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[CANCER RESEARCH 33, 1862-1865, August 1973] Isolation and Purification of L-Methionine-a-deamino--y mercaptomethane-Lyase (L-Methioninase) from Cbs tridiu m sporo genes' Willi Kreis and Catherine Hession Memorial Sloan-Kettering Cancer Center, New York, New York 10021 SUMMARY the point of high purity, as indicated by a single band in the disc electrophoretic assay. In an attempt irreversibly to deplete biological systems of the essential amino acid L-methionine, L-methionine a-dcam mo- -y-mercaptomethane-lyase was isolated from MATERIALS AND METhODS Clostridium sporogenes (ATCC 7955), highly purified, and characterized. All the investigations undertaken indicate C. sporogenes (ATCC 7955) was grown for 18 hr in a a double function: release of the a-amino- and the ‘y medium containing Bacto cooked meat medium, 0.35% methanethiol group by a single protein unit. It is possible (Difco Laboratories, Detroit, Mich.); Bacto tryptone, that this bifunctional activity is brought about by gene 1.0% (Difco); proteose peptone, 1.0% (Difco); sodium fusion. thioglycollate, 0. 1% (Matheson Coleman and Bell, East Rutherford, N. J.); and Ucon lubricant LB625, 0.01% (Union Carbide Corp., Pearl River, N. Y.). Batches of INTRODUCTION 4 g of cells were suspended in 3.5 ml of 0. 1 M sodium: potassium phosphate buffer, pH 7.4, containing 0.5 mM This laboratory reported that a methylhydrazin@ de neutralized 2 (Fluka, Buchs, Switzerland) and 5 mM rivative, I-methyl-2-(p-isopropylcarbamoyl) benzylhydra mercaptoethanol (NKPPM buffer) (Sigma Chemical Co., zinc hydrochloride, (NSC 77213), with clinically demon St. Louis, Mo.) were treated in an ultrasonic disinte strated activity in Hodgkin's disease and lymphosarcoma, grator, Model 60W (Measuring and Scientific Equipment showed characteristic disturbance of the normal methyla Ltd., London, England) 3 times for 3 mm over 15 mm tion of RNA, especially tRNA in P815 mouse neoplasms at 20 kc/sec. The cell suspension was kept in an alcohol: (8). This disturbance consisted of a significant in vivo ice mixture throughout the sonic disruptions. All extrac methylation of RNA-guanine at position 7 and a consid tion steps were performed at 0—4°.Thedisrupted suspen erable reduction of the normal methylation of cytoplasmic sion was centrifuged at 30,000 x g for 30 mm, the pre RNA of these cells. The effect preceded the inhibition of cipitate was washed 2 times with NKPPM buffer, and RNA synthesis and the therapeutic activity of the com the supernatant and the 2 washings of the precipitate pound could be prevented by massive doses of L-methi were then combined. Protamine sulfate (Nutritional Bio onine administered 2 hr before the drug (7). If indeed the chemicals Corp., Cleveland, Ohio), 0.5% in NKPPM undermethylation of RNA (and possibly also DNA) con buffer, was added to a final concentration of 0.25% and tributes to the inhibition of RNA synthesis, it was con the precipitate was discarded after centrifugation. Am cluded that by depletion of the source of methyl groups, monium sulfate was added to 42% saturation and the en i.e., L-methionine, this effect could be achieved more ef zyme-inactive material was removed by filtration through fectively. It was hoped that an L-methionine-degrading glass wool. Additional ammonium sulfate (to 75% satura enzyme would further the studies in this direction, would tion) precipitated all enzyme-active material. After cen help to elaborate on the biological significance and trifugation the precipitate was dissolved in and dialyzed mechanism of DNA and RNA methylation in normal against NKPPM buffer. Two successive gel filtrations and neoplastic tissues, and might be beneficial in tumor on a Sephadex G-200 (Pharmacia Fine Chemicals, Inc., chemotherapy. Piscataway, N. J.) column (50 x 3.0 cm), conditioned and Clostridium sporogenes was used as the source of an cluted with NKPPM buffer, were then performed. One enzyme that degrades 1-methionine to methanethiol, am enzyme-active protein peak was observed. Following monia, and ct-ketobutyric acid. Such an enzyme in crude filtration through the Sephadex G-200 column, the en preparations of C. sporogenes was first described by Wie zyme-active fractions were pooled, precipitated with sendanger and Nisman (21). We carried the isolation to 1 This work was supported in part by Grant CA 08748 from the ‘The abbreviations used are: PPH, pyridoxal phosphate; NKPPM, National Cancer Institute. sodium : potassium phosphate buffer, pH 7.4, containing 0.5 m@i neu Received December 27, 1972; accepted April 18, 1973. tralized PPH and 5 mM mercaptoethanol. 1862 CANCER RESEARCH VOL. 33 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1973 American Association for Cancer Research. L-MethiOninase ammonium sulfate (5.2 g/lO ml), and dissolved in and Radioactive methanethiol released from L-methionine la dialyzed against NKPPM buffer. This pooled sample beled with either 35S or ‘4Cin the methyl group (Amer was filtered a 2nd time through the Sephadex G-200 sham/Searle) showed good quantitative agreement (33.6 column. The enzyme-active fractions from the 2nd Seph and 33.8 nmoles). No radioactive material was trapped adex G-200 were pooled, and the precipitation and when the substrate was labeled with ‘4Cin positions I or dialysis steps were repeated. The resulting enzyme-ac 1, 2, 3, and 4 of L-methionine (Amersham/Searle). The tive sample was applied to a DEAE-Sephadex A-SO 3rd reaction product, a-ketobutyric acid, was identified as (Pharmacia) column (45 x 3. 1 cm). The proteins were the 2 ,4-dinitrophenylhydrazonc derivative by paper eluted with a linear gradient of 0 to 0.4 M KCI in NKPPM [Whatman No. 1, system, 1-butanol :ethanol :water without PPH. The 1st of 2 major peaks contained all the (4 : I : 5)] and thin-layer chromatography (silica gel, enzyme activity. After precipitation with ammonium sul system, 75% phenol) and by its melting point and melting fate (5.2 g/ 10 ml), the protein was dissolved in I mM p0- point of mixture with the derivative of authentic a-keto tassium phosphate buffer, pH 6.5, containing 5 mM mer butyric acid. Minor amounts of other reaction products captoethanol. Final purification was achieved on a hy have not yet been analyzed. droxylapatite (Bio-Gel HI) (Bio-Rad Laboratories, Rich Methanethiol in the incubation medium was identified mond, Calif.) column (9 x 3 cm), using a linear gra by a color reaction for mercaptans ( I5) which consisted dient of I mM to 0.2 M phosphate buffer, pH 6.5, which of the addition of 0. 1 ml of sodium nitroprussiate solu contained 5 mM 2-mercaptoethanol. For protection of the tion [1.5 g sodium nitrosopentacyanofcrratc(III) dis enzyme activity in the last 2 columns, 2 ml of 0.5 M solved in 5 ml of 2 N HC1 and followed by the addition of phosphate buffer containing 2.5 mM PPH were added to 95 ml methanol and 10 ml concentrated ammonia] to the all the tubes used for the collection of lO-ml fractions. reaction product after incubation as above and immediate When methanethiol and ammonia were assayed si spectrophotometric determination of the developed multaneously in each individual fraction of the first purple color at 530 nm. The test is, at best, semiquan and last column, there was good qualtitative and quanti titative. tative coincidence. Acceptable coincidence was also Disc electrophoresis was performed on polyacrylamide found for the ratio of methanethiol to ammonia in all the gel at 5°according to the methods of Ornstein (13) and combined fractions during the isolation procedure (Table Davis (4). Reagents and conditions were as described I). The overall purification was 120-fold (Table I). earlier (9). About 98% of the enzyme activity was lost during the The molecular weight, as determined by gel filtration process. Disc electrophoretic monitoring of the pro on Sephadex G-200 and by ultracentrifugation and sedi teins present at several steps of the extraction procedure mentation analysis with Rayleigh interferometry (16), was gave evidence of the stepwise purification (Chart I). The about 150,000. enzymatically active fractions of the last column Activity of the enzyme was optimal in the pH range of (Step 6) produced only I band in the disc electrophoresis. 7_S to 8.5 for both functions and absent below pH 6.0. The @ For the assay of methanethiol and ammonia, 50 of Km values under the standard conditions of the enzyme L-methionine-methyl- ‘@C(1.33 zmoles; specific activ assay were similar for the 2 functions: 90 mM for the re ity, 0.0379 mCi/mmole; Amersham/Scarle, Arlington lease of methanethiol and 78 mM for the release of am Heights, Ill.); 50 j.zl of sodium PPH (0.472 ,umole); SO monia. Maximal enzyme activity was dependent upon the to 90 @lof sodium : potassium phosphate buffer, pH 7.4 presence of PPH. The approximate Km value for the (0.5 M); and 10 to 50 zl of enzyme were mixed in pre latter was 10 @MPPH when measured in the methane cooled Pyrex ignition tubes (10 x 70 mm) each containing thiol assay, indicating that L-methionine is bound loosely 1 small glass bead. Each tube was then “plugged―in and the cofactor more strongly to the enzyme. Dialysis dividually with a fluted paper disc (Whatman No. 1, of the enzyme against 10 mM hydroxylaminc in NKPPM 26-mm diameter) which was moistened with 2 drops of a buffer decreased the activity exponentially to 5% within 3 saturated mercuric chloride solution. The tubes were hr and to less than 2% within 18 hr. Dialysis against plain incubated in a shaking water bath for 15 mm at 37°,and buffer reduced the enzyme activity within the same time the reaction was stopped by cooling in ice.

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