Antiproliferative activity of L-asparaginase of Tetrahymena pyriformis on human breast cancer cell lines

Purified L-asparaginase of Tetrahymena pyriformis is a multi-subunit enzyme exhibiting protein kinase activity as well. The enzyme's L-asparaginase activity is affected by its phosphorylation state. Both native and dephosphorylated L-asparaginase show antiproliferative activity on three breast cancer cell lines (T47D, BT20 and MCF-7) and on Walker 256 cells. These cells do not possess measurable L-asparaginase or L-asparagine synthetase activity. When T47D cells are treated for different times with L-asparaginase and then placed in fresh medium, the growth of cells treated for 1, 3, or 6 hours is initiated and parallels control curve, while the growth of cells treated for 24 or 48 hours with L-asparaginase stays at the same inhibitory level (24 h treatment) or continues to drop (48 h treatment). Addition of D-asparagine, a competitive inhibitor of T. pyriformis L-asparaginase, counteracts the antiproliferative activity of L-asparaginase, indicating that L-asparaginase and not the kinase activity is responsible for that effect.     

Effects on specific antibodies on the catalytic activity of L-asparaginase from Serratia marcescens and Escherichia coli.

Rabbit antisera against highly purified L-asparaginase from Serratia marcescens and from Escherichia coli showed up to 60% inhibition of the catalytic amidohydrolysis of L-asparagine when combined with the homologous enzyme. This inhibition was diminished somewhat against the heterologous enzyme. Kinetic studies in the presence of these antisera showed an increased Kmapp for both homologous and heterologous enzymes using L-asparagine as substrate. In contrast, kinetic studies employing the poor substrate, L-glutamine, showed activation attributable to specific antibodies. This was seen in lower Kmapp values and up to twofold increases in the Vmax over the normal rabbit serum controls. The high degree of cross-inhibition (approximately 80%) and the low degree of cross-reactivity in the quantitative precipitin test (approximately 34%) suggest that these two enzymes possess structural similarities located mainly in the regions of the catalytic sites.     

L-asparaginase of Tetrahymena pyriformis is associated with a kinase activity

Most of L-asparaginase activity of Tetrahymena pyriformis was found to be present in microsomal membranes from which it has been purified to homogeneity (Tsirka, S.A.E. and Kyriakidis, D.A. Mol. Cell. Biochem. 83: 147–155, 1988). The native enzyme has a relative molecular weight of approximately 200 kDa, while under denaturing conditions the enzyme exhibits. a subunit size of 39 kDa. Aminoacid analysis and an oligopeptide from N-terminal sequence have been determined. Dephosphorylation of L-asparaginase by alkaline phosphatase results in an activation of its catalytic activity. This enzyme also exhibits intrinsic phosphorylation activity with a Km value for ATP of 0.5 mM. Autophosphorylation with -^32P ATP of purified L-asparaginase results in the phosphorylation of tyrosine residues as well as in loss of its activity. Mg²⁺ and Ca²⁺ added together act synergistically to stimulate the kinase activity by more than 160%. The polyamines putrescine, spermidine and spermine activate the kinase approximately 100%, while neither cAMP or cGMP have any effect. These results indicate that this membrane protein with dual L-asparaginase/kinase activity must play an important role in regulating the intracellular levels of L-asparagine in Tetrahymena pyriformis.     

Hydrophobic formate dehydrogenase from Pseudomonas sp. 101 in the system of reverse micelles of aerosol OT in octane

NAD(+)-dependent formate dehydrogenase (FDH) was hydrophobized with palmitoyl chloride to give the samples with various modification degrees (2-10). The native and modified FDHs were comparatively studied in the system of reverse micelles of Aerosol OT in octane. Like the native, the modified enzyme displayed three maxima in the curve of dependence of its catalytic activity on the degree of surfactant hydration (the micelle size), which reflect the enzyme functioning in the form of a monomer, dimer, or octamer. The peak corresponding to the functioning of the FDH dimer was found to decrease along with an increase in the modification degree. Thus, the modified enzyme mainly functions in the form of monomer and octamer. The modified FDH displayed membranotropy and revealed the dependence of catalytic activity on surfactant concentration.     

Production,purification, characterization,antioxidant and antiproliferative activities of extracellular L-asparaginase produced by Fusarium equiseti AHMF4

L-Asparaginase is an antileukemic agent that depletes L-asparagine “an important nutrient for cancer cells” through the hydrolysis of L-asparagine into L-aspartic acid and ammonia leading to leukemia cell starvation and apoptosis in susceptible leukemic cell populations. Moreover currently, bacterial L-asparaginase has been limited by problems of lower productivity, stability, selectivity and a number of toxicities along with the resistance towards bacterial L-asparaginase. Then the current work aimed to provide pure L-asparaginase with in-vitro efficacy against various human carcinomas without adverse effects related to current L-asparaginase formulations. Submerged fermentation (SMF) bioprocess was applied and improved to maximize L-asparaginase production from Fusarium equiseti AHMF4 as alternative sources of bacteria. The enzyme production in SMF was maximized to reach 40.78 U mL⁻¹ at the 7th day of fermentation with initial pH 7.0, incubation temperature 30 °C, 1.0% glucose as carbon source, 0.2% asparagine as nitrogen source, 0.1% alanine as amino acid supplement and 0.1% KH₂PO₄. The purification of AHMF4 L-asparaginase yielded 2.67-fold purification and 48% recovery with final specific activity of 488.1 U mg⁻¹ of protein. Purified L-asparaginase was characterized as serine protease enzyme with molecular weight of 45.7 kDa beside stability at neutral pH and between 20 and 40 °C. Interestingly, purified L-asparaginase showed promising DPPH radical scavenging activity (IC₅₀ 69.12 μg mL⁻¹) and anti-proliferative activity against cervical epitheloid carcinoma (Hela), epidermoid larynx carcinoma (Hep-2), hepatocellular carcinoma (HepG-2), Colorectal carcinoma (HCT-116), and breast adenocarcinoma (MCF-7) with IC₅₀ equal to 2.0, 5.0, 12.40, 8.26 and 22.8 μg mL⁻¹, respectively. The enzyme showed higher activity, selectivity and anti-proliferative activity against cancerous cells along with tiny cytotoxicity toward normal cells (WI-38) which indicates that it has selective toxicity and it could be applied as a less toxic alternative to the current formulations.     

Hydrophobized Formate Dehydrogenase from Pseudomonas sp. 101 in the System of Reverse Micelles of Aerosol OT in Octane

NAD⁺-dependent formate dehydrogenase (FDH) was hydrophobized with palmitoyl chloride to give the samples with various modification degrees (2–10). The native and modified FDHs were comparatively studied in the system of reverse micelles of Aerosol OT in octane. Like the native, the modified enzyme displayed three maxima in the curve of dependence of its catalytic activity on the degree of surfactant hydration (the micelle size), which reflect the enzyme functioning in the form of a monomer, dimer, or octamer. The peak corresponding to the functioning of the FDH dimer was found to decrease along with an increase in the modification degree. Thus, the modified enzyme mainly functions in the form of monomer and octamer. The modified FDH displayed membranotropy and revealed the dependence of catalytic activity on surfactant concentration.     

Purification of an L-asparaginase-atrial natriuretic peptide fusion protein expressed in Escherichia coli

A novel fusion protein designed to facilitate protein purification was expressed in Escherichia coli and purified separately by two different chromatography methods. L-Asparaginase from Erwinia chrysanthemi is fused to the N-terminus of a model peptide, alpha-human atrial natriuretic peptide (alpha-hANP). L-Asparaginase was chosen because of its selective affinity for L-asparagine and because of its unusually high isoelectric point(8.6). A gene construction without the L-asparaginase native signal sequence caused expression at a level of 8% of total cell protein, while gene construction with the native signal sequence resulted in over five time less expression. The hybrid protein expressed without the signal sequence was purified from clarified cell lysate byeither L-asparagine affinity chromatography or cation exchange chromatography. After digestion of the fusion protein with factor Xa protease, a peptide with a molecular weight corresponding to the theoretical molecular weight of alpha-hANP was observed by coupled HPLC/mass spectrometry. (c) 1995 John Wiley & Sons Inc.     

多聚唾液酸对L-天冬酰胺酶的修饰及修饰酶特性研究

来源于大肠杆菌 (Ｅ .ｃｏｌｉ)的Ｌ 天冬酰胺酶是治疗淋巴性白血病和恶性淋巴肿瘤的有效酶制剂 ,已应用于临床。该酶与其他蛋白质类药物一样 ,在临床应用中存在两个常见问题 :一是酶制剂在体内易被降解 ,导致半衰期短 ;二是免疫原性。为了解决上述问题 ,人们用亲水性的大分子如血清蛋白、右旋糖苷和单甲氧基聚乙二醇 (ｍＰＥＧ)对该酶进行修饰。其中ｍＰＥＧ[1] 修饰后的Ｌ 天冬酰胺酶的抗原抗体结合能力完全消失 ,免疫原性下降 ,且体内半衰期延长 ;但酶活力只有天然酶的 8%～ 14% ,且ｍＰＥＧ在人体组织中无法降解 ,目前尚难评估长期使用…     

L-asparaginase: a promising chemotherapeutic agent

This article comprises detailed information about L-asparaginase, encompassing topics such as microbial and plant sources of L-asparaginase, treatment with L-asparaginase, mechanism of action of L-asparaginase, production, purification, properties, expression and characteristics of l-asparaginase along with information about studies on the structure of L-asparaginase. Although L-asparaginase has been reviewed by Savitri and Azmi (2003), our effort has been to include recent and updated information about the enzyme covering new aspects such as structural modification and immobilization of L-asparaginase, recombinant L-asparaginase, resistance to L-asparaginase, methods of assay of L-asparagine and L-asparaginase activity using the biosensor approach, L-asparaginase activity in soil and the factors affecting it. Also, side-effects of L-asparaginase treatment in acute lymphoblastic leukemia (ALL) have been discussed in the current review. L-asparaginase has been and is still one of the most widely studied therapeutic enzymes by researchers and scientists worldwide.     

Physicochemical properties and antiproliferative activity of recombinant Yersinia pseudotuberculosis L-asparaginase

The physicochemical, catalytic, and antiproliferative activity of a recombinant L-asparaginase from Yersinia pseudotuberculosis (YpA) have been studied. The following results were obtained: the K _M value for L-asparagine is 17 ± 0.9 ??M, the optimal temperature is 60°C, pH is 8.0, pI is 5.4 ± 0.3, the L-glutaminase activity is no more than 5?C6% of the L-asparaginase activity, and the antiproliferative activity on the Fisher L5178y lymphadenosis cell line comprised T/C = 136% (p < 0.001) at a 15% recovery rate. The described characteristic allows one to regard YpA as an antitumor enzyme with biological features similar to the L-asparaginase of E. coli.     

L-asparaginase of Thermus thermophilus: Purification,properties and identificaation of essential amino acids for its catalytic activity

L-asparaginase EC 3.5.1.1 was purified to homogeneity from Thermus thermophilus. The apparent molecular mass of L-asparaginase by SDS-PAGE was found to be 33 kDa, whereas by its mobility on Sephacryl S-300 superfine column was around 200 kDa, indicating that the enzyme at the native stage acts as hexamer. The purified enzyme showed a single band on acrylamide gel electrophoresis with pI = 6.0. The optimum pH was 9.2 and the Km for L-asparagine was 2.8 mM. It is a thermostable enzyme and it follows linear kinetics even at 77°C. Chemical modification experiments implied the existence of histidyl, arginyl and a carboxylic residues located at or near active site while serine and mainly cysteine seems to be necessary for active form.     

L-Asparaginase: A Promising Chemotherapeutic Agent

ABSTRACT

This article comprises detailed information about L-asparaginase, encompassing topics such as microbial and plant sources of L-asparaginase, treatment with L-asparaginase, mechanism of action of L-asparaginase, production, purification, properties, expression and characteristics of l-asparaginase along with information about studies on the structure of L-asparaginase. Although L-asparaginase has been reviewed by , our effort has been to include recent and updated information about the enzyme covering new aspects such as structural modification and immobilization of L-asparaginase, recombinant L-asparaginase, resistance to L-asparaginase, methods of assay of L-asparagine and L-asparaginase activity using the biosensor approach, L-asparaginase activity in soil and the factors affecting it. Also, side-effects of L-asparaginase treatment in acute lymphoblastic leukemia (ALL) have been discussed in the current review. L-asparaginase has been and is still one of the most widely studied therapeutic enzymes by researchers and scientists worldwide.     

Purification and characterization of L-asparaginase with anti-lymphoma activity from Vibrio succinogenes.

Homogeneols L-asparaginase with anti-lymphoma activity was prepared from Vibrio succinogenes, an anaerobic bacterium from the bovine rumen. An overall yield of pure L-asparaginase of 40 to 45% and a specific activity of 200 +/- 2 IU per mg of protein was obtained. The pure enzyme can be stored at -20 degrees for at least 3 months with no loss of activity. The isoelectric point of the L-asparaginase is 8.74. No carbohydrate, phosphorus, tryptophan, disulfide, or sulfhydryl groups were detected. The enzyme has a molecular weight of 146,000 and a subunit weight of approximately 37,000. The Km of the enzyme for L-asparagine is 4.78 X 10(-5) M and the pH optimum of the L-asparaginase reaction is 7.3. D-Asparagine was hydrolyzed at 6.5% of the rate found with the L isomer. L-Glutamine and a variety of other amides were not hydrolyzed at significant rates; the activity of the enzyme for L-glutamine was 130- to 600-fold less than that of other therapeutically effective L-asparaginases of bacterial origin. The L-asparaginase from V. succinogenes is immunologically distinct from the L-asparaginase (EC-2) of Escherichia coli.     

Construction and structural modeling of a single-chain Fv-asparaginase fusion protein resistant to proteolysis

In this study, we construct a fusion protein composed of L-asparaginase (ASNase; from Escherichia coli AS 1.357) and a protective single-chain Fv (scFv), which was selected from a phage-display scFv library from our previous studies. The antibody moiety of the fusion protein was fused to the N-terminus of the enzyme moiety via a linker peptide, (Gly(4)Ser)(6). Recombinant plasmid pET-SLA was constructed to express scFv-ASNase fusion to high levels in E. coli and the expressed product was found to form inclusion bodies. We obtained a soluble fusion protein by refolding and purification. The soluble fusion protein exhibited about 82% of the enzymatic activity of the native ASNase at the same molar concentration, and had a K(m) value similar to that of the native enzyme for the substrate L-asparagine. Importantly, the fusion protein was more stable than native ASNase. In addition: (1) following treatment with trypsin, alpha-chymotrypsin, and rennet, at 37 degrees C for 30 min, scFv-ASNase fusion retained 94.0%, 88.8%, and 84.5% of its original activity, respectively, whereas native ASNase became inactive; and (2) ScFv-ASNase fusion had a much longer in vitro half-life (9 h) in serum than the native enzyme (2 h). The three-dimensional structure of the fusion protein was obtained by modeling with the Homology and Discover modules of the INSIGHT II software package. On the basis of the structural evidence and biochemical properties, we propose that the scFv moiety of the fusion protein may confer ASNase moiety resistance to proteolysis as a result of both steric hindrance and a change in the electrostatic surface of the enzyme.     

Characterization and partial purification of L-asparaginase from Corynebacterium glutamicum

A high L-asparaginase (L-asparagine amidohydrolase: EC 3.5.1.1) activity was found under conditions of lysine overproduction in cultures of Corynebacterium glutamicum. L-Asparaginase was purified 98-fold by protamine sulphate precipitation. DEAE-Sephacel anion exchange, ammonium sulphate precipitation and Sephacryl S-200 gel filtration. The asparaginase protein was subjected to PAGE under non-denaturing conditions, identified by an in situ reaction and eluted from the gel in an active form. The estimated Mr from gel filtration and SDS-PAGE was 80,000. The L-asparaginase activity was inhibited by the L-asparagine analogue 5-diazo-4-oxo-L-norvaline. Neither D-asparagine nor L-glutamine was a substrate for the enzyme. L-Asparaginase was produced constitutively: its role may be that of an overflow enzyme, converting excess asparagine into aspartic acid, the direct precursor of lysine and threonine.     

Purification and properties of an L-asparaginase from Cylindrocarpon obtusisporum MB-10

An L-asparaginase producing mesophilic fungus Cylindrocarpon obtusisporum MB-10 was isolated from soil. The constitutive intracellular L-asparaginase from the organism was purified. The enzyme after 65-fold purification with an overall yield of 11% and specific activity of 100 unit.mg-1 seemed to be homogeneous in native, SDS-PAGE and thin layer isoelectric focusing gel. The apparent Mr of the enzyme was 216,000, and it constituted four identical subunits. The pI of the enzyme was 5.5. It was a conjugate protein with 37.3% (w/w) carbohydrate. The enzyme was stable to storage at -20 degrees C and to repeated freezing and thawing. The L-asparaginase from the organism was very much specific for L-asparagine and did not hydrolyze D-asparagine and L-glutamine. The pH and temperature optima for the enzyme activity were 7.4 and 37 degrees C, respectively. The Km of the L-asparaginase was found to be 1 x 10(-3)M. Metal ions, such as Zn2+, Fe2+, Cu2+, Hg2+ and Ni2+ potentially inhibited the enzyme activity, while metal chelators like EDTA, CN-, cysteine, etc., enhanced the activity indicating that the enzyme was not a metalloprotein. Its activity was also enhanced in the presence of reduced glutathione but not with dithiothreitol and 2-mercaptoethanol. Differential inhibition of the enzyme activity was observed with iodoacetamide and p-chloromercuribenzoate, thus indicating possible involvement of free-SH group in the enzyme catalysis.     

Conformational flexibility of a model protein upon immobilization on self-assembled monolayers

The present study reports on the retention of conformational flexibility of a model allosteric protein upon immobilization on self-assembled monolayers (SAMs) on gold. Organothiolated SAMs of different compositions were utilized for adsorptive and covalent attachment of bovine liver glutamate dehydrogenase (GDH), a well-characterized allosteric enzyme. Sensitive fluorimetric assays were developed to determine immobilization capacity, specific activity, and allosteric properties of the immobilized preparations as well as the potential for repeated use and continuous catalytic transformations. The allosteric response of the free and immobilized forms towards ADP, L-leucine and high concentrations of NAD(+), some of the well-known activators for this enzyme, were determined and compared. The enzyme immobilized by adsorption or chemical binding responded similarly to the activators with a greater degree of activation, as compared to the free form. Also loss of activity involving the two immobilization procedures were similar, suggesting that residues essential for catalytic activity or allosteric properties of GDH remained unchanged in the course of chemical modification. A recently established method was used to predict GDH orientation upon immobilization, which was found to explain some of the experimental results presented. The general significance of these observations in connection with retention of native properties of protein structures upon immobilization on SAMs is discussed.     

Molecular characterization of the soybean L-asparaginase gene induced by low temperature stress

L-asparaginase (EC 3.5.1.1) catalyzes the hydrolysis of the amide group of L-asparagine, releasing aspartate and NH4+. We isolated a low temperature-inducible cDNA sequence encoding L-asparaginase from soybean leaves. The full-length L-asparaginase cDNA, designated GmASP1, contains an open reading frame of 1,258 bp coding for a protein of 326 amino acids. Genomic DNA blotting and fluorescence in situ hybridization showed that the soybean genome has two copies of GmASP1. GmASP1 mRNA was induced by low temperature, ABA and NaCl, but not by heat shock or drought stress. E. coli cells expressing recombinant GmASP1 had 3-fold increased L-asparaginase activity. A possible function of L-asparaginase in the early response to low temperature stress is discussed.     

PEGylated recombinant L-asparaginase from <Emphasis Type="Italic">Erwinia carotovora</Emphasis>: Production,properties, and potential applications

N-hydroxysuccinimide ester of monomethoxy polyethylene glycol hemisuccinate was synthesized. It acylated amino groups in a molecule of recombinant L-asparaginase from Erwinia carotovora. A method of L-asparaginase modification by the obtained activated polyethylene glycol derivative was developed. The best results were produced by modification of the enzyme with a 25-fold excess of reagent relative to the enzyme tetramer. The modified L-asparaginase was isolated from the reaction mixture by gel filtration on Sepharose CL-6B. The purified bioconjugate did not contain PEG unbound to the protein, demonstrated high catalytic activity, and exhibited antiproliferative action on cell cultures.     

Glycosylation of Escherichia coli L-asparaginase.

Reductive coupling with sodium cyanoborhydride has been used with lactose and N-acetylneuraminyl lactose to prepare glycosylated Escherichia coli L-asparaginase. A substantial degree of modification can be achieved without significant loss of enzyme activity. The lactosylated enzyme shows increased thermal stability and resistance to proteolytic cleavage and is cleared more rapidly from the plasma of mice, compared to native asparaginase. The effect on clearance varies directly with the degree of lactosylation. Asparaginase modified with N-acetylneuraminyl lactose, in contrast, with approximately 13.6 mol of N-acetylneuraminyl lactose/mol of enzyme, is cleared more slowly, with a t 1/2 that is approximately twice that of the native enzyme.