Isolation, purification and physicochemical properties of glutamin-asparaginase from Pseudomonas boreopolis 526

A new homogeneous enzyme which is capable of catalyzing the hydrolysis of both glutamine and asparaginase has been purified from extracts of Pseudomonas boreopolis 526 by the improved method. Purification involves few stages. The ratio of glutaminase to asparaginase activity is approximately 1.5:1.0. The enzyme is stable on storage and has a wide pH optimum of action (6-8.5). The molecular weight is about 134 000-145 000 D and the subunit molecular weight is about 34 000 D. No free SH-groups have been detected in the enzyme molecule.     

Biological properties of glutamin-(asparagin-)ase from Pseudomonas boreopolis 526

Glutamine(asparagine)ase from Ps. boreopolis 526 has an antineoplastic effect on lymphoid leukemia P-388. The enzyme half-life in the mouse serum is 8.5 hours. Glutamine(asparagine)ase has no cross-antigenicity with L-asparaginase from E. coli (Bayer, FRG). Specific antibodies against L-asparaginase (Bayer, FRG) do not influence the activity of glutamine(asparagine)ase.     

Isolation, purification and stability of a glutamin(asparagin)ase preparation from Pseudomonas boreopolis 526

A technique for purification of glutamine asparaginase from Pseudomonas boreopolis 526 is described which provides a 37% yield of the enzyme homogeneous according to electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate. The effect of pH, freezing, thawing and lyophilic drying on the stability of glutamine asparaginase was studied. The enzyme is rather stable at pH 4.8 and 4 degrees C. Lyophilic drying of the homogeneous enzyme without addition of stabilizers resulted in a decrease of its activity an is accompanied by formation of protein conglomerates with molecular weights of 280,000 and 660,000 D. Freezing and thawing decreased the activity of the nature enzyme by 40-50%.     

Control of glutaminase synthesis in Escherichia coli

Summary Glutaminase levels in E. coli B after growth on various media are presented. The results show that glutaminase synthesis is repressible and suggest glutamine as the corepressor.     

Characteristics of Triticale glutaminase]

The glutaminase (EC 3.5.1.2) isolated from seedlings of triticale (Triticale sp.) had a pH optimum of about 8, was inhibited with excess substrate (glutamine), and reaction products (glutamate and NH4+). A monovalent anion (Cl-) and a multivalent anion (phosphate) were shown to activate the glutaminase. Some features of the glutaminase from triticale were similar to those of animal glutaminase activated by phosphate and were different from features of the enzyme from Escherichia coli.     

Improvement in the persistence of microbial asparaginase and glutaminase in the circulation of the rat by chemical modifications

Three enzymes used in cancer chemotherapy (asparaginases from Escherichia coli and Erwinia carotovora and glutaminase from Achromobacter) were each reacted with four amino specific reagents (ethyl acetimidate, O-methylisourea, succinic anhydride, and formaldehyde/sodium borohydride). The half-lives of the modified enzymes measured in the blood of rats showed that guanidation, acetimidation and reductive alkylation were more likely to increase the persistence of the native enzymes than succinylation. However, the improvement in the persistence of any one enzyme after any one modification could not be predicted from the results with the others. It was concluded that changes in persistence caused by each modification were due to the different effects on the tertiary structure of each native enzyme. The advantages of chemical modification for increasing the persistence of enzymes over other methods such as encapsulation or aggregation are discussed.     

Engineering the substrate specificity of Escherichia coli asparaginase. II. Selective reduction of glutaminase activity by amino acid replacements at position 248

The use of Escherichia coli asparaginase II as a drug for the treatment of acute lymphoblastic leukemia is complicated by the significant glutaminase side activity of the enzyme. To develop enzyme forms with reduced glutaminase activity, a number of variants with amino acid replacements in the vicinity of the substrate binding site were constructed and assayed for their kinetic and stability properties. We found that replacements of Asp248 affected glutamine turnover much more strongly than asparagine hydrolysis. In the wild-type enzyme, N248 modulates substrate binding to a neighboring subunit by hydrogen bonding to side chains that directly interact with the substrate. In variant N248A, the loss of transition state stabilization caused by the mutation was 15 kJ mol(-1) for L-glutamine compared to 4 kJ mol(-1) for L-aspartic beta-hydroxamate and 7 kJ mol(-1) for L-asparagine. Smaller differences were seen with other N248 variants. Modeling studies suggested that the selective reduction of glutaminase activity is the result of small conformational changes that affect active-site residues and catalytically relevant water molecules.     

Cloning, sequence analysis, and expression of ansB from Pseudomonas fluorescens, encoding periplasmic glutaminase/asparaginase

A gene (ansB) encoding a class II glutaminase/asparaginase has been cloned from Pseudomonas fluorescens and characterized by DNA sequencing, promoter analysis and heterologous expression in Escherichia coli. We show that ansB is monocistronic and depends on the alternate sigma factor sigma 54 for expression. A second open reading frame located downstream of ansB is highly homologous to a number of bacterial genes that encode secreted endonucleases of unknown function.     

A Note: Serological study of four pathovars of Pseudomonas syringae: Ps. syr. aptata, Ps. syr. tabaci, Ps. syr. mors-prunorum and Ps. syr. phaseolicola

The antigenic reactions of 35 strains of four pathovars of Pseudomonas syringae (Ps. syr. aptata, Ps. syr. tabaci, Ps. syr. mors-prunorum and Ps. syr. phaseolicola ) were studied by double diffusion and indirect immunofluorescent staining, and anti-whole-cell and anti-LPS-extract sera. It had already been shown that the precipitating lines in Ouchterlony double-diffusion tests, due to bacterial LPS, were suitable for the distinction of O-serogroups. The investigation of serological cross-reactions between the 35 strains and 20 antisera revealed that three pathovars were serologically homogeneous: Ps. syr. aptata, Ps. syr. tabaci and Ps. syr. phaseolicola. They could fit into three O-serogroups formerly described: namely APTPIS, TAB and PHA. The O-serogroups APTPIS and TAB showed some common antigens. The 10 strains of Ps. syr. mors-prunorum studied were distributed into two O-serogroups (eight strains belonging to the O-serogroup MOP1, one strain to MOP2, and the last strain failed to react with any of the serogroups).