Physical properties and subunit structure of l-asparaginase isolated from Erwinia carotovora

Abstract

1. l-Asparaginases from Erwinia carotovora and Escherichia coli (EC2 enzyme) are both capable of inhibiting and eliminating certain types of tumour cells. The Er. carotovora enzyme is a more basic protein, however, and in contrast with the EC2 enzyme it contains neither tryptophan nor cystine, and disulphide bonds are therefore absent. The molecule is very stable in solution from pH3.0 to about pH12.0, and is somewhat more stable at alkaline pH than is the Esch. coli enzyme. Calculations based on a s(0) (20,w) 7.43S and a sedimentation-equilibrium molecular weight of 135000+/-10000 give a frictional ratio (f/f(0)) of 1.08. The molecular conformation is therefore very compact in solution, and the electron microscope shows the negatively stained molecules as almost spherical particles with a diameter of 7.2+/-0.7nm. 2. Sedimentation-velocity and equilibrium ultracentrifugation, in 5-8m solutions of urea and guanidinium chloride, and also electrophoresis in sodium dodecyl sulphate-polyacrylamide gel, reveal a dissociation of the native protein molecule into four subunits of similar molecular weight in the range 32500-38000. The enzymically inactive subunits can be physically reassembled into an active tetramer when urea is removed by dialysis. Although the subunit structures of the Er. carotovora enzyme and the Esch. coli enzyme molecules are similar, the secondary bonding forces holding the subunits together in the tetramer are somewhat stronger in the Er. carotovora enzyme. 3. The optical-rotatory-dispersion (o.r.d.) parameters that characterize the Cotton effects arising from ordered structure in the molecule are [m'](233)=-3522+/-74 degrees and [m'](200)=9096+/-1700 degrees . These show very marked changes as the secondary structure is disrupted and the molecule dissociates into subunits. A correlation pathway was traced on the basis of o.r.d. parameters and enzyme activity as the polypeptide chains were denatured and renatured (and reconstituted) into active molecules after the dilution of solutions in urea. Subunits resulting from treatment with sodium dodecyl sulphate do not show the typically disordered o.r.d. profile, but nevertheless they are inactive.

One‐step purification and kinetic properties of the recombinant l‐asparaginase from Erwinia carotovora

“…We think that the most adequate solution is to measure Erwinia asparaginase concentrations by the Biuret or modified Lowry [18] methods, where the aromatic-residue content is not significant. We used the latter method in our study and the activity of recombinant asparaginase (630 i.u./mg) corresponded well with that of the wild-type enzyme (550 i.u./ mg) when protein concentration had been determined with amino acid Technicon AutoAnalyser [25].…”

“…The reported activities of wild-type L-asparaginase from Erw. carotovora vary from 310 [21] to 550 i.u./mg [25]. For ErA activities this range is even larger, namely from 200 [27] to 615 i.u./mg [22].…”

Bacterial recombinant L-asparaginases: properties, structure and anti-proliferative activity

“…carotovora [61][62][63][64][65][66], P. vulgaris [67], V. succinogenes [26], S. marcencens [68] Для получения бесклеточного экстракта на первом этапе очистки клетки разрушают ультразвуком. Полученный после ультрацентрифугирования (30000 g) супернатант последовательно подвергается хроматографии на колонках с Q-Sepharose и DEAE-Toyopearl 650m (L-аспарагиназы RrA и YpA) [52,53] или SP-Sepharose (L-аспарагиназа Нра) [54].…”

“…Действительно, некоторые L-аспарагиназы характеризуются значительной структурной устойчивостью в денатурирующих условиях, которая может показаться удивительной для фермента с нековалентно связанными субъединицами. Например, после 48-часовой инкубации в 0,2 М фосфатном буфере, (рН 7,4), содержащем 8 М мочевину, диссоциируют только 50% молекул ErA [61]. Интересно отметить, что олигомеризация L-аспарагиназы является субстрат-зависимым процессом, что предполагает возможность аллостерической регуляции каталитической активности.…”

L‐Asparaginase: A Review

“…Using DONV with absorbance at 274 nm as an alternative substrate, and also using a double-sector cell that allowed layering of enzyme into both sectors at once, thus canceling enzyme absorbance, Holcenberg et al (79) determined the specific activities of various portions of the sedimentation velocity profile. They found that the slower-sedimenting species had less than 4% of the activity of the tetramer, Cammack et al (39) also report that the subunits in Erwinia asparaginase are inactive. In fact, there is no evidence so far to suggest that the subunits of any asparaginase have activity.…”