The preparation of endotoxin-free L-methionine-alpha-deamino-gamma-mercaptomethane-lyase (L-methioninase) from Pseudomonas putida.

Many types of human and animal tumors have an absolute requirement for methionine. This requirement can be satisfied by homocysteine only in normal cells and tissues. Therefore, methionine may be an important target in cancer therapy. To attack this target we have purified endotoxin-free methioninase from Pseudomonas putida by a novel and simple procedure. This procedure involves (1) a heat step of the cell extract at 60 degrees C for 8 min, (2) DEAE-Toyopearl ion-exchange chromatography, (3) DEAE-Sephadex A50 ion-exchange gel filtration chromatography, and (4) affinity chromatography on Acticlean to remove the endotoxin bound to the enzyme. The yield for this purification was up to 80%. The methioninase has four subunits of approximate molecular weight 43 kDa. This is the first methodology for methioninase that allows rapid purification with high yield and separation from endotoxin suitable for in vivo efficacy testing against methionine-dependent tumors in animal models.

Depletion of serum methionine by methioninase in mice.

Methionine dependence is a tumor-specific metabolic defect found in human cancer cell lines as well as in fresh human tumor specimens. Methionine dependent tumors cease growing when deprived of methionine, unlike normal cells which can substitute homocysteine for methionine for their growth requirement. We have previously purified a stable, endotoxin-free methioninase from the bacterium, Pseudomonas putida. We demonstrate in this report that purified methioninase can lower the serum levels of methionine in normal and nude mice from 60 microM to approximately 5 microM within 1 hour. The circulating half-life of methioninase is approximately 100 minutes in mice after i.v. injection. The enzyme therefore seems to be a good candidate as an antitumor agent for methionine-dependent tumors.