[Bacterial L-asparaginase and glutamin(asparagin)ase: some properties, structure and anti-tumor activity]. / Bakterial'nye L-asparaginazy i glutamin(asparagin)azy: nekotorye svoistva, stroenie i protivoopukholevaia aktivnost'.

Experimental material on structurally and functional organization, regulation of biosynthesis and activity, mechanism of action, genetic determinants, heterologous expression of bacterial L-asparaginases is accumulated. The modern approaches to isolation and purification of these enzymes, some questions of practical using in oncology in the schedules combined chemotherapy of leukemia the native and modified forms of L-asparaginases are discussed. The some results before carried out in the IBMC RAMS and number institutes of the Russia on study bacterial L-asparaginases and glutamine(asparagine)ases are summarized.

[Miniature thin layer biosensors which are sensitive to glutamate and glutamine]. / Miniatiurnye tonkoplenochnye biosensory, chuvstvitel'nye k glutaminovoi kislote i glutaminu.

Integrated thin film biosensors for simultaneous measurement of L-glutamine and L-glutamate in a micro-flow cell were developed. Due to a novel glutaminase with an activity optimum in the neutral pH range a direct monitoring of glutamine in mammalian cell culture medium could be performed. The glutamine bienzyme sensor was prepared by coimmobilization of glutaminase with glutamate oxidase within a photo patterned poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogel membrane. The sensor response is linear in the concentration range of 55 mmol to 10 mmol glutamine/l. Additionally a glutamate biosensor is integrated on the sensor chip for difference measurement of possible glutamate interferences. The sensor chip can be used for at least 300 measurements without any alteration in the performance of its sensors. A new sensor chip with an integrated flow cell provides the possibility of simultaneous measurement of four different parameters at a cell volumina of 1 ml. Completing the microsystem a battery operated surface mounted device (SMD) potentiostat was developed to get a "lab on chip".

Miniaturized thin film glutamate and glutamine biosensors.

Integrated thin film biosensors were developed for the simultaneous measurement of L-glutamine and L-glutamate in a mu-flow cell. Due to a novel glutaminase with an activity optimum in the neutral pH range, direct monitoring of glutamine in a mammalian cell culture medium could be performed. The glutamine bienzyme sensor was prepared by co-immobilization of glutaminase with glutamate oxidase within a photopatterned poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogel membrane. The sensor response was linear in the concentration range of 50 mumol to 10 mmol glutamine/l. Additionally, a glutamate biosensor was integrated on the sensor chip for difference measurement of possible glutamate interferences. The sensor-chip could be used for at least 300 measurements without any alteration in the performance of its sensors. A new sensor-chip with an integrated flow cell provided the possibility of simultaneous measurement of four different parameters at a cell volume of 1 microliter. In order to complete the microsystem, and in order to obtain a "lab on chip", a battery operated surface mounted device (SMD) potentiostat was developed.

[Isolation, various physico-chemical and catalytic properties of L-methionine-gamma-lyase from Pseudomonas taetrolens]. / Vydelenie, nekotorye fiziko-khimicheskie i kataliticheskie svoistva L-metionin-gamma-liazy iz Pseudomonas taetrolens.

Homogeneous preparation of L-methionine gamma-lyase was isolated from Ps. taetrolens. As shown by gel filtration and gradient polyacrylamide gel electrophoresis molecular mass of the native L-methionine gamma-lyase was 130-135 kDa. Polyacrylamide gel electrophoresis in presence of 0.1% SDS showed that L-methionine gamma-lyase proved to be a tetramer, which consisted of identical subunits with a molecular mass of 34 kDa. Pyridoxal-5'-phosphate was bound to the enzyme in the ratio of four moles of the cofactor per a mole of protein. The absorption spectrum of the enzyme exhibited maximal values at 420 nm, which is specific for a number of pyridoxal phosphate-containing enzymes. L-methionine gamma-lyase from Ps. taetrolens was found to be dissimilar in its physicochemical and catalytic properties to the same enzymes from other sources.

[Isolation, purification and stability of a glutamin(asparagin)ase preparation from Pseudomonas boreopolis 526]. / Vydelenie, ochistka i stabil'nost' preparata glutamin(asparagin)azy iz 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%.

[Biological properties of glutamin-(asparagin-)ase from Pseudomonas boreopolis 526]. / Biologicheskie svoistva glutamin(asparagin)azy iz 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 physicochemical properties of glutamin-asparaginase from Pseudomonas boreopolis 526]. / Vydelenie, ochistka i nekotorye fiziko-khimicheskie svoistva glutaminasparaginazy iz 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.

[The effect of preparations of microbial glutamin(asparagin)ase on cultured tumor cells]. / Deistvie preparatov glutamin[asparagin]azy iz mikroorganizmov na opukholevye kletki v kul'ture.

Glutamin (asparagin)ase from Pseudomonas boreopolis 526 is shown to produce a cytotoxic effect on the human ovary carcinoma cells (CaOv line), Fisher lympholeukemia (L-8 line) and Burkitt's lymphoma cells (P3HR line). A significant inhibition of the DNA synthesis is found in L-8 and P3HR cell lines. The enzyme is less active in the cells of the CaOv line.

[Effect of glutamin(asparagin)ase preparations from microorganisms on DNA synthesis in tumor cells]. / Vliianie preparatov glutamin(asparagin)azy iz mikroorganizmov na sintez DNK v opukholevykh kletkakh.

The effect of glutamin (asparagin)ase from Pseudomonas fluorescens and Pseudomonas boreopolis 526 on DNA synthesis by tumor cells, lines CaOv and L8, has been studied. The L8 cells have been demonstrated highly sensitive to the enzyme.

[Improved procedure for isolation and purification of methionine gamma-lyase from Pseudomonas putida]. / Usovershenstvovannyi metod vydeleniia i ochistki metionin-gamma-liazy iz Pseudomonas putida.

An improved, simplified and relatively rapid procedure is developed for isolation and purification of a new antitumor enzyme--methionine gamma-lyase from Pseudomonas putida. The method includes five steps instead of seven steps in previous procedure with a good yield of the enzyme. The purified enzyme was shown to be homogeneous by the criteria of disc gel electrophoresis. The highly homogeneous preparations of the enzyme exhibited the absorption maxima at 280 and 420 nm. The detailed studies on antileukemic activity of the methionine gamma-lyase are currently in progress.

[Cytotoxic effect of methionine-gamma-lyase on neoplastic cells in culture]. / Tsitotoksicheskii éffekt metionin-gamma-liazy na rakovye kletki v kul'ture.

The influence of methionine-gamma-lyase from Pseudomonas putida on DNA synthesis by CaOv and L-8 cell lines has been studied. The agent has been demonstrated to inhibit the incorporation of 3H-thymidine into L-8 cell line and to have no effect on CaOv cells.