Physical properties and subunit structure of L-asparaginase isolated from Erwinia carotovora.
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.
D000596:Amino Acids / Q000032:analysis; D001215:Asparaginase / Q000032:analysis / Q000302:isolation & purification; D002499:Centrifugation, Density Gradient; D002942:Circular Dichroism; D003902:Detergents; D004355:Drug Stability; D004586:Electrophoresis; D004589:Electrophoresis, Disc; D004885:Erwinia / Q000201:enzymology*; D004926:Escherichia coli / Q000201:enzymology; D006146:Guanidines; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008433:Mathematics; D008854:Microscopy, Electron; D008956:Models, Chemical; D008970:Molecular Weight; D009905:Optical Rotatory Dispersion; D011487:Protein Conformation; D011489:Protein Denaturation; D013053:Spectrophotometry; D013464:Sulfuric Acids; D014461:Ultracentrifugation; D014466:Ultraviolet Rays; D014508:Urea
D000596:Amino Acids; D003902:Detergents; D006146:Guanidines; D046911:Macromolecular Substances; D013464:Sulfuric Acids; D014508:Urea; D001215:Asparaginase