Abstract:
A novel arginine deiminase of an approximately 45,000 molecular weight derived from mycoplasma having an ability to decompose arginine, and the method of manufacturing this novel enzyme from mycoplasma. This enzyme is an effective anti-cancer agent, as it shows anti-cancer activities both in vitro and in vivo.

Description:
This application is a continuation, of application Ser. No. 08/037,713, filed Mar. 24, 1993, now abandoned which is a continuation, of application Ser. No. 07/558,158, filed Jul. 26, 1990, now abandoned. 
    
    
     BACKGROUND OF THE INVENTION 
     The present invention relates to a novel arginine deiminase and the manufacturing method thereof. Further, the present invention relates to an anti-cancer agent containing this arginine deiminase as an effective ingredient. 
     DESCRIPTION OF THE RELATED ART 
     Recently, increasing attention is focused on the fresh attempt to cure cancer by using enzymes on the basis of the requirement of nourishment by cancer cells. In other words, this treatment attempts to inhibit the growth of, or necrotize, the cancer cells by decomposing the nourishment required by the cancer cells and thus by shutting out the source of nourishment from the reach of the cancer cells. Among these anti-tumor enzymes, L-asparaginase (EC 3.5.1.1) [see Nature, 229, 168(1971)] and arginase (EC 3.5.3.1) [Br. J. Cancer, 19, 379(1965)] are known. However, L-asparaginase is effective only to a few types of cancer such as leukemia and malignant lymphoma because there are a small number of cancers dependent on L-asparagine, while arginase fails to show anti-cancer activities in vivo although it shows inhibiting activities against the growth of a variety of cancer cells in vitro [Br. J. Cancer, 30, 50(1974)]. 
     Moreover, although there have been reports that arginine deiminase is obtainable from mycoplasma and other microorganisms [J. Biol. Chem., 241, 2228-2236 (1966),ibid., 252, 2615-2620 (1987), ibid., 250, 4580-4583 (1975), Arch. Biochem. Biophys. 69, 186-197 (1957), etc.], no reports have mentioned the anti-cancer activity of this enzyme. 
     SUMMARY OF THE INVENTION 
     It is an object of the present invention to provide a novel arginine deiminase. Another object is to provide a method of manufacturing this novel arginine deiminase from mycoplasma. Further, the other object of the present invention is to provide an anti-cancer agent containing arginine deiminase as an effective ingredient. 
     The present inventors conducted research on many new types of arginine deiminase derivable from Pseudomonas, Streptococcus, mycoplasma and other microorganisms. As a result, the present invention was accomplished when it was found that a cell extract of mycoplasma contains arginine deiminase, which has its optimum pH in the physiological range, shows a high stability, and consequently provides strong inhibiting activities against the growth of cancer cells. 
     Arginine deiminase (EC 3.5.3.6.) is an enzyme that hydrolyzes amidino group of L-arginine into L-citrulline and ammonia, and is called by a general name of arginine dihydrolase, arginine desiminase, or guanidinodesiminase. The resultant L-citrulline is decomposed into ornithine and carbamoyl phosphate, and this carbamoyl phosphate is further decomposed into ammonia and water. ATP is produced in this process. In other words, arginine deiminase is an enzyme involved in the first step of the pathway leading to the production of ATP in connection with the metabolism of the amidino groups of arginine in bacteria and yeasts. 
     The arginine deiminase of the present invention has the following physicochemical properties: 
     (a) Function 
     Hydrolyzes the amidino groups of L-arginine into L-citrulline and ammonia. 
     (b) Optimum pH: 6.0-7.5 
     (c) Stable pH: 4.5-9.0 
     (d) Optimum temperature: Approx. 50° C. 
     (e) Km value: Approx. 0.2 mM 
     (f) Isoelectric point (PI): Approx. 4.7 
     (g) Molecular weight: Approx. 45,000 
     (by SDS-polyacrylamide gel electrophoresis method) 
     Approx. 90,000 
      (by gel filtration HPLC method) 
     (h) Amino acid sequence from N-terminal 
     Ser-Val-Phe-Asp-Ser-Lys-Phe-Lys-Gly-Ile-His-Val-Tyr-Ser-Glu- 
     Also, the arginine deiminase of the present invention comprises the amino acid sequence shown in FIG. 4. 
     The arginine deiminase of the present invention is prepared from mycoplasma by the following method: 
     Any mycoplasma having an ability to produce the arginine deiminase of the present invention can be used. These types of mycoplasma include M. arginini, M. hominis, M. salivarium, M. gallinarum and M. orale. The most suitable strain is the M. arginini [IFO Catalog No. 14476, ATCC Catalog No. 23838 or NCTC Catalog No. 10129) available from the Institute of Fermentation, Osaka, ATCC or NCTC. 
     In the present invention, the above mycoplasma is cultured in a liquid medium added with arginine, at a temperature of about 37° C. for one to several days in a standing condition. This mycoplasma is a facultative anaerobic bacterium and can be grown under an anaerobic condition or in the presence of oxygen. A PPLO meat broth or the like is used, and arginine is added to the medium at a ratio of 1-10 g to 1 liter of medium. 
     After culturing, the cells are collected, then are suspended in a buffer solution, for example, such as a phosphate buffer solution, and are disrupted by such means as sonication; finally, the precipitates are removed through centrifugal separation or a similar method. The supernatant solution is thus obtained as a cell extraction, which undergoes a process of purification by chromatography such as gel filtration chromatography, ion-exchange chromatography, affinity chromatography and the like. As a result, an arginine deiminase having the above-mentioned physicochemical properties is obtained. 
     The arginine deiminase of the present invention is clearly different from the known types of arginine deiminase reported in various literature, as shown in Table 1. 
     
                                           TABLE 1__________________________________________________________________________                             Archives of         J.B.C. J.B.C.                      J.B.C. Biochemistry         252, 2615-                241, 2228-                      250, 4580-                             and Biophy-  (Present         2620   2236  4583   sics 69, 168-Reference  invention)         (1979) (1966)                      (1975) 197 (1957)__________________________________________________________________________Source M. arginini         M. arthritidis                M. hominis                      Pseudomonas                             Strepto-                      putida faecalisMolecular  45,000 49,000 78,300                      54,000 not knownweight (SDS-PAGE)         (SDS-        (SDS-90,000        PAGE)        PAGE)  (gel filt-  ration  HPLC)Isoelectric  4.7    7.0    not   6.13   not knownpoint                knownkm     0.2 mM 4 μM                0.1-0.4 mM                      0.2 mM 0.15 mMVmax   50 μ/mg         18 μ/mg                53 μmg                      58.8 μ/mg                             41 μ/mgOptimum pH  6.7-7.5         not    6.5-6.7                      6.0    6.8         knownAmino acid  Ser    Ala    not   not    not knownat N-                known knownterminal__________________________________________________________________________ 
    
     Further, the homology search was done between the N-amino acid sequence of the arginine deiminase of the present invention and registered proteins in the computer database, but it was found that no protein has an amino acid sequence similar to that obtained by the present invention. For these reasons, it has been concluded that the arginine deiminase of the present invention is a novel arginine deiminase. 
     In addition, an attempt was made in the present invention to clone the gene of arginine deiminase of M. arginini from the genome DNA of M. arginini. By DNA sequence analysis of the cloned gene, a nucleotide sequence coding for the arginine deiminase was determined, as illustrated in FIG. 4. Accordingly, the arginine deiminase of the present invention was found to contain the 409 amino acids and have a molecular weight of 46375, nearly identical with the molecular weight of proteins obtained by the SDS-polyacrylamide gel electrophoresis. 
     As indicated by Experiments 1 through 6, the arginine deiminase of the present invention shows a superb effect of prolonging the lives of mice carrying such cancer cells as leukemia cells, fibro-sarcoma cells and colon carcinoma cells and the like. For this reason, it is evident that the arginine deiminase of the present invention is effective in the treatment of a variety of tumors, specially malignant tumors, in humans. 
     The activity of arginine deiminase can be determined by causing it to produce citrulline using the method reported by Fenske et. al. [J. Bacteriol. 126, 501-510 (1976)], and by measuring the amount of the yields using the method given by Archibald [J. Biol. Chem. 156, 121-142 (1944)]. 
     Specifically, 20 μl of arginine deiminase solution is added to 1.0 ml of 10 mM arginine [dissolved in 0.1M phosphate buffer of pH 7.0], then an enzyme reaction is allowed to take place at 37° C. for 10 minutes, and the reaction is terminated by adding 1 ml of mixed acid solution (H 2  SO 4  :H 3  PO 4  =1:3). Then, the resultant reactive solution is added with 25 μl of 3% aqueous diacetylmonooxim solution, heated at 100° C. for 20 minutes under a light-shielded condition, left to cool under a room temperature for 30 minutes, and then the amount of the citrulline yield is measured quantitatively by measuring the absorbence at 490 nm. The activity of arginine deiminase producing 1 μmol of citrulline in one minute is defined as 1 unit. 
     With regard to an acute toxicity of arginine deiminase, there was no case of mouse deaths due to the administration of not more than 1 mg/mouse of arginine deiminase orally or intravenously in the tail. Further, the dissection of the administered animals showed no irregularities in their organs, thus affirming the high safety of arginine deiminase. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 shows a gel filtration chromatogram of the M. arginini cell extract of the present invention, FIG. 2 an ion-exchange chromatogram of the fractions 42-52 thereof, and FIG. 3 affinity chromatogram of the fractions 50-53 thereof. FIGS. 4a-4c show the complete amino acid sequence of the arginine deiminase, and the nucleotide sequence which codes for the enzyme. FIG. 5 through FIG. 8 show the nucleotide sequence and amino acid sequence thereto of the oligonucleotide used as probes. 
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
     Examples of the present invention are shown below. 
     EXAMPLE 1 
     Preparation of arginine Deiminase 
     (1) Culturing of M. arginini 
     A strain of M. arginini (IFO Catalog No. 14476) obtained from the Institute of Fermentation, Osaka was inoculated into a liquid medium [composed of 21 g of PPLO broth w/o CV (Difco), 10 g of L-arginine, 200 ml of horse serum, 100 ml of 25% freshly prepared yeast extract, 5 ml of 0.4% phenol red solution and 700 ml of distilled water, and adjusted pH to 7.0], and then cultured in a 5% CO 2  incubator at 37° C. for 2 days in a standing condition. 
     (2) Separation and purification of arginine deiminase 
     (A) Preparation of M. arginini cell extract 
     2 g of M. arginini cells were separated by centrifuging 2 liters of the liquid medium obtained according to the above (1) step, at a 7,000 rpm for 20 minutes. These M. arginini cells were suspended in 30 ml of 10 mM phosphate buffer of pH 7.0. The cells contained in this suspension were then disrupted by sonication, and the insoluble matter is removed by centrifugal separation. Finally, the resultant supernatant was obtained as a M. arginini cell extract. 
     (B) The purification of arginine deiminase 
     Arginine deiminase was purified from the M. arginini cell extract obtained in the above (A) step, by applying the following three different types of chromatography. 
     i) Gel filtration chromatography 
     The above M. arginini cell extract was applied to gel filtration chromatography under the following condition, using a Cellulofine GCL-2000 m [Chisso Corporation]: 
     
         ______________________________________Column size:     2.6 × 90 cmVolume of gel:   480 mlFlow rate:       24 ml/hrVolume of fraction:            6 ml eachEluent:          10 mM potassium phosphate            buffer (pH 7.0), + 0.5 M            sodium chloride______________________________________ 
    
     The absorbance at 280 mm and arginine deiminase activity of each fraction thus obtained were measured by the aforementioned methods, and the results were as shown in FIG. 1. It was found that the arginine deiminase was contained in the fractions 42-52. 
     ii) Ion-exchange chromatography 
     The fractions 42-52 obtained in the above i) step were dialyzed for 24 hours against a 10 mM phosphate buffer. Then, the inner dialyzed solution was applied to ion-exchange chromatography under the following condition, using a DEAE-Toyopearl [Toso Co.]: 
     
         ______________________________________Column size:     2.6 × 30 cmVolume of gel:   320 mlFlow rate:       60 ml/hrVolume of fraction:            10 ml eachEluent:          10 mM phosphate buffer            (pH 7.0)            (linear gradient of sodium            chloride from 0 to 0.5M)______________________________________ 
    
     In other words, the dialyzed fractions containing the arginine deiminase were added into a DEAE-Toyopearl column equilibrated with the same buffer as that used in the dialysis in order to absorb arginine deiminase, and 10 ml of each fraction was collected by elution under the condition of a linear gradient of the sodium chloride concentration from 0M to 0.5M in the buffer at a flow rate of 60 ml/hr. 
     As in the above i) step, the absorbance at 280 nm and arginine deiminase activity of each fraction were measured, and the results were as shown in FIG. 2. It was found that the arginine deiminase was contained in the fractions 50-53. 
     iii) Affinity chromatography 
     The fractions 50-53 obtained in the above ii) step were dialyzed for 24 hours against a 10 mM potassium phosphate buffer. Then, the dialyzed solution was applied to affinity chromatography under the following condition, using a Arginine-Sepharose 4B [Pharmacia Co.]: 
     
         ______________________________________Column size:     2.2 × 10 cmVolume of gel:   38 mlFlow rate:       50 ml/hrVolume of fraction:            8 ml eachEluent:          10 mM phosphate buffer            (pH 7.0)            (linear, gradient of            sodium chloride from 0 to            1.0M)______________________________________ 
    
     In other words, the dialyzed fractions containing the arginine deiminase were added into a Arginine-Sepharose column equilibrated with the same buffer as that used in dialysis in order to absorb arginine deiminase, and 8 ml of each fraction was collected by elution under the condition of a linear gradient from 0M to 1.0M of the sodium chloride concentration in the buffer at a flow rate of 50 ml/hr. 
     As in the above ii) step, the absorbance at 280 nm and arginine deiminase activity of each fraction were measured, and the results were as shown in FIG. 3. It was found that the arginine deiminase was contained in the fractions 71-81. 
     iv) SDS-polyacrylamide gel electrophoresis 
     The fractions 71-81 obtained in the above iii) step were analyzed by SDS-polyacrylamide gel electrophoresis according to the method reported by Laemmli et. al. [Nature, 727, 680-685 (1970)]. The purification of arginine deiminase was confirmed, as a single band was observed at a molecular weight of 45,000. 
     (C) Physicochemical properties of arginine deiminase 
     The following measurements were made with the enzyme solution obtained in the above (B) iii) step: 
     i) Optimum pH, optimum temperature, stable pH and stable temperature 
     The optimum pH range is 6.0-7.5, and the stable pH range when treated at 4° C. for 24 hours is 4.5-9.0. The highest activity is obtained in the 45°-55° C. temperature range, and as for stability against temperature, the activity is completely lost for treatment at 95° C. for 5 minutes or at 60° C. for 30 minutes. 
     ii) Substrate specificity 
     The arginine deiminase hydrolyzes L-arginine to produce L-citrulline and ammonia. 
     iii) Molecular weight 
     Measurement by the SDS-polyacrylamide gel electrophoresis indicated a molecular weight of approximately 45,000. 
     Measurement by the gel filtration HPLC method using a TSK G3000SW XL  [Toso Co.] indicated a molecular weight of approximately 90,000. 
     iv) Isoelectric point 
     Measurement of the isoelectric point by the electrofocusing method indicated an isoelectric point of approximately 4.7. 
     v) Km value and Vmax value 
     By the Lineweaver-Burk plot analysis Km and Vmax were determined as 0.2 mM and 50 U/mg, respectively. 
     vi) The amino acid sequence from the N-terminal was determined by a peptide sequencer [Applied Biosystem Co.] as follows: 
     Ser-Val-Phe-Asp-Ser-Lys-Phe-Lys-Gly-Ile-His-Val-Tyr-Ser-Glu- 
     vii) Biological Activity 
     The arginine deiminase showed an inhibiting activity against the growth of cancer cells in vitro, and indicated a superb effect of prolonging the lives of cancer-carrying mice in vivo. 
     EXAMPLE 2 
     (1) Preparation of mycoplasma genome DNA 
     M. arginini [IFO Catalog No. 14476] was lysed by SDS, and then was treated first by protinase K and then by RNase A. Then, the solution was extracted with phenol and then with chloroform; finally, genome DNA was prepared by dialyzing the resultant aqueous layer against 10 mM tris-hydrochloric acid (pH 8.0) in 1 mM EDTA. 
     (2) Preparation of mycoplasma genes library 
     The mycoplasma genome DNA prepared in the above (1) step was digested by appropriate restriction enzymes, then was inserted to a plasmidvector pUC19 [Toyobo Co.]; finally, this vector was introduced into E. coli HB101 [purchased as competent cells from Takara Shuzo Co.], to provide a mycoplasma genes library. 
     (3) Design and synthesis of oligonucleotide for hybridization probe 
     It was previously reported that the DNA of mycoplasma contains only a small amount of GC [Proc. Natl. Acad. Sci., 84, 166-169 (1987)]. In view of this finding, an oligonucleotide having 24 bases (a mixture of 4 types, and see FIG. 5 through 8 for their sequences) was designed from the N-terminal amino acid sequence of the arginine deiminase, and was synthesized by a DNA synthesizer [ABI model 308 B]. 
     (4) Screening by colony hybridization 
     The oligonucleotide synthesized in the above (3) step was labeled by 32p, and was used as a probe for screening the mycoplasma gene library produced in the (2) step. Thus, positive clones were isolated. 
     (5) Determination of nucleotide sequences 
     Plasmid DNA was prepared from the clones obtained in the above (4) step, and the DNA fragment to be hybridized with the probe was integrated into M13mp19 [Toyobo Co.]. Using the dideoxy method, the nucleotide sequence was determined for both strands of the cloned gene. The results are shown in FIG. 4. 
     A 1230 bp open reading frame including a nucleotide sequence coding for the N-terminal, 30  amino acids sequence of the arginine deiminase was found in the cloned gene, and the 1227 bp genes, following the initiation codon ATG, were identified as the arginine deiminase gene of M. arginini (see FIG. 4). Since it is known that the TGA codon is recognized as a tryptophan codon in mycoplasma [Proc. Natl. Acad. Sci., 82, 2306-2309 (1985)], the TGA codons existing at 5 different positions in the arginine deiminase genes were considered as tryptophan codons, and the analysis was performed on the assumption that there were only 2 termination codons, TAA and TAG. 
     The molecular weight derived from the total 409 amino acids was 46375, which is nearly identical with the molecular weight obtained by the protein SDS-polyacrylamide gel electrophoresis. 
     EXAMPLE 3 
     Preparation of formulated arginine deiminase 
     The fractions 71-81 obtained in the Example 1 (2) iii) step were dialyzed for 24 hours against a phosphate buffered saline (PBS) (pH 7.4), and the dialyzed product was diluted by PBS in a 0.2-2.0 mg/ml solution. Then, the solution was sterilized by filtration with a 0.2 μm filter, to prepare an aqueous solution-type arginine deiminase preparation. 
     EXAMPLE 4 
     Expression of Arginine Deiminase by Transformant 
     (1) Production of Mutant Gene 
     In accordance with the Kunkel method [Proc. Natl. Acad. Sci. USA, 82, 488 (1985)], point mutation was carried out to replace all of the 5 TGA codons of the arginine deiminase gene (shown in FIG. 4) by TGG codons which are the tryptophan codons of E. coli, using 5 different types of oligonucleotide as primers. Then, clones in which all of the 5 TGA codons have been replaced by TGG codons were selected by the plaque hybridization method using the above oligonucleotide as a probe. The selected clones were sequenced to make sure that all of the 5 TGA codons had been replaced by TGG codons. 
     (2) Preparation of Arginine Deiminase expressing Vector (pAD 12) 
     Plasmid pAD 12 was prepared by inserting, into the Sac I-Hind III site of plasmid pVC 19, genes containing the structural genes and regulatory genes 240 bq upstream therefrom of the mutant arginine deiminase genes obtained in the above (1) step. The resultant plasmid pAD 12 was found to have a promoter sequence of mycoplasma arginini arginine deiminase, and an analysis of the base sequence of the pAD 12 indicated the presence of a consensus sequence (SD, -10, -35 sequence) of the procaryotic cell promoter. Thus, it was considered possible that the plasmid pAD 12 serve as a vector expressing arginine deiminase of E. coli. 
     (3) Culturing of Transformants and preparation of Cell Extract 
     The pAD 12 prepared in the above (2) step was introduced to E. coli HB 101 (Takara Shuzo Co.), and the resultant transformant was cultured overnight at 37° C. in a 5 ml LB medium [1.0% Bacto Tryptone (DIFCO Co.), 0.5% Bacto Yeast Extract (DIFCO Co.), 0.2% glucose, 1.0% NaCl, pH 7.5]. Then, 1 ml of the culture medium was added to 250 ml of LB medium to be cultured overnight at 37° C. The cell collected from the culture by centrifugation was washed twice with physiological saline, and were suspended in 2 ml of 0.1M potassium phosphate buffer (pH 7.0). Then, the cells were disruped by sonication, and the supernatant after the removal of insolubles by centrifugation was obtained as the cell extract. The same process was repeated to prepare a cell extract of E. coli HB 101 retaining the pUC 19 which did not contain arginine deiminase genes. This extract was used as the control. 
     (4) Measurement of Arginine Deiminase Activity 
     The HB 101 cell extract retaining pAD 12 and the HB 101 cell extract retaining pUC 19 (control) both obtained in the (3) step were diluted 10 times by a 0.1M potassium phosphate buffer (pH 7.0), and 10 μl of each resultant solution was incubated at 37° C. for 5 hours. Then, the amount of citrulline yield was measured by the aforementioned method. The amount of arginine deiminase expression in the HB 101 retaining pAD 12 was calculated from the difference between the amount of the citrulline yield in the HB 101 retaining pAD 12 and that in the HB 101 retaining pUC 19. The result indicated 1.2 unit of arginine deiminase activity for every 250 ml of LB medium. 
     In addition, test results concerning the anti-cancer effects of the arginine deiminase of the present invention are presented in the experiments below. 
     Experiment: Anti-cancer effects of arginine deiminase 
     Experiment 1 
     Method Using Cultured Strains of Human Cancer Cells 
     First, 1×10 4  cells of each type of human cancer cells were inoculated in a 24-well microplate containing 1.0 ml of DME medium with a 10% serum of bovine fetal serum, and the arginine deiminase preparation obtained in the above step was added to this medium, in such an amount that the concentration of this preparation in the medium was 5 ng/ml or 10 ng/ml. Then, after culturing in a 5% CO 2  incubator at 37° C. for 3 days, the number of cancer cells were counted using an automated cell counter (Coultor counter) [Coultor Co.]. As the control, the same amount of PBS was added to the medium instead of the arginine deiminase preparation. The inhibiting activity against cancer cell growth was indicated in terms of the number of cancer cells in samples, relative to the corresponding number for the control which was set as 100. The results are as shown in Table 2. 
     
                       TABLE 2______________________________________              No. of cells              (% of control)Type of cell         5 ng/ml  10 ng/ml______________________________________Human liver cancer cell (HLE)                16.7     6.7Human malignant fibro-sarcoma                20.1     10.3cell (B32)Human squamous carcinoma                31.6     22.9cancer cell (Caski)Human squamous carcinoma                57.4     41.8cancer cell (HSC-4)Human malignant melanoma                41.7     23.4cells (VMRC)Human nasopharyngeal 57.1     46.9carcinoma cell (KB)Human lung carcinoma 42.3     45.8cells (A549)______________________________________ 
    
     Experiment 2 
     Method Using Mouse Leukemia Cells 
     A total of 21 male CDF 1  mice (BALB/c♀×DBA/B♂), 7 weeks old, were used, as subjects, and 1×10 5  cells of leukemia cells L1210 were transplanted i.p. to each mouse. Then, 9 mice were randomly selected as a control group, and the other were equally devided into 2 test groups. The aforementioned arginine deiminase preparations were chronically injected i.p. to the test groups daily for 8 days. The control group animals were administered with PBS. 
     Experiment 3 
     Method Using Mouse Fibro-sarcoma Meth A 
     A total of 27 male CDF 1  mice (BALB/c♀×DBA/2♂), 7 weeks old, were used as subjects, and 1×10 6  cells of fibro-sarcoma cells Meth A were transplanted i.p. to each mouse. Then, 9 mice were randomly selected as a control group, and the others were equally devided into 2 test groups. The arginine deiminase preparations were chronically injected i.p. to the test groups daily for 10 days. The control group animals were administered with PBS. 
     Experiment 4 
     Method Using Mouse Colon carcinoma Cells Colon 26 
     A total of 25 male CDF 1  mice (BALB/c♀×DBA/2♂), 7 weeks old, were used as subjects, and 1×10 6  cells of colon carcinoma cells Colon 26 were transplanted i.p. to each mouse. Then, 9 mice were randomly selected as a control group, and the others were equally devided into 2 test groups. The arginine deiminase preparations were chronically injected i.p. to the test groups daily for 14 days. The control group animals were administered with PBS. 
     Experiment 5 
     Method Using Mouse Sarcoma 180 
     A total of 24 male ICR mice, 7 weeks old, were used as subjects, and 1×10 6  cells of Sarcoma 180 cells were transplanted i.p. to each mouse. Then, 8 mice were randomly selected as a control group, and the others were equally devided into 2 test groups. The arginine deiminase preparations were chronically injected i.p. daily for 14 days to the test groups. The control group animals were administered with PBS. 
     Experiment 6 
     Method Using Mouse Ascites Hepatoma MH 134 
     A total of 26 male C3H/HeN mice, 7 weeks old, were used as subjects, and 1×10 6  cells of Ascites Hepatoma MH 134 were transplanted i.p. to each mouse. Then, 10 mice were randomly selected as a control group, and the others were equally devided into 2 groups. The arginine deiminase preparations were chronically injected i.p. daily for 14 days to the test groups. The control group animals were administered with PBS. 
     The anti-cancer effect was indicated in terms of the percentage (T/C %) of the average number of days lived by the test groups in comparison with that by the control group. The results obtained from the above Experiments 2 through 6 are shown in Tables 3 through 7, respectively. 
     
                       TABLE 3______________________________________(L1210)Group          Ave. No. of days lived                         T/C (%)______________________________________Control (N = 9)          8.4 ± 0.7   (100)0.25 mg/mouse (N = 6)          9.3 ± 0.5   1110.5 mg/mouse (N = 6)          10.3 ± 1.4  126______________________________________ 
    
     
                       TABLE 4______________________________________(Meth A)Group          Ave. No. of days lived                         T/C (%)______________________________________Control (N = 9)          10.6 ± 1.6  (100)0.1 mg/mouse (N = 9)          10.7 ± 0.7  1010.5 mg/mouse (N = 9)          12.6 ± 1.7  118______________________________________ 
    
     
                       TABLE 5______________________________________(Colon 26)Group          Ave. No. of days lived                         T/C (%)______________________________________Control (N = 9)          11.1 ± 1.3  (100)0.1 mg/mouse (N = 8)          11.9 ± 1.4  1070.5 mg/mouse (N = 8)          21.8 ± 4.6  196______________________________________ 
    
     
                       TABLE 6______________________________________(Sarcoma 180)Group          Ave. No. of days lived                         T/C (%)______________________________________Control (N = 8)          21.3 ± 9.9  (100)0.04 mg/mouse (N = 8)           35.3 ± 13.3                         1660.2 mg/mouse (N = 8)          48.4 ± 9.0  228______________________________________ 
    
     
                       TABLE 7______________________________________(MH 134)Group          Ave. No. of days lived                         T/C (%)______________________________________Control (N = 10)          25.0 ± 6.7   1000.04 mg/mouse (N = 8)          &gt;75            &gt;3000.2 mg/mouse (N = 8)          &gt;75            &gt;300______________________________________