Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

1. L-asparaginase from M. phlei was purified about 170-fold with an 11% yield. The purification procedure consisted of: fractionation with ammonium sulphate; adsorption of contaminating proteins on calcium phosphate gel; chromatography on Sephadex G-150 and DEAE-cellulose. The specific activity of the final preparation was 32.6 i.u./mg protein. 2. Molecular weight of the enzyme as determined by Sephadex G-100 filtration amounted to 126 000. Optimum pH was 8.8-9.2. The enzyme did not hydrolyse L-glutamine over the pH range 4-9, and was inhibited by D-asparagine. The apparent Michaelis constant for L-asparagine was 0.7 mM; energy of activation, 9800 cal/mole. 3. On polyacrylamide-gel electrophoresis the final preparation revealed two protein bands, one of which was coincident with the enzyme activity.
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PMID:Purification and properties of L-asparaginase from Mycobacterium phlei. 0 91

The fluorescence lifetimes and relative quantum yields of several derivatives of tyrosine are reported. The quenching of the fluorescence of these compounds by phosphate, caesium and iodide ions has been investigated; the encounter rate constants, calculated from the quenching parameters and lifetimes, show a clear dependence on the charges borne by the quenchers and fluorophores. The ratio of the Stern-Volmer constants of iodide and caesium, ions of similar size, defines an electrostatic parameter sensitive to the charge of the fluorophore which can be evaluated without knowledge of the fluorescent lifetimes. The mean of the encounter rate constants for caesium and iodide ions defines a rate constant which is largely charge-independent and is used to establish a steric parameter. The two parameters are used to investigate the tyrosine environment in bovine ribonuclease A (EC 3.1.4.23) and Erwinia carotovora L-asparaginase (EC 3.5.1.1). The quantum yield of L-asparaginase (0.12) is very high for a class A protein and may be associated with the absence of disulphide bridges. There was no evidence for more than one type of tyrosine residue from the quenching experiments with either enzyme, an observation which is attributed to efficient energy transfer amongst tyrosine residues. At pH values close to the isoelectric points of the enzymes the electrostatic parameter suggests that the environment of the quenchable tyrosines in L-asparaginase is somewhat more positive than in ribonuclease. In 1% sodium dodecyl sulphate the tyrosine environment of L-asparaginase becomes markedly negative as expected. The steric parameter indicates a lower accessibility of the tyrosine residues in L-asparaginase than in ribonuclease; an illustrative calculation is provided linking the steric parameter with the number of exposed tyrosine residues by taking into account the greater collision frequency of the larger protein molecules and the encounter distance for quenching determined from charge effects on the quenching of the model compounds. The calculation suggests that three tyrosyl residues are accessible in ribonuclease, in good agreement with other studies, but in L-asparaginase the number increases from 0.4 at pH 5.73 to 0.8 at pH 9.16 suggesting a loosening of the enzyme structure at high pH.
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PMID:An investigation of the electronic and steric environments of tyrosyl residues in ribonuclease A and Erwinia carotovora L-asparaginase through fluorescence quenching by caesium, iodide and phosphate ions. 98 70

Over 20 cytoenzymochemical tests were carried out in 152 patients with different types of acute leukemia to estimate the effects of some antiblastic drugs such as L-asparaginase, Purinethol, Methotrexate, Endoxan, Vinchristine, Cytosine Arabinoside a.o. The patients selected for the study were carefully examined before treatment at different moments during and/or at the end of the treatment. The effects of these drugs on the blast cells were mild when the cellular populations had a low rate of nucleic acid synthesis, high glycogenic score and high amounts of lipids or an important oxidative enzymatic activity. The enzymatic prediction tests: the acid phosphate deviation test and the succinic dehydrogenase inhibition test including the variant suggested by some of the authors - the latic dehydrogenase inhibition test - gave satisfactory results only in certain cases of acute leukemia.
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PMID:Cytoenzymochemical effects of some antiblastic drugs and prediction of response to chemotherapy in acute leukemias. 105 33

L-Asparaginase of Tetrahymena pyriformis is a lipoprotein with relative M(r) approximately 200 kDa and one subunit size of 39 kDa. This enzyme also exhibits protein kinase activity and it is autophosphorylated in tyrosine residues. Phosphorylation-dephosphorylation of L-asparaginase resulted in complete loss or activation by more than 10-fold of its catalytic activity. Both native and dephosphorylated forms of L-asparaginase are inactivated by phospholipase C and this inactivation can be reversed by the addition of lipids. Based on these results a working hypothesis is suggested that L-asparaginase of T. pyriformis exists in four interconvertible forms: Form A, phosphorylated complexed with lipids, form HA, dephosphorylated (highly active), form I, free of lipids, (inactive) and form B, free of lipids and phosphate.
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PMID:A model for the regulation of the activity of L-asparaginase/ kinase enzyme of Tetrahymena pyriformis. 250 74

A large-scale process was developed to purify L-asparaginase from submerged cultures of Erwinia carotovora. Cells from 880 L of fermentation broth were harvested and washed using a plate and frame type filter press. A cellular acetone powder was prepared from the washed cells by suspending the cells twice in acetone and the residual acetone was removed by washing the acetone powder in the filter press with 10 mM phosphate buffer (pH 7.0). The cellular acetone powder was extracted with 10 mM borate buffer at pH 9.5. The enzyme-rich borate extract was recovered by filtration and clarified by an in-line bag filter. The filtrate was adjusted to pH 7.5 and filtered through a 1-micron bag filter precoated with Celite and then through a 0.22-micron cartridge filter. The cell-free extract, containing 21 x 10(6) IU of enzyme and 448 g of total protein, was applied to an L-asparagine Sepharose 6 Fast Flow affinity column (9 L) using a bag filter loaded with Cell Debris Remover as an in-line prefilter. The affinity gel was prepared by coupling L-Asn at pH 9.0 to epoxy-activated Sepharose 6 Fast Flow beads. A total of 14 x 10(6) IU of enzyme (35 g protein) was eluted at pH 9.0 in 10.5 L. The eluted enzyme was determined to be greater than 90% pure using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The total process time from whole broth to affinity column elution was 68 h and the enzyme yield was 38%. This improved process for the 880 L fermentation broth produced a cell-free extract of high specific activity, shortened the process time, increased the column capacity, and yielded a product with high purity.
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PMID:L-asparaginase from Erwinia carotovora. An improved recovery and purification process using affinity chromatography. 280 97

L-Asparaginase activity reaches maximal values at the stationary phase of growth of Tetrahymena pyriformis and fluctuates upon the growth conditions and the composition of the medium. Most of the L-asparaginase activity (80%) is associated with the endoplasmic reticulum, and the remaining with the pellicles. Detergents either alone or in combination with NaCl up to 0.5 M concentration failed to solubilize L-asparaginase. Solubilization can be accomplished by means of either the chaotropic agents KSCN and NaClO4, or 0.1 M sodium phosphate buffer pH 8.0, following pretreatment of the particulates with 2% w/v Triton X100. L-Asparaginase has been purified to near homogeneity by hydrophobic and gel filtration chromatography. The native enzyme has a relative molecular weight of 230,000. It is a multiple subunit enzyme, with subunit size of 39,000. Its isoelectric point is at pH 6.8. It acts optimally at pH 8.6 with a Km of 2.2 mM. It does not hydrolyse L-glutamine and its reaction is inhibited competitively by D-aspartic acid and D-asparagine as well as by L-asparagine analogues with substituents at the beta position.
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PMID:Purification and properties of a membrane-bound L-asparaginase of Tetrahymena pyriformis. 313 90

A large-scale process was developed to purify gram quantities of a therapeutic enzyme, L-asparaginase, from submerged cultures of Erwinia carotovora. Cells were harvested from 150 L of fermentation broth and washed. A cellular acetone powder was prepared and extracted with pH 9.5 borate buffer. After continuous centrifugation and filtration to remove cell debris, the acetone powder extract was adjusted to pH 7.7 and adsorbed onto a 16-L CM-Sepharose Fast Flow column, with a precolumn packed with Cell Debris Remover. The enzyme was desorbed from the catin-exchange column at pH 9.0 and further purified with an affinity column of L-asparagine Sepharose CL-4B. After dialysis-concentration to remove buffer salt, the enzyme was depyrogenated, formulated, sterile filled, and lyophilized as a single-dose final product. The final-product evaluation included analysis of the content of protein, sodium chloride, glycine, sodium, glucose hydrate, phosphate, and endotoxin, as well as reconstitution, potency, pH, specific activity, uniformity of fill, and sterility. The product was further subjected to visual examination, sodium dodecyl sulfate polyacrylamide gel electrophoresis, native gel electrophoresis, isoelectric focusing, amino acid analysis, N-terminal sequencing, peptide mapping, and immunological comparison.
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PMID:Large-scale recovery and purification of L-asparaginase from Erwinia carotovora. 375 84

Cultural and nutritional requirements for maximum l-asparaginase synthesis were determined. Conventional aerobic and anaerobic fermentations were not satisfactory. The former yielded larger quantities of cells containing minimal amounts of l-asparaginase, whereas the latter supplied only minute amounts of bacteria that contained an abundance of enzyme. However, the combination of these classical methods, i.e., allowing growth to proceed aerobically until the mid to late exponential phase and then forcing the facultative microbial cells toward anaerobic metabolism by static incubation, produced 2.6 international units of enzyme per ml of fermentation broth when glucose was present. Enzyme synthesis was not induced by terminating aeration-agitation in the absence of glucose, nor was it induced in the presence of glucose when aeration was continued. Use of 0.2 m phosphate buffer resulted in a constant pH near the optimum value of 7.5 during l-asparaginase formation. Addition of 0.05% l-asparagine prior to induction was also beneficial, but other amino acids or their catabolites failed to increase biosynthesis of l-asparaginase.
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PMID:Effect of glucose and low oxygen tension on L-asparaginase production by a strain of Escherichia coli B. 492 8

In a series of four experiments, asparaginase and glutaminase activity was measured in liver and kidney tissue of 7- to 19-day-old male broiler chicks. In Experiment 1, chicks were fed purified amino acid diets with 14.8 and 44.6% protein equivalents (PE) with 1, 3, or 5% added sodium bicarbonate. In Experiments 2, 3, and 4 the chicks were fed a 23% protein basal control diet, basal diet containing 5% ammonium chloride, and basal diet containing 5% ammonium chloride with 5 or 10% sodium bicarbonate, asparagine, or glutamine. In Experiments 2 and 4 the chicks were also fed 25, 50, or 75% protein-isolated soy-purified diets. The 44.6% PE diet increased liver and kidney asparaginase activity in chicks as compared to chicks fed a 14.8% PE diet. The addition of sodium bicarbonate to the 44.6% PE amino acid diet decreased the kidney asparaginase activity equivalent to kidney asparaginase activity of chicks fed the 14.8% PE diet. Asparaginase activity increased 4-fold in the kidneys of chicks fed the 23% protein basal diet containing 5% ammonium chloride and the pH of the urine from the chicks was 4.9. Chicks fed basal diets with 5% ammonium chloride plus 10% sodium bicarbonate or asparagine had the same kidney asparaginase activity and urine pH as chicks fed the 23% protein basal control diet. Glutamine added to chick diets containing 5% ammonium chloride did not decrease the kidney asparaginase activity or the urine acidity. Liver asparaginase activity was not increased in acidotic chicks fed diets with 5% ammonium chloride. The asparaginase activity of liver and kidney tissue were both significantly increased in chicks fed 75% protein-isolated soy purified diets and the pH of their urine was 5.6. The increase in liver asparaginase of chicks fed 75% protein or 44.5% PE diets was probably due to an endocrine gluconeogenic response producing increased catabolism of the majority of amino acids. The increase in kidney asparaginase of chicks fed 75% protein, 44.5% PE diets, and 23% protein basal diets with 5% ammonium chloride was primarily related to metabolic acidosis. Phosphate-dependent glutaminase (PDG) activity was localized in chick kidney mitochondria and was heat sensitive (55 C for 30 sec). The phosphate-independent glutaminase (PIG) activity was primarily localized in chick kidney mitochondria but was stable to a temperature of 55 C for 30 sec.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Asparagine and glutamine metabolism in chicks. 632 68

Crude extracts of Thermoactinomyces vulgaris 13 M.E.S. were prepared by different procedures and their L-asparaginase activity was compared. The optimum conditions for the enzyme activity in the crude extract was exerted at pH 8.8, using borate or tris-HCl buffer, or at pH 7.4, using phosphate buffer, after a reaction time of 30 minutes at 50-55 degrees C, using 1.35-5.4 mg protein of the crude extract. The enzyme showed an apparent km value of 4.5 x 10(-3), was more thermostable in crude extracts than in whole cells, and was inhibited by HgCl2, KCN, DL-asparagine, L-aspartic acid, and ammonium hydroxide. Enzyme purification by alcohol precipitation and gel filtration was attempted.
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PMID:L-asparaginase activity in cell-free extracts of Thermoactinomyces vulgaris 13 M.E.S. 664 56


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