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1.
A new approach to the regulation of catalytic properties of medically relevant enzymes has been proposed using the novel recombinant preparation of L-asparaginase from Erwinia carotovora (EwA), a promising antitumor agent. New branched co-polymers of different composition based on chitosan modified with polyethylene glycol (PEG) molecules, designated as PEG-chitosan, have been synthesized. PEG-chitosan copolymers were further conjugated with EwA. In order to optimize the catalytic properties of asparaginase two types of conjugates differing in their architecture have been synthesized: (1) crown-type conjugates were synthesized by reductive amination reaction between the reducing end of the PEG-chitosan copolymer and enzyme amino groups; (2) multipoint-conjugates were synthesized using the reaction of multipoint amide bond formation between PEG-chitosan amino groups and carboxyl groups of the enzyme in the presence of the Woodward’s reagent. The structure and composition of these conjugates were determined by IR spectroscopy. The content of the copolymers in the conjugates was controlled by the characteristic absorption band of C-O-C bonds in the PEG structure at the frequency of 1089 cm?1. The study of catalytic characteristics of EwA preparations by conductometry showed that at physiological pH values the enzyme conjugates with PEG-chitosan with optimized structure and the optimal composition demonstrated 5–8-fold higher catalytic efficiency (k cat/K m) than the native enzyme. To certain extent, this can be attributed to favorable shift of pH-optima in result of positively charged amino-groups introduction in the vicinity of the active site. The proposed approach, chito-pegylation, is effective for regulating the catalytic and pharmacokinetic properties of asparaginase, and is promising for the development of prolonged action dosage forms for other enzyme therapeutics.  相似文献   

2.
L-Asparaginase from Escherichia coli A-1-3 was modified with activated polyethylene glycols (2-0-methoxypolyethylene glycol-4,6-dichloro-s-triazine) with molecular weights of 750, 1900 and 5000. The modification of asparaginase to 73 amino groups out of the total 92 amino groups in the molecule with polyethylene glycol of 5000 daltons gave rise to a complete loss of the binding ability towards anti-asparaginase serum from rabbit. This modified asparaginase retained the enzymic activity (7%) and had a resistivity against trypsin. Asparaginases modified with polyethylene glycols of 750 and 1900 daltons did not show a substantial change of the immunogenic properties.  相似文献   

3.
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.  相似文献   

4.
Two enzymes that catalyze the hydrolysis of l-asparagine have been isolated from extracts of Pseudomonas geniculata. After initial salt fractionation, the enzymes were separated by chromatography on diethylaminoethyl-Sephadex and purified to homogeneity by gel filtration, ion-exchange chromatography, and preparative polyacrylamide electrophoresis. The enzymes differ markedly in physicochemical properties. One enzyme, termed asparaginase A, has a molecular weight of approximately 96,000 whereas the other, termed asparaginase AG, has a molecular weight of approximately 135,000. Both enzymes are tetrameric. The asparaginase A shows activity only with l-asparagine as substrate, whereas the asparaginase AG hydrolyzes l-asparagine and l-glutamine at approximately equal rates and it is also active with d-asparagine and d-glutamine as substrates. The asparaginase A was found to be devoid of antitumor activity in mice, whereas the asparaginase AG was effective in increasing the mean survival times of both C3H mice carrying the asparagine-requiring Gardner 6C3HED tumor line and Swiss mice bearing the glutamine-requiring Ehrlich ascites tumor line. These differences in antitumor activity were related to differences in the K(m) values for l-asparagine for the two enzymes. The asparaginase A has a K(m) value of 1 x 10(-3) M for this substrate whereas the corresponding value for the AG enzyme is 1.5 x 10(-5) M. Thus the concentration of asparagine necessary for maximal activity of the asparaginase A is very high compared with that of the normal plasma level of asparagine, which is approximately 50 muM.  相似文献   

5.
A series deactivation model is utilized to theoretically examine the influence of different modifying agents on enzyme deactivation kinetics. A form of the Hill-type equation is used to describe the effect of the modifying agents on the model parameters. Modification-induced inactivation equations are presented for the acetylation and succinylation of E. Coli asparaginase, for the site-specific reagent and substrate modification of flavocytochrome b(2) from Baker's yeast, and for the guanidinium chloride inactivation of cathepsin D. The analysis of more data for these and other enzymes would help further substantiate the technique presented and enhance the applicability of the model.  相似文献   

6.
Bioconjugation protocols have been recently used to improve the therapeutic properties of the anti‐leukemic enzyme L ‐asparaginase. In this study, we study the variation of reaction factors, such as mass ratio, pH value, reaction temperature and time, and the concentration of cross‐linker, in the modification reaction of L ‐asparaginase with silk fibroins. The optimum reaction parameters were obtained as follows: pH 7.0, mass ratio of silk fibroin to L ‐asparaginase 5:1, reaction time 8 h, and temperature 4°C. The rate of ε‐amino group modification was 57.88% and the recovery of modified L ‐asparaginase was 66.58% under these reaction conditions. We isolated and purified the silk‐L ‐asparaginase conjugates with two consecutive chromatography steps: anion‐exchange (XK 16×20, Q Sepharose FF) and gel filtration (Tricorn 10×600, Sephcryl S‐300 HR) chromatography. Finally, the stabilities of the enzymes were investigated. The results showed that modified L ‐asparaginase had a higher thermostability and higher resistance to trypsin digestion.  相似文献   

7.
Enzymes in cancer: Asparaginase from chicken liver   总被引:2,自引:1,他引:1  
1. A procedure for partial purification of asparaginase from chicken liver is presented. 2. The bulk of the enzyme is located in the soluble fraction of chicken liver. 3. Molecular weights of chicken-liver asparaginase and of the guinea-pig serum enzyme, estimated by gel filtration, were 306000 and 210000 respectively. The Michaelis constants (Km) at 37° and pH8·5 were 6·0×10−5m and 7·2×10−5m respectively. 4. At 50° the chicken-liver enzyme was moderately stable, some activity being lost by aggregation; in dilute electrolyte solutions the activity rapidly diminished. 5. The anti-lymphoma effect of guinea-pig serum in mice carrying the 6C3HED tumour was confirmed. Chicken-liver asparaginase also showed an effect but in this case the enzyme preparation had to be administered repeatedly. 6. Guinea-pig serum asparaginase was stable for several days in mouse blood, after intraperitoneal injection, whereas chicken-liver asparaginase rapidly disappeared. 7. Aspartic acid β-hydrazide was shown to be a competitive inhibitor of chicken-liver asparaginase with Ki approx. 5·6×10−4m. In mice it produced an anti-lymphoma effect, as reported previously.  相似文献   

8.
A modification of the diphenylamine procedure of Giles and Myers (2) is described which has 30% higher sensitivity and a lower reagent blank than the parent procedure, and is more conveniently carried out. Acetaldehyde in the procedure is replaced by paraldehyde, which is easier to store and which, unlike acetaldehyde, can be included in the diphenylamine reagent with little or no loss of reagent stability. The reagent is useful for about 1 month after preparation. The extraction method of Abraham et al. (5) for further increasing sensitivity is applicable to this modified procedure.  相似文献   

9.
It has been demonstrated that the activity of asparaginase A from Ps. fluorescens AG is completely inhibited by 10(-4) M p-chloromercurybenzoate and by 70-85% by Zn2+, Ca2+ and Cu2+ (2.10(-2) M). Iodoacetate, iodoacetamide, N-ethylimide of maleic acid and 5,5'-dithiobis-(2-nitrobenzoic acid) do not decrease the enzyme activity. Dithiothreitol and beta-mercaptoethanol reactivate the enzyme. L-asparagine, the substrate of asparaginase, protects the enzyme in a large degree against the inhibitory action of p-chloromercurybenzoate. p-chloromercurybenzoate induces a sharp increase in the asparaginase inactivation rate at acidic (6.5--5.5) and alkaline (7.5-8.5) values of pH. The enzyme modification by p-chloromercurybenzoate does not change the Km value for L-asparagine, but decreases Vmax. Thus it may be assumed, that asparaginase from Ps. fluorescens AG contains sulfhydryl groups essential for the enzyme activity.  相似文献   

10.
Rabbit antiserum was raised against potassium-independent asparaginase purified from Lupinus polyphyllus. A survey of 54 lines of Lupinus showed that only 11 contained the enzyme in maturing cotyledons with activities > 0.5 μmol/hr per g fr. wt. Potassium-dependent asparaginase activity was detected in a number of the remaining varieties.  相似文献   

11.
l-Asparaginase (EC 3.5.1.1.) activity has been detected in crude extracts of Lupinus arboreus young leaves, root tips, flower buds, and developing seeds. The enzyme was also present in Lupinus angustifolius root tips, developing nodules, and developing seeds. The asparaginase from each of these tissues had the same electrophoretic mobility on polyacrylamide gels and a Km of 6–8 mm for asparagine. In extracts other than those of the developing seeds, asparaginase activity was dependent upon the inclusion of K+ ion and a sulfhydryl protectant in the extraction buffer. No asparaginase activity was detected in mature leaves, in the plant fraction of nodules that were fixing nitrogen, nor in root tissue further than 1.5 cm from the root tip. Asparaginase has been purified 326- and 230-fold from L. arboreus and L. angustifolius developing seeds, respectively. A molecular weight of 75,000 was obtained by gel filtration. An apparent Km of 6.6 and 7.0 mm for asparagine was determined for the purified L. arboreus and L. angustifolius asparaginases, respectively. Of the amides, nitriles, and hydroxamates examined, the L. arboreus enzyme hydrolyzed only l-asparagine and dl-aspartyl hydroxamate. This same enzyme was inhibited by d-asparagine, 5-diazo-4-oxo-l-norvaline, dl-aspartyl hydroxamate, d-and l-aspartate, 3-cyano-l-alanine, glycine, and cysteine. Glutamine, glutamine analogs, and a number of other amino acids, amides and amines did not inhibit the L. arboreus asparaginase.  相似文献   

12.
An L-asparaginase has been purified some 250-fold from extracts of Klebsiella aerogenes to near homogeneity. The enzyme has a molecular weight of 141,000 as measured by gel filtration and appears to consist of four subunits of molecular weight 37,000. The enzyme has high affinity for L-asparagine, with a Km below 10(-5) M, and hydrolyzes glutamine at a 20-fold lower rate, with a Km of 10(-3) M. Interestingly, the enzyme exhibits marked gamma-glutamyltransferase activity but comparatively little beta-aspartyl-transferase activity. A mutant strain lacking this asparaginase has been isolated and grows at 1/2 to 1/3 the rate of the parent strain when asparagine is provided in the medium as the sole source of nitrogen. This strain grows as well as the wild type when the medium is supplemented with histidine or ammonia. Glutamine synthetase activates the formation of L-asparaginase. Mutants lacking glutamine synthetase fail to produce the asparaginase, and mutants with a high constitutive level of glutamine synthetase also contain the asparaginase at a high level. Thus, the formation of asparaginase is regulated in parallel with that of other enzymes capable of supplying the cell with ammonia or glutamate, such as histidase and proline oxidase. Formation of the asparaginase does not require induction by asparaginase and is not subject to catabolite repression.  相似文献   

13.
Crystals of an L-asparaginase from Vibrio succinogenes were obtained with the hanging drop method from ammonium sulphate-containing solutions. The crystals belong to the orthorhombic space group P22(1)2(1) with unit cell dimensions of a = 71.3 A, b = 85.8 A, c = 114.0 A, and contain two tetrameric enzyme molecules per unit cell. There are two subunits in the asymmetric unit; a molecular dyad is coincident with the crystallographic dyad. The crystal lattice is similar to that reported for an Escherichia coli asparaginase. Rotation function calculations have revealed that the V. succinogenes enzyme has 222 point group symmetry in the crystal. The second and third molecular dyads differ, however, from the corresponding E. coli asparaginase dyads by approximately 40 degrees. The crystals diffract to at least 2.2 A resolution and are suitable for X-ray crystallographic structure determination.  相似文献   

14.
The chloroplastic glyceride isoform of dihydroxyacetone phosphate reductase (Gly-DHAPR) in the photosynthetic unicellular green algae, Dunaliella, plays key role in the synthesis of glycerol-P and glycerides. A four-step procedure has been developed to purify the Gly-DHAPR from the chloroplasts of Dunaliella tertiolecta. The enzyme was purified 462-fold to apparent electrophoretic homogeneity by precipitation of Rubisco by polyethylene glycol-4000, and successive chromatography on DEAE cellulose, Sephacryl S-200, and Red Agarose. The overall yield of the purified enzyme was 5.1% with a specific activity of 425 μmol. min?1. mg?1 protein, and a subunit molecular mass of 37 kD. The Gly-DHAPR had little preference for NADH or NADPH, but was highly specific for DHAP. The purified enzyme was slightly stimulated by 50 mM NaCl, KCl or by 25 mM MgCl2. Detergents, lipids, fatty acids, or long-chain acyl-CoA derivatives inhibited the Gly-DHAPR. The Gly-DHAPR differs in properties from the other chloroplastic osmoregulatory isoform of DHAP reductase from Dunaliella, but has significant similarities with the glyceride isoforms from higher plants for glycerol-P and triglyceride synthesis.  相似文献   

15.
Three enzymes which catalyze the hydrolysis of L-asparagine have been identified in extracts of Citrobacter freundii. One of these (asparaginase-glutaminase (EC 3.5.1.1) also shows substantial glutaminase activity. This enzyme is extremely labile, is sensitive to inactivation by p-chloromercuribenzoate, and is not protected by dithiothreitol. A second enzyme (asparaginase B) is also sensitive to mercurials but is protected from inactivation by dithiothreitol. This enzyme has a relatively low affinity for L-asparagine (Km = 1.7-10(-3) M). The third enzyme (asparaginase A) is insensitive to inactivation by mercurials, is stable upon long term storage and has a relatively high affinity for L-asparagine (Km = 2.9-10(-5) M). This enzyme has been purified to homogeneity and has a molecular weight of approx. 140 000; the subunit weight being approx. 33 000. The C. freundii asparaginase A produced significant increases in the survival time of C3H/HE mice carrying the 6C3HED lymphoma tumor.  相似文献   

16.
Bacterial asparaginases (EC 3.5.1.1) have attracted considerable attention because enzymes of this group are used in the therapy of certain forms of leukemia. Class II asparaginase from Escherichia coli (EcA), a homotetramer with a mass of 138 kDa, is especially effective in cancer therapy. However, the therapeutic potential of EcA is impaired by the limited stability of the enzyme in vivo and by the induction of antibodies in the patients. In an attempt to modify the properties of EcA, several variants with amino acid replacements at subunit interfaces were constructed and characterized. Chemical and thermal denaturation analysis monitored by activity, fluorescence, circular dichroism, and differential scanning calorimetry showed that certain variants with exchanges that weaken dimer–dimer interactions exhibited complex denaturation profiles with active dimeric and/or inactive monomeric intermediates appearing at low denaturant concentrations. By contrast, other EcA variants showed considerably enhanced activity and stability as compared to the wild-type enzyme. Thus, even small changes at a subunit interface may markedly affect EcA stability without impairing its catalytic properties. Variants of this type may have a potential for use in the asparaginase therapy of leukemia.  相似文献   

17.
A new homogeneous enzyme which is capable of catalyzing the hydrolysis of both glutamine and asparaginase has been purified from extracts of Pseudomonas boreopolis 526 by the improved method. Purification involves few stages. The ratio of glutaminase to asparaginase activity is approximately 1.5:1.0. The enzyme is stable on storage and has a wide pH optimum of action (6-8.5). The molecular weight is about 134 000-145 000 D and the subunit molecular weight is about 34 000 D. No free SH-groups have been detected in the enzyme molecule.  相似文献   

18.
Asparaginase is an important antileukemic agent extensively used worldwide but the intrinsic glutaminase activity of this enzymatic drug is responsible for serious life threatening side effects. Hence, glutaminase free asparaginase is much needed for upgradation of therapeutic index of asparaginase therapy. In the present study, glutaminase free asparaginase produced from Enterobacter cloacae was purified to apparent homogeneity. The purified enzyme was found to be homodimer of approximately 106 kDa with monomeric size of approximately 52 kDa and pI 4.5. Purified enzyme showed optimum activity between pH 7–8 and temperature 35–40°C, which is close to the internal environment of human body. Monovalent cations such as Na+ and K+ enhanced asparaginase activity whereas divalent and trivalent cations, Ca2+, Mg2+, Zn2+, Mn2+, and Fe3+ inhibited the enzyme activity. Kinetic parameters Km, Vmax and Kcat of purified enzyme were found to be 1.58×10−3 M, 2.22 IU μg-1 and 5.3 × 104 S-1, respectively. Purified enzyme showed prolonged in vitro serum (T1/2 = ~ 39 h) and trypsin (T1/2 = ~ 32 min) half life, which is therapeutically remarkable feature. The cytotoxic activity of enzyme was examined against a panel of human cancer cell lines, HL-60, MOLT-4, MDA-MB-231 and T47D, and highest cytotoxicity observed against HL-60 cells (IC50 ~ 3.1 IU ml-1), which was comparable to commercial asparaginase. Cell and nuclear morphological studies of HL-60 cells showed that on treatment with purified asparaginase symptoms of apoptosis were increased in dose dependent manner. Cell cycle progression analysis indicates that enzyme induces apoptosis by cell cycle arrest in G0/G1 phase. Mitochondrial membrane potential loss showed that enzyme also triggers the mitochondrial pathway of apoptosis. Furthermore, the enzyme was found to be nontoxic for human noncancerous cells FR-2 and nonhemolytic for human erythrocytes.  相似文献   

19.
The aspartase and asparaginase genes of an industrial strain of Pseudomonas (PO7111) were cloned in the wide host range cosmid vector pJRD203 and identified by complementation of aspartase and asparaginase mutants. Subclones containing the asparaginase gene overproduced the enzyme 9-fold, but aspartase clones were highly unstable suggesting that aspartase overproduction conferred a severe growth disadvantage.  相似文献   

20.
Organophosphorous hydrolase (OPH) was physically and covalently immobilized within photosensitive polyethylene glycol (PEG)-based hydrogels. The hydroxyl ends of branched polyethylene glycol (b-PEG, four arms, MW = 20,000) were modified with cinnamylidene acetate groups to give water-soluble, photosensitive PEG macromers (b-PEG-CA). The b-PEG-CA macromers underwent photocrosslinking reaction and formed gels upon UV irradiation (>300 nm) in the presence of erythrosin B. Native OPH was pegylated with cinnamylidene-terminated PEG chains (MW = 3400) to be covalently linked with the b-PEG-CA macromers during photogelation. The effect of pegylation on the stability of the enzyme was determined. Furthermore, the effect of enzyme concentration, wavelength of irradiation, and photosensitizer on the stability of the entrapped enzyme was also investigated. The pegylated OPH was more stable than the native enzyme, and the OPH-containing gels exhibited superior stability than the soluble enzyme preparations.  相似文献   

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