Properties Of Asparaginase Research Articles

Application of PEG-chitosan copolymers for regulation of catalytic properties of enzymes for medical application using recombinant Erwinia carotovora L-asparaginase as an example

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.

61: Pharmacokinetic, pharmacodynamic and immunogenicity profiles of a pegylated recombinant Erwinia chrysanthemi-derived L-asparaginase in Mice

This chapter presents the assay, purification, and properties of asparaginase from guinea pig serum, Escherichia coli, and Serratia marcescens. The enzyme activity of L- asparaginase from guinea pig serum may be measured by the determination of ammonia or L-aspartic acid. The assay procedure is based on the direct nesslerization of ammonia. Two different assays for L-aspartic acid have been described, one based on the amount of aspartate-14C formed from 14C-labeled asparagines and the other on the change in absorption at 340 mμ after the addition of aspartate–oxaloacetate transaminase, malic dehydrogenase, and nicotinamide adenine dinucleotide phosphate (NADH). The enzyme is stable for at least six months at -20°, to repeated freezing and thawing, and to heating at 55° for 10 minutes. It is also stable to dialysis in the cold but is labile under conditions that promote surface denaturation. The same assay methods and definition of units apply to L-Asparaginase from Escherichia coli. Escherichia coli asparaginase is inhibited specifically by 5-diazo-4-oxo-L-norvaline (DONV). The inhibition can be reversed by L-asparagine.

Production and properties of asparaginase from a new Erwinia sp.

Asparaginase production by a mesophilic strain of Erwinia sp. was examined; the maximum of activity was found at 40 degrees C and pH 8.5. Among the various carbon sources, mannitol was shown to be the best for production of activity. Inorganic nitrogen sources were better than the organic ones. The enzyme activity was not inhibited by 10 mmol/L metal ions (Na+, K+, Mg2+, Ca2+, Ba2+, Co2+, Ni2+, Zn2+); the activity was strongly inhibited by addition of EDTA. L-Arginine, DL-alanine, L-asparagine and L-glutamine stimulated the L-asparaginase production by 3.9, 1.7, 4.3 and 4.0 fold, respectively. The combination of L-arginine, L-asparagine and L-glutamine synergistically stimulated the asparaginase up to 5.8 fold.

Purification and properties of asparaginase from the testa of immature seeds of pea (Pisum sativum L.)

A K+-dependent asparaginase (E.C. 3.5.1.1.) was purified 1328-fold from the testas of immature pea seeds (Pisum sativum L., var. Bolero) and characterized. Antibodies raised against purified asparaginase cross-reacted with the putative asparaginase band in Western blot analyses of semi-purified extracts. However, for crude extracts of pea testas, a cross-reaction was obtained with at least four protein bands, one of which was asparaginase protein. Affinity-purified antibodies to the four strongest bands of crude extracts were fairly specific for the bands from which they were purified, suggesting a mixture of specific antibodies. The Mr of asparaginase was 69,000 by Sephacryl S200 chromatography and also by mobility on native PAGE relative to BSA. There was no evidence for dissociation into subunits on SDS-PAGE, suggesting a monomeric protein of Mr 69,000. Other properties include an apparent Km of 2.4 mM, pI between 4.5 and 5, and competitive inhibition by aspartate and glycine.

The purification and properties of asparaginase from Lupinus species

Asparaginase, which catalyses the conversion of asparagine to ammonia and aspartate, has been purified from maturing Lupinus seeds. The enzyme has a high K m for asparagine (12.2 mM) and is able to utilise a number of asparagine analogues as substrates although glutamine and its analogues are not reactive. The possible route for the reassimilation of the liberated ammonia is discussed.

Immunochemical properties of asparaginase modified by chemical substitutions

Immunochemical properties of asparaginase modified by chemical substitutions

Asparaginase clearance: influence of the LDH-elevating virus.

SummaryAsparaginase, injected in conventional therapeutic doses into mice previously inoculated with the lactate dehydrogenase-elevating virus (LDH-virus), disappeared from the peripheral blood at a significantly slower rate than in corresponding virus-free mice. Direct testing of the transplanted tumors and leukemias employed in the earlier studies on the antitumor properties of asparaginase have indicated that the virus, was unknowingly present. The evidence indicates that the virus, through its impairment of host enzyme clearance and possibly other factors, played a significant role in the observed therapeutic effects of EC-2 l-asparaginase against sensitive mouse leukemia and lymphosarcoma.We thank J. D. Loveless, M. A. Fitzmaurice, and Edith R. Shapiro for assistance.

Purification and properties of asparaginase from escherichia coli B.

Purification and properties of asparaginase from escherichia coli B.