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)

Obvious protection of the catalytic activity of Esch. coli L-asparaginase by alpha 2-macroglobulin (alpha 2M) was observed under conditions otherwise propitious to the dissociation of the tetrameric molecule into inactive subunits, i.e. very diluted enzyme solutions or the presence of either SDS or urea. The degree of protection depended on enzyme and alpha 2M concentrations respectively, and on the preincubation time of the alpha 2M-enzyme mixture prior to substrate addition. The formation of a catalytically active complex between alpha 2M and L-asparaginase was confirmed by gel filtration on a Sephadex-G column and by polyacrylamide gel electrophoresis. The fact that the migration distance of the active complex corresponded to the migration of alpha 2M and the absence in that case of a migration band corresponding to the intact molecule suggest that complexing of the enzyme with alpha 2M prevented its dissociation into subunits and thus its inactivation. Addition of alpha 2M to the already dissociated enzyme molecule did not restore its catalytic activity. Alpha2-macroglobulin was shown to have an inhibiting effect on the proteolytic activity of almost all proteases and no effect on their esterolytic activity. Furthermore, it prevents the inhibition of esterolytic activity by some natural compounds. The effect of alpha 2M on other types of catalytic activity has not been investigated enough to afford a generalization of the possible role of this macroglobulin in the control of enzyme activity in the body. This paper reports the results of an in vitro study of the effect of alpha 2M on the catalytic activity of an important amidase, i.e. L-asparaginase (L-asparagine amidohydrolase 3.5.1.1), which in recent years has been used in the treatment of acute lymphocytic leukemia in children.
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PMID:Interaction of alpha 2-macroglobulin with L-asparaginase. 9 Mar 34

The denaturation and reconstitution of Erwinia carotovora and Escherichia coli L-asparaginases has been followed by optical rotatory dispersion, circular dichroism and analytical ultracentrifugation. Denaturation in urea results in dissociation of the native enzyme (mol. wt. 140 000 approx.) to produce unfolded subunits (mol. wt. 35 000 approx.); the Erwinia L-asparaginase subunits can be refolded by dilution or dialysis in alkaline conditions, pH 10.5, without aggregation to the active tetramer, to give a rather unstable solution of a monomer possibly in equilibrium with dimer. These alkaline-reconstituted subunits undergo a conformational change to a more ordered state in the presence of sodium dodecylsulphate, similar to those produced by the action of sodium dodecylsulphate on the native enzyme. If the denatured subunits are reconstituted in the pH range 5.0-7.5, the enzymically active tetramer is reformed in up to 80% yield, depending upon the conditions of temperature and concentration. Kinetic data for these various transitions suggest that dissociation is a rate-limiting step while conformational changes of the polypeptide chains are relatively much more rapid. The possible significance of these different rates of change to therapeutic considerations is discussed.
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PMID:Comparative study on conformational stability and subunit interactions of two bacterial asparaginases. 23 38

Effect of urea on the activity of serum L-asparaginase in outbred guinea pigs (Hartley) was examined for comparison between heat-resistant and heat-sensitive types. The heat-resistant serum L-asparaginase was much more stable to urea-treatment than the heat-sensitive serum enzyme, and the urea-inactivation of the serum enzyme was protected by Na+ or K+. Liver L-asparaginase of the guinea pig, in which the serum enzyme was resistant to heat, was also resistant to heat, and vice versa, although liver enzyme was much more sensitive to heat than serum enzyme. The heat inactivation of liver enzyme was also protected by Na+. Similar results were also demonstrated on purified serum L-asparaginase, although the amino acid compositions between the two purified enzyme preparations were slightly different. When the animal having heat-resistant serum L-asparaginase were crossed with each other, serum enzyme of the resultant progenies was also resistant to heat, and vice versa. The serum enzyme of two inbred strains (JY-1, Hartley/F) was thermostable and the enzyme of the other (Strain 2, Strain 13) was thermolabile.
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PMID:Effect of strain differences on heat-susceptibility of L-asparaginase in the guinea pig. 71 34

L-Asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) from Escherichia coli B was modified by treatment with 2,4,6-trinitrobenzene-1-sulfonic acid at pH 7.5. The introduction of 13 trinitrophenyl groups into one mol of the tetrameric enzyme (TNP 13-asparaginase) results in a loss of 67% of the catalytic activity while the presence of 20 groups (TNP 20-asparaginase) reduces the enzymatic activity by 88%. The modified proteins are homogeneous as judged by disc gel electrophoresis and by the monodisperse boundary in the analytical ultracentrifuge having a sedimentation coefficient of 7.2 S. The rate of dissociation of the TNP 13-asparaginase is twice as fast and TNP 20-asparaginase three times as fast as that of unmodified asparaginase in 4 M urea. Trinitrophenylated subunits in 8 M urea can reassociate into the tetramer after removal of urea by dialysis or by dilution. hybridization of unmodified and TNP subunits indicates that that trinitrophenyl derivatives qualify as suitable variants for studying subunit interactions in oligomeric proteins.
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PMID:The effect of trinitrophenylation on subunit interactions in L-asparaginase. 78 47

Immobilized asparaginase was prepared by embedding asparaginase (which is effective for remission in children with leukemia) into fibrin polymer formed by fibrinogen-fibrin conversion in the presence of thrombin. The immobilized asparaginase film did not dissolve in 6 mol/1 urea, suggesting that blood coagulation factor XIII participates in the cross-linking between fibrins and between fibrin and asparaginase.
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PMID:Immobilized L-asparginase embedded in fibrin polymer. 109 44

Purified L-asparaginase from Serratia marcescens had an apparent-weight average molecular weight of 171,000 to 180,000 as determined by electrophoresis on polyacrylamide gels and by sedimentation equilibrium at low speed in an analytical ultracentrifuge. A subunit molecular weight of 31,500 +/- 1,500 was estimated for the enzyme after treatment with sodium dodecyl sulfate and urea and electrophoresis on polyacrylamide gels; a similar value was obtained by high-speed sedimentation equilibrium in the presence of guanidine hydrochloride. Our data indicate that the Serratia enzyme could have five or six subunits of 32,000 daltons, compared to four subunits of 32,000 daltons in the Escherichia coli enzyme. The Serratia L-asparaginase also appears to be a larger molecule than the enzyme from Erwinia carotovora, Proteus vulgaris, Acinetobacter glutaminasificans, and Alcaligenes eutrophus. The Serratia enzyme, like that from E. caratovora, was more resistant than the E. coli enzyme to dissociation by sodium dodecyl sulfate. This resistance could be due to the finding that the Serratia enzyme had a relatively high hydrophobicity, similar to the enzyme from E. caratovora, when compared with the hydrophobicity of the E. coli enzyme. The isoelectric point of the Serratia enzyme was approximately 5.2. The influence of certain physical characteristics of the enzyme on the biological properties is discussed.
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PMID:Physical properties of L-asparaginase from Serratia marcescens. 110 30

Site-specific mutagenesis has been used to probe amino acid residues proposed to be critical in catalysis by Escherichia coli asparaginase II. Thr12 is conserved in all known asparaginases. The catalytic constant of a T12A mutant towards L-aspartic acid beta-hydroxamate was reduced to 0.04% of wild type activity, while its Km and stability against urea denaturation were unchanged. The mutant enzyme T12S exhibited almost normal activity but altered substrate specificity. Replacement of Thr119 with Ala led to a 90% decrease of activity without markedly affecting substrate binding. The mutant enzyme S122A showed normal catalytic function but impaired stability in urea solutions. These data indicate that the hydroxyl group of Thr12 is directly involved in catalysis, probably by favorably interacting with a transition state or intermediate. By contrast, Thr119 and Ser122, both putative target sites of the inactivator DONV, are functionally less important.
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PMID:Probing the role of threonine and serine residues of E. coli asparaginase II by site-specific mutagenesis. 128 59

Site-specific mutagenesis was used to replace the three histidine residues of Escherichia coli asparaginase II (EcA2) with other amino acids. The following enzyme variants were studied: [H87A]EcA2, [H87L]EcA2, [H87K]EcA2, [H183L]EcA2 and [H197L]EcA2. None of the mutations substantially affected the Km for L-aspartic acid beta-hydroxamate or impaired aspartate binding. The relative activities towards L-Asn, L-Gln, and l-aspartic acid beta-hydroxamate were reduced to the same extent, with residual activities exceeding 10% of the wild-type values. These data do not support a number of previous reports suggesting that histidine residues are essential for catalysis. Spectroscopic characterization of the modified enzymes allowed the unequivocal assignment of the histidine resonances in 1H-NMR spectra of asparaginase II. A histidine signal previously shown to disappear upon aspartate binding is due to His183, not to the highly conserved His87. The fact that [H183L]EcA2 has normal activity but greatly reduced stability in the presence of urea suggests that His183 is important for the stabilization of the native asparaginase tetramer. 1H-NMR and fluorescence spectroscopy indicate that His87 is located in the interior of the protein, possibly adjacent to the active site.
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PMID:Site-specific mutagenesis of Escherichia coli asparaginase II. None of the three histidine residues is required for catalysis. 152 38

Photo-switchable ion and enzyme sensors were fabricated by the use of glassy carbon electrode coated with nonactindoped or enzyme modified poly(vinyl chloride) (PVC) membranes. The ion sensor with nonactin-doped PVC membrane, which contained spirobenzopyran as the photosensitive dye, exhibited a potentiometric photoresponse to NH4+ ion in the solution. The dynamic range of the NH4+ ion sensor was 10(-7)--10(-3) M. Urea, adenosine, and asparagine sensors were prepared by coating the surface of the NH4+-ion sensor with urease, adenosine deaminase, and asparaginase membranes, respectively. These enzyme sensors could be used for determining the substrates at the micro mole level. The performance characteristics of these sensors were compared with those previously prepared membrane electrode sensors.
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PMID:Photo-switchable ion and enzyme sensors. Photoinduced potentiometric response of glassy carbon electrode coated with polymer or polymer/enzyme dual membrane. 263 77

L-Asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) from Erwinia carotovora undergoes extensive dissociation from active tetramer to inactive monomers when freeze-dried. The monomeric state is stabilized by reconstitution of the freeze-dried enzyme with buffers of high pH and high ionic strength. Some compounds, particularly sugars and sugar derivatives, prevent dissociation on freeze-drying, whereas others, such as urea and chaotropic ions, increase dissociation. The effects of additives are not related to water retention. The dissociation is completely reversible on reconstitution at neutral pH, but the alkali-stabilized monomer only partially reassociates when the pH is brought back to neutrality.
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PMID:The effect of freeze-drying on the quaternary structure of L-asparaginase from Erwinia carotovora. 665 94


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