Glutaminase‐free asparaginase from vibrio succinogenes: An antilymphoma enzyme lacking hepatotoxicity

Abstract

Microbiology 04-4 and Pathology R-1, Miami, FL. USA.Successful treatment of neoplastic disease has been impeded by the lack of therapeutic agents which specifically destroy tumor cells. Enzymes which selectively deplete substrates required by tumor cells, but not by normal tissue, could improve therapeutic indices dramatically. Presently, microbial asparaginases are used clinically for treating acute lymphocytic leukemia. While these enzymes should destroy neoplastic cells and spare normal tissues, their use is accompanied by many toxic effects and immunosuppression. The administration of Escherichia colior Erwinia carotovora asparaginase depletes circulating glutamine as well as asparagine. It has been suggested that this glutaminase activity may be responsible for the observed toxicities. We have isolated a glutaminase-free asparaginase from Vibrio succinogenes with potent antilymphoma activity. Previously, we demonstrated that administration of Vibrio asparaginase does not cause the immunosuppression of humoral or cell-mediated responses assaciated with treatment by other microbial enzymes. In the present communication we have evaluated the hepatotoxic effects of different asparaginases since liver damage is the major toxicity associated with treatment by these microbial enzymes. BALBk mice treated with 50 IU of E. coli asparaginase daily for 4 days exhibited diffuse microfatty infiltration within hepatocytes throughout the liver. Cross-sections of liver from V. succinogenes asparaginase-treated mice appeared normal as compared to specimens from control animals. Quantitation of the total amount of extractable lipid from the livers of E. coli asparaginase-treated animals indicated a 45 % and I27 % increase in lipid concentration as compared to controls after 4 and 5 days of treatment, respectively. The Vibrio enzyme did not cause a change in extractable lipid concentration as compared to control animals. Plasma antithrombin 111 activity and albumin, triglyceride and cholesterol concentrations all decreased in E. coliasparaginase-treated mice, confirming hepatotoxicity. No modifications in plasma proteins were observed in mice treated with asparaginase from V. succinogenes. The plasma lipids did decrease minimally but only the levels of cholesterol were shown to be statistically significant from those of controls. The data strongly support the concept that specific asparagine depletion is not significantly hepatotoxic. More importantly, the asparaginase from V. succinogenes may serve as a potent antileukemic agent without causing damage to normal tissues.

Comparative Studies on Effect of Carbon and Nitrogen Sources on L -Asparaginase Production

“…The purification of enzyme was carried out by using crude enzyme extract (Distasio et al, 1982).The enzyme purification was performed by ammonium sulphate precipitation. Finely powdered ammonium sulfate was added to the crude extract.…”

Overview on L-Asparaginase

“…K+independent plant L-Asparaginases were reported by Michalska and coworkers. [41] They found that the αβ subunits arise from auto-proteolytic cleavage of two copies of precursor protein and the heterodimer is in αββα configuration. Thr193 acts as the nucleophile and is part of the active site.…”

“…[9] L-asparaginase with two distinct bands of molecular weights 40.2 and 39.8 kDa were purified from Erwinia carotovora MTCC 1428. [41] A three step puridfication of asparaginase has also been described. Crude extract was precipitated by 70% ammonium sulphate after which further purification was carried out with Sephadex G-200 using HPLC system.…”

Microbial Amidases and their Industrial Applications: A Review

“…While Roberts et al [21] isolated Achromobacteraceae from the soil samples and observed that this family has L-glutaminase and L-asparaginase activity in a ratio of 1.2:1. There are also many reports on the production of the intracellular L-asparaginase as observed from E. aroideae [34] E.carotovora [35] Serratia marcescens [36] ,Vibrio succinogenes [37], E. coli [38], Pseudomonas7A (Jakob et al, 1996) [39] and P.fluorescens [40].…”