elastin (not shown). The amidolytic activity of earthworm autolysate was investigated with several synthetic amide substrates. As shown in Table 1, the most sensitive substrate was found to be the thrombin substrate H-o -Phe -P ip - -Arg -pNA, with lesser effects for the urokinase substrate pyro--Glu--Gly-Arg-pNA, the kailikrein substrate H - D - V a I - L e u - A r g - p N A , the

tissue plasminogen activator substrate H - o - l l e - P r o - A r g - p N A , the plasmin substrate H - D - V a I - L e u - L y s - p N A and the trypsin substrate BZ-L-Arg-pNA. There was virtually no activity on the elastase sub- strate p y r o - G l u - P r o - V a l - p N A .

This was confirmed as a thrombin-like enzyme by the fact that its addition to 0.4% fibrinogen solution caused the solution to coagulate. When diluted to a concentration of 2-13 or less, the stock enzyme sol- ution, added to the equivalent volume of fibrinogen, still caused coagulation (this activity is equivalent to that of 400 U/ml or more of thrombin). Furthermore, coagulation also occurred after the enzyme was added to human or rat plasma; it was found that addition of 10 ~1 to 0.28 ml of human plasma resulted in reduced r values and increased M a values in TEG.

Table 1. Comparative amidolytic activity of earthworm autolysate with several synthetic substrates

Substrate hydrolysis Substrate (nmols/min)

H-D-Phe-Pip-Arg-pNA (S-2238) 1,460 pyro-Glu-Gly-Arg-pNA (S-2444) 922 H-D-VaI-Leu-Arg-pNA (S-2266) 928 H-D-Ile-Pro-Arg-pNA (S-2288) 848 H-D-Val-Leu-Lys-pNa (S-225 I) 360 BZ-L-Arg-pNA 21 MeOSuc-AIa-AIa-Pro-VI-pNA 3 BZ-L-Tyr-pNA 0 pyro-Glu-Pro-Val-pNA (S-2484) 0

The reaction mixture (1 ml) contained 25-100/~1 of earthworm autolysate, partially purified by Cellulofine GCL-2,000 gel filtration. 2.5 x 10 4M substrate and 0.01 M phosphate buffer containing 0.15 M NaCI (pH 7.2). After incubation for 10 min at 37°C, the p-nitroanifine liberated was determined from the absorption at 405 nm. The results are expressed as nmoles of substrate hydrolyzed per rain per ml of autolysate. Each value is the mean of three determinations.

Earthworm fibrinolytic enzyme 765

In contrast, when the enzyme solution was diluted to a concentration of 2 -9 or more, lysis became much more prominent than clotting activity, and M~ values decreased. The fibrinolysis potency was inhib- ited with l m M of DFP, 10mg/ml of SBTI or 10 KIU/ml of aprotinin. No inhibition was observed with 10mM E-ACA or with 10mM t-AMCHA. Although in a crude state, this fibrinolytic enzyme was relatively stable at neutral pH but was gradually inactivated above 60°C. After five rounds of freezing and thawing, more than 95% of the activity re- mained. At pH values of 5.0-11.0, it was stable for 20 min at room temperature, but it was labile below pH 4.0.

Figure 2 showed the zymography pattern of earth- worm autolysate. The main components revealed mol. wts of about 40,000 and 21,000. An additional minor component with a mol. wt of 15,000 was also detected. The major enzyme with the highest activity was further purified with the guidance of fibrinolysis. Ion-exchange chromatography and gel filtration pro- duced a preparation purified to such a degree as to show a single protein zone in analytical polyacryl- amide electrophoresis. Its molecular weight in SDS-PAGE was 35,500. After treatment with DFP, the enzyme was found to have an N-terminal se- quence of I le-Val-Gly~31y-Ile-Glu-Ala-Arg-Pro- Tyr~31u-Phe-Pro-Trp-Gln-Val- , as determined with a sequencer.

DISCUSSION

It has been known for many years that earthworms contain several proteases which disintegrate casein, gelatin and albumin (Keilin, 1920; Pak, 1979). The present authors have also reported that earthworms contain a fibrinolytic enzyme, which we called lumbrokinase (Mihara et al., 1983, 1990, 1991). However, the existence of such a protease of better stability, as shown in the present study, was not known. Since most of the common proteins were completely disintegrated in the earthworm sample allowed to stand for 7 years, it may be easy to isolate and purify a stable fibrinolytic enzyme alone as residue. In the present study, the authors purified the major enzyme and demonstrated the amino acid sequence of 16 residues from the N- terminal, which showed homology with swine pancreatic proelastase (elastase 2 precursor) and human pancreatic proelastase (elastase 2A precursor) (66.7 and 60.0%, respectively). The earthworm autolysate had strong fibrinolytic activity, hydrolyz- ing casein and various amide substrates, especially thrombin substrates such as H-D-Phe-Pip-Arg- pNA; however, its reaction to fibrinogen and reduced r values in TEG revealed that the autolysate was very similar to thrombin. In its molecule, thrombin possesses a Kringle structure similar to that of plasmin or plasminogen activator. In addition, genetically, it is closely related to

Fig. 2. Zymography of earthworm autolysate. About 20/~l (a) and 5 ~ul (b) of autolysate were used. Numbers on the right side indicate molecular weight (5 x l04) using uroki-

nase as the standard.

fibrinolytic enzyme. Although the molecular structure of the earthworm enzyme has, thus far, not been reported, the present results indicate that it may correspond to the very classic nature of today's plasmin. After all, no report has been published on animal-derived protease, which is stable in solution. Further study is needed on the structure of the enzyme itself, or on some stabilizing components found in earthworms, to confirmed them as orally effective.

Acknowledgements--The authors are indebted to Dr H. Tsushima, Kawasaki Medical College, for cooperating in amino acid sequence determination, and to Dr K. Tanisawa, Industry Science Institute, Osaka University, for examining the homology of the primary structure of purified enzyme and other proteins.

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