Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.1.1 (asparaginase)
 2,695 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The antileukemic activity of L-asparaginase (ASNase), an important component of therapy for acute lymphoblastic leukemia, is thought to result from depletion of serum L-asparagine (Asn). In studies of the pharmacological effects of ASNase, investigators have reported prolonged reduction in the serum concentration of Asn after the administration of ASNase. Such measurements may not be valid because ASNase present in the blood sample may hydrolyze Asn before its determination. We examined recovery of [U-14C]Asn from blood samples with and without various concentrations of added ASNase. In the presence of greater than or equal to 0.01 IU/ml of ASNase, the amount of [U-14C]Asn recovered was less than 15% of that without ASNase. Utilizing this assay, we studied the effect of 2 known inhibitors of ASNase in an attempt to improve Asn recovery. In the presence of aspartic beta semialdehyde (ASA), or 5-diazo-4-oxo-L-norvaline (DONV), and up to 1.0 IU/ml ASNase, Asn levels remained at greater than 90% of control. ASA prevented the hydrolysis of exogenous Asn in blood samples drawn from patients after ASNase injection. We also developed a method to determine Asn in serum utilizing high pressure liquid chromatography. Using this method, we found that the Asn level was greater than 90% of a normal level in the presence of 40 mM DONV and 1.0 IU/ml ASNase. Examination of serum from 4 patients treated with ASNase showed that Asn is detectable 7-19 days sooner when DONV is present in the blood collection system than in its absence. We conclude that: (a) as little as 0.01 IU/ml ASNase can hydrolyze Asn added to blood; (b) continued hydrolysis of Asn by ASNase ex vivo can result in falsely low serum Asn measurements; (c) ASA or DONV present in the collection tubes obviates the problem of continued ASNase activity; and (d) the degree and duration of Asn depletion after ASNase therapy is much less than previously believed. Thus, for accurate measurements of the duration and degree of Asn depletion by ASNase, an ASNase inhibitor such as ASA or DONV should be present in the blood collection system.
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PMID:Measurement of serum L-asparagine in the presence of L-asparaginase requires the presence of an L-asparaginase inhibitor. 174 29

Studies on L-asparaginase synthesis in V. proteus showed increased synthesis in cultures grown under conditions of moderate aeration (P less than 0.005) after oxygen had been used up from the medium. Addition of sodium lactate to the medium at a concentration of 80 mu mole/ml, stimulated L-asparaginase synthesis (2.2 times over control) in moderately-aerated cultures (P less than 0.001). The substrate L-asparagine induced enzyme synthesis when growth conditions were made anaerobic or lactate was incorporated into the medium (3.8 times increased enzyme synthesis over control).
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PMID:Induction of L-asparaginase synthesis in Vibrio proteus. 177 15

Two patients with hematological malignancies were successfully treated with monomethoxypolyethylene glycol-conjugated Escherichia coli L-asparaginase (PEG2-ASP), which reportedly lacks both antigenicity and immunogenicity but retains catalytic activity as well as slow clearance in an experimental animal model. A 20-year-old male patient with leukemic lymphoma was refractory to conventional chemotherapy but responsive to L-asparaginase (L-ASP) followed, however, by severe adverse effects. On relapse, an intravenous infusion of 100-200 IU/day dose of PEG2-ASP alone led to a complete remission 2 months later without hypersensitivity or other significant adverse reactions. Surprisingly, he remained in a complete remission for over one year with a regular weekly infusion of PEG2-ASP, combined with a weekly small dose of Ara-C. During this period, blood asparagine was not detectable. The other patient, a 64-year-old woman with chronic myelogenous leukemia in blast crisis achieved, within 6 weeks, a complete remission with twice-weekly infusions of PEG2-ASP. Thus, PEG2-ASP is a highly effective antitumor agent overcoming the limitations in therapeutic use of L-ASP.
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PMID:High efficacy of monomethoxypolyethylene glycol-conjugated L-asparaginase (PEG2-ASP) in two patients with hematological malignancies. 186 35

L-Asparaginase of T. pyriformis is a membrane-bound enzyme with an active site situated on the outside surface of the membrane. When radioactive L-asparagine was incubated with T. pyriformis cells in the L-asparaginase assay medium, the hydrolysis was 240 higher than the uptake of this amino acid. In a similar experiment performed in salt medium (Wagner's solution), the hydrolysis was linearly increased and reached after one hour of incubation a value of 60 nmol/10(6) cells, while the uptake after 20 min of incubation reached a plateau with a value of 15 nmol/10(6) cells. The uptake of L-leucine under these conditions was 44 nmol/10(6) cells/hr, while no measurable transport of aspartic acid was observed. That L-aspartic acid is not migrated into T. pyriformis cells is in agreement with the finding that no efflux of this amino acid takes place as well. The uptake of L-asparagine is pH and K+ dependent, whereas Na+ ions strongly inhibit this uptake. The Km and Vmax values of L-asparagine uptake is 1.43 mM and 0.7 nmol/min, respectively. The half life of L-asparagine "protein transport system" was 40 min, a value which is very close to the half life of the membrane-bound L-asparaginase of this microorganism. Ouabain and vanadate inhibit the uptake of L-asparagine by more than 80%, while ouabain or vanadate inhibit in vivo 5% or 95% the activity of L-asparaginase, respectively. This indicates the lack of interrelationship between the L-asparagine "protein transport system" and the L-asparaginase protein molecule.
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PMID:Transport of L-asparagine in Tetrahymena pyriformis ecto-L-asparaginase is not related to L-asparagine-protein transport system. 193 Feb 47

Since fumarate and nitrate are not usually available in the oral ecosystem, it was investigated whether aspartate and asparagine could be used as alternative electron acceptors by Wolinella recta, which is strictly dependent on a respiratory metabolism with formate or H2 as electron donors. Both aspartate and asparagine were indeed shown to support growth of W. recta with formate as electron donor. Fermentative growth with aspartate alone was not possible. Succinate was the major end-product and was formed in equimolar quantities with respect to the amount of formate consumed. The consumption of aspartate and asparagine, on a molar basis, was 10-30% higher than that of formate. Cell-free extracts were prepared from cells grown with formate + fumarate, formate + aspartate, formate + asparagine, and formate + fumarate + aspartate. All these extracts contained high activities of asparaginase, aspartate ammonia-lyase and fumarate-reductase, but no significant activity of aspartate aminotransferase was detected, indicating that fumarate was synthesized directly from aspartate and subsequently reduced to succinate. Based on these results it seems likely that aspartate and asparagine can serve as natural electron acceptors for W. recta in periodontal lesions in which proteolytic bacteria abound.
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PMID:Aspartate and asparagine as electron acceptors for Wolinella recta. 194 91

L-Asparaginase has been encapsulated in Swiss mouse or human erythrocytes by hypotonic haemolysis followed by isotonic resealing and reannealing. The details of incorporation and properties of carrier erythrocytes are presented. When L-asparaginase loaded into 51Cr-labelled erythrocytes, was infused intravenously, the same half-life was found for asparaginase and 51Cr. In addition, L-asparaginase loaded into erythrocytes was much more effective in eliminating plasma asparagine compared with the same dose of free L-asparaginase injected in solution, during a sustained period (14 days).
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PMID:Erythrocytes as carriers for L-asparaginase. Methodological and mouse in-vivo studies. 198 Feb 86

The apparent active site of human leukocyte glycoasparaginase (N4-(beta-acetylglucosaminyl)-L-asparaginase EC 3.5.1.26) has been studied by labeling with an asparagine analogue, 5-diazo-4-oxo-L-norvaline. Glycoasparaginase was purified 4,600-fold from human leukocytes with an overall recovery of 12%. The purified enzyme has a Km of 110 microM, a Vmax of 34 mumol x l-1 x min-1, and a specific activity of 2.2 units/mg protein with N4-(beta-N-acetylglucosaminyl)-L-asparagine as substrate. The carbohydrate content of the enzyme is 15%, and it exhibits a broad pH maximum between 7 and 9. The 88-kDa native enzyme is composed of 19-kDa light (L) chains and 25-kDa heavy (H) chains and it has a heterotetrameric structure of L2H2-type. The glycoasparaginase activity decreases rapidly and irreversibly in the presence of 5-diazo-4-oxo-L-norvaline. At any one concentration of the compound, the inactivation of the enzyme is pseudo-first-order with time. The inhibitory constant, K1, is 80 microM and the second-order rate constant 1.25 x 10(3) M-1 min-1 at pH 7.5. The enzyme activity is competitively protected against this inactivation by its natural substrate, aspartylglucosamine, indicating that this inhibitor binds to the active site or very close to it. The covalent incorporation of [5-14C]diazo-4-oxo-L-norvaline paralleled the loss of the enzymatic activity and one inhibitor binding site was localized to each L-subunit of the heterotetrameric enzyme. Four peptides with the radioactive label were generated, purified by high performance liquid chromatography, and sequenced by Edman degradation. The sequences were overlapping and all contained the amino-terminal tripeptide of the L-chain. By mass spectrometry, the reacting group of 5-diazo-4-oxo-L-norvaline was characterized as 4-oxo-L-norvaline that was bound through an alpha-ketone ether linkage to the hydroxyl group of the amino-terminal amino acid threonine.
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PMID:Glycosaparaginase from human leukocytes. Inactivation and covalent modification with diazo-oxonorvaline. 200 22

Three ACNU-resistant clones (R1, R3, and R12) were isolated from 9L rat glioma cells under selection pressure of ACNU in vitro. The authors have investigated the mechanisms of resistance and characteristics of these clones at the cellular level by studying cross-resistance patterns to chemical and physical agents. Although these resistant sublines showed complete cross-resistance to methyl-chloroethylnitrosourea (MCNU), no cross-resistance was observed for other alkylating agents, while each of the resistant sublines showed partial cross-resistance to structurally dissimilar toxic agents (vinblastine, Adriamycin, and VP-16). No difference in ACNU uptake was observed between 9L and R3 cells, and resistance patterns among alkylating agents suggested that the mechanism of ACNU resistance was specific to bifunctional nitrosoureas. Based on a transport study, this multidrug resistance could be explained by reduced intracellular uptake of these drugs, but there seemed little possibility that membrane P-glycoprotein, which usually is observed in typical multidrug-resistant cells, was expressed in these ACNU-resistant cells because enhanced drug efflux was not found in ACNU-resistant sublines. Significant collateral sensitivity to L-asparaginase indicated that ACNU might disturb the asparagine synthetic pathways by its mutagenic action. The increased level of total glutathione in the resistant sublines may be one mechanism of radiation or ACNU resistance.
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PMID:Cross-resistance patterns in ACNU-resistant glioma sublines in culture. 207 67

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.
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PMID:Purification and properties of an L-asparaginase from Cylindrocarpon obtusisporum MB-10. 208 Sep 24

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 [gamma-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.
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PMID:L-asparaginase of Tetrahymena pyriformis is associated with a kinase activity. 211 26


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