Biochemical criteria for the evaluation of drug efficiency on adjuvant arthritis and nephrotoxic serum nephritis in the rat: studies with phenylbutazone, L-Asparaginase, colchicine, lysine acetylsalicylate, and pyridinol carbamate.

The levels of serum orosomucoid, haptoglobin, and seromucoid were evaluated as possible quantitative criteria for the estimation of drug efficiency in adjuvant arthritis and nephrotoxic serum nephritis. In adjuvant arthritis, haptoglobin, seromucoid, and chiefly orosomucoid serum levels were generally very sensitive to anti-inflammatory agents such as phenylbutazone and pyridinol carbamate, and to immunosuppressive agents such as L-asparaginase. There was a significant correlation between the serum levels of these glycoproteins and the arthritis scores. In nephrotoxic serum nephritis, seromucoid levels were correlated with the proteinuria of the autologous phase and were found to be a good complementary criterion for the analysis of the efficiency of pyridinol carbamate, colchicine, iysine acetylsalicylate, and L-asparaginase.     

Asparagine catabolism in bryophytes: Purification and characterization of two L-asparaginase isoforms from Sphagnum fallax

Nitrogen represents a critical nutrient in raised bogs. In Sphagna , dominating this habitat, the prevalent storage amino acid asparagine is catabolized predominantly by the enzyme L-asparaginase (EC 3.5.1.1). L-asparaginase activity has been detected in each of 10 Sphagnum species investigated. In Sphagnum fallax Klinggr. (Klinggr. clone 1) cultivated under axenie conditions in continuous feed bioreactors, the enzyme displayed a light dependent increase in activity. We separated two isoforms, designated L-asparaginase 1 and 2, characterized by their different elution patterns from an anion-exchange column. In stem segments only L-asparaginase 2 could be detected, whereas in capitulae L-asparaginase 1 represented the dominating isoform. Purified chloroplasts displayed no L-asparaginase activity. Almost the entire activity was located in the cytosohc fraction. L-asparaginase 1 and 2 have been purified 82-fold and 188-fold, respectively, by ion-exchange, size-exclusion and hydrophobic interaction chrornatography. Identical pH optima (8.2) and molecular weights (126 000) were determined. The K_m values for asparagine (7.4 m M for L-asparaginase 1 and 6.2 m M for L-asparaginase 2) were in the range of those described for higher plants. On the other hand Sphagnum L-asparaginase is comprised of four subunits as are the L-asparaginases of microorganisms. So, the characteristics of the bryophyte enzyme appear to be intermediate between those from higher plants and those from microorganisms.     

A fluorometric assay for L-asparaginase activity and monitoring of L-asparaginase therapy

The antineoplastic enzyme L-asparaginase is commonly used for the induction of remission in acute lymphoblastic leukemia (ALL). There is no simple method available for measuring the activity of this highly toxic drug. We incubated L-asparaginase from Erwinia chrysanthemi with L-aspartic acid beta-(7-amido-4-methylcoumarin) and measured the release of 7-amino-4-methylcoumarin fluorometrically for 30-300 min. The rate of the hydrolysis of the substrate was linear over a 50-fold range of the concentration of the enzyme. With increasing substrate concentration, the enzyme showed a saturable kinetic pattern with V(max) of 0.547 (SD 0.059) microM/min/mg of enzyme (n = 3) and Km of 0.302 (SD 0.095) mM (n = 3). This assay enables rapid analysis of L-asparaginase activity in biological samples and it can be used, for example, for monitoring of L-asparaginase activity in serum of ALL patients during their L-asparaginase therapy.     

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.     

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.     

CHEMICAL MODIFICATION OF L-ASPARAGINASE FROM Cladosporium sp. FOR IMPROVED ACTIVITY AND THERMAL STABILITY

L-Asparaginase (ASNase), an antileukemia enzyme, is facing problems with antigenicity in the blood. Modification of L-asparaginase from Cladosporium sp. was tried to obtain improved stability and improved functionality. In our experiment, modification of the enzyme was tried with bovine serum albumin, ovalbumin by crosslinking using glutaraldehyde, N-bromosuccinimide, and mono-methoxy polyethylene glycol. Modified enzymes were studied for activity, temperature stability, rate constants (k_d), and protection to proteolytic digestion. Modification with ovalbumin resulted in improved enzyme activity that was 10-fold higher compared to native enzyme, while modification with bovine serum albumin through glutaraldehyde cross-linking resulted in high stability of L-asparaginase that was 8.5- and 7.62-fold more compared to native enzyme at 28°C and 37°C by the end of 24 hr. These effects were dependent on the quantity of conjugate formed. Modification also markedly prolonged L-asparaginase half-life and serum stability. N-Bromosuccinimide-modified ASNase presented greater stability with prolonged in vitro half-life of 144 hr to proteolytic digestion relative to unmodified enzyme (93 h). The present work could be seen as producing a modified L-asparaginase with improved activity and stability and can be a potential source for developing therapeutic agents for cancer treatment.     

Protein analysis with tetra-substituted sulfonated cobalt phthalocyanine by the technique of Rayleigh light scattering

This is the first report of cobalt-tetrasulfonatophthalocyanine (CoTSPc) as a probe of Rayleigh light scattering (RLS) to determine proteins at nanogram levels. A highly sensitive method has been developed for the determination of proteins by the light scattering technique on a common spectrofluorimeter, based on the fact that the weak RLS of CoTSPc can be greatly enhanced in the presence of proteins. Under optimum conditions, the linear ranges of the calibration curves were 0.10-34.3 microg x mL(-1) for both human serum albumin and bovine serum albumin, with detection limits of 15.5 and 13.9 ng x mL(-1), respectively. Moreover, there is almost no interference of any amino acids and metal ions. The method has been applied to the direct determination of total proteins in human serum samples, and the results were satisfactory with clinical data provided.     

Pancreatic endocrine function in leukemic children treated with L-asparaginase

The effect of arginine infusion on blood glucose and plasma levels of insulin, C-peptide and glucagon has been studied in leukemic children before and after treatment with L-asparaginase (10,000 U/m2/day for 10 days). Therapy induced a significant reduction in basal and peak blood glucose, insulin and C-peptide levels, while glucagon was unmodified. The conserved C-peptide-insulin molar ratio suggests the interference of L-asparaginase with proinsulin synthesis. In conclusion our results prove a decreased insulin reserve with a preserved, although reduced, beta-cell function.     

Amino acid content of L5178Y and L5178Y/L-ASE cells after L-asparaginase treatment

The amino acid contents of tumor cells that are either sensitive or resistant to treatment with L-asparaginase were measured. These amino acid concentrations were measured as a function of incubation time with L-asparaginase or as a function of the L-asparaginase dose. The cell types compared were the mouse leukemia lines L5178Y (sensitive to L-asparaginase treatment) and L5178Y/L-ASE (resistant to L-asparaginase treatment). Upon L-asparaginase treatment both cell lines lost most of their cellular asparagine but, whereas the resistant cells exhibited the ability to rebound to about 50% of initial values, the sensitive cells did not. While previous work had suggested that asparagine-dependent glycine synthesis was essential for sensitive cells (but not in resistant cells), we found no difference in the glycine content of either of the two cell lines as a function of either time or dose that would support this hypothesis. Major differences between the two cell lines were seen in the content of the essential amino acids before treatment with L-asparaginase. After incubation without L-asparaginase the contents of the two cell lines became similar. These results are discussed in terms of possible mechanisms of L-asparaginase sensitivity and resistance.     

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.     

The primary structure of L-asparaginase from Escherichia coli

The carboxymethylated L-asparaginase from Escherichia coli A-1--3 was fragmented with cyanogen bromide and the resulting peptides were isolated by using gel filtration on Sephadex G-50 and column chromatography on DE-52. The amino acid sequences of the 7 cyanogen bromide peptides thus obtained were established completely or partially by further fragmentation with trypsin, chymotrypsin and pepsin, and the Dansyl Edman method. Based on the above results and the complete sequences of the tryptic peptides from the carboxymethylated L-asparaginase reported in the previous paper, the whole sequence of the enzyme was established. The reported sequence consists of 321 amino acid residues and its calculated molecular weight is 34 080.     

L-asparaginase-induced apoptosis in ALL cells involves IP3 receptor signaling

L-asparaginase treatment is used in the clinic to treat acute lymphoblastic leukemia (ALL) patients. Lee et al. (2019, Blood 133:2222-2232) demonstrated that L-asparaginase induces apoptosis by activating inositol 1,4,5-trisphosphate (IP₃)-induced Ca²⁺ signaling in a Huntingin-associated protein 1 (HAP1)-dependent manner. Moreover, HAP1 levels inversely correlate with the sensitivity of the ALL cells to L-asparaginase. HAP1 can therefore be used as biomarker for evaluating L-asparaginase resistance.     

Monitoring lysin motif-ligand interactions via tryptophan analog fluorescence spectroscopy

The lysin motif (LysM) is a peptidoglycan binding protein domain found in a wide range of prokaryotes and eukaryotes. Various techniques have been used to study the LysM-ligand interaction, but a sensitive spectroscopic method to directly monitor this interaction has not been reported. Here a tryptophan analog fluorescence spectroscopy approach is presented to monitor the LysM-ligand interaction using the LysM of the N-acetylglucosaminidase enzyme of Lactococcus lactis. A three-dimensional model of this LysM protein was built based on available structural information of a homolog. This model allowed choosing the amino acid positions to be labeled with a Trp analog. Four functional single-Trp LysM mutants and one double-Trp LysM mutant were constructed and biosynthetically labeled with 7-azatryptophan or 5-hydroxytryptophan. These Trp analogs feature red-shifted absorption spectra, enabling the monitoring of the LysM-ligand interaction in media with a Trp background. The emission intensities of four of the five LysM constructs were found to change markedly on exposure to either L. lactis bacterium-like particles or peptidoglycan as ligands. The method reported here is suitable to monitor LysM-ligand interactions at (sub)micromolar LysM concentrations and can be used for the detection of low levels of peptidoglycan or microbes in solutions.     

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.     

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.     

Direct determination of tryptophan using high-performance anion-exchange chromatography with integrated pulsed amperometric detection

Here we present a new method to rapidly quantify tryptophan (Trp) in proteins, animal feed (Mehaden fishmeal), cell cultures, and fermentation broths. Trp is separated from common amino acids by anion-exchange chromatography in 12min and directly detected by integrated pulsed amperometry. The estimated lower detection limit for this method is 1pmol. Alkaline (4M NaOH) hydrolysates can be directly injected, and therefore we used this method to determine the optimum alkaline hydrolysis conditions for the release of Trp from a model protein, bovine serum albumin (BSA). This method accurately determined the Trp content of BSA and fishmeal. High levels of glucose (2%, w/w) do not interfere with the chromatography or decrease recovery of Trp. We used this method to monitor free Trp during an Escherichia coli fermentation.     

Optimization of culture media for L-asparaginase production by newly isolated bacteria,Bacillus sp. GH5

The enzyme L-asparaginase has been extensively studied by many researchers mainly because of its considerable therapeutic properties. Producing a convenient quantity of L-asparaginase can be conducted either by discovering new microbial sources with higher enzyme production or by manipulating the medium components for known microbial sources. The present paper discusses the studies carried out in order to enhance the production of L-asparaginase by newly isolated bacteria, Bacillus sp. GH5. Based on the results obtained from media optimization studies, a modified media was developed for optimal L-asparaginase production. Concisely, screening of the nutrients using a proper statistical design showed that tapioca starch, gelatin, ammonium oxalate, CaCO₃, and L-asparagine were respectively the most important sources for carbon, organic nitrogen, inorganic nitrogen, mineral salt, and amino acids. The composition of the optimized medium was the following (per 1 L): 5.0 g L-asparagine; 0.5 g MgSO₄ · 7H₂O; 6.0 g NaHPO₄ · 2H₂O; 3.0 g (NH₄)₂C₂O₄; 0.5 g CaCO₃; 0.014 g CaCl₂ · 2H₂O; 2.0% w/v tapioca starch; 5.0 g gelatin; and 15.0 g agar.     

Amino acids in the plasma of fasting Japanese monkeys (Macaca fuscata fuscata andM. f. yakui)

The plasma levels of 26 amino acids and related compounds were determined in five male and five female adult members of each of the two subspecies of Japanese monkeys,Macaca fuscata fuscata andM. f. yakui. Activities of L-asparaginase and histaminase in plasma were also measured.Numerous differences in amino acid levels between the sexes in the subspeciesfuscata were noted, with the female consistently exhibiting lower values. Few differences were observed between the sexes of the subspeciesyakui or between the two subspecies of Japanese monkeys. These monkeys were similar to other previously studied nonhuman primates in exhibiting measurable levels of 3-methylhistidine in plasma. There were numerous quantitative differences among Japanese monkeys and stump-tailed macaques, rhesus monkeys, chimpanzees, and man, with the Japanese monkeys usually exhibiting higher plasma levels.L-asparaginase activity was not detectable in these Japanese monkeys. Histaminase activity was similar to that previously measured in pig-tailed macaques and chimpanzees, lower than that in rhesus monkeys and stump-tailed macaques, and higher than that in man.     

pH dependence of the kinetic parameters of L-asparaginase.

The concentration dependence of the rate of hydrolysis of L-asparagine by Escherichia coli L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) has been measured over the range pH 4.5 to pH 9.1 by a direct spectrophotometric assay at 220 nm and by a coupled assay utilizing glutamate dehydrogenase to detect the ammonia produced. The velocity of the hydrolysis reaction at saturating levels of substrate is independent of pH over this interval. The plot of V/km over the same interval is bell-shaped, being dependent on pKa values of 6.58 and 8.69. The higher pKa is attributed to the amino group of asparagine. The lower pKa is associated with the enzyme active site and is probably due to an imidazole group.