Physical Properties of Acinetobacter Glutaminase-Asparaginase with Antitumor Activity

Acinetobacter glutaminase-asparaginase has been shown to consist of 4 subunits (molecular weight 33,000) by sedimentation equilibrium in 5.5 m guanidine HCl and electrophoresis in sodium dodecyl sulfate on polyacrylamide gels after cross-linking the protein with dimethyl suberimidate. Moving boundary velocity experiments showed that most of the native enzyme sediments as the tetramer (s20,w = 7.42 ± 0.03 S). On the other hand, equivalent boundary calculations always showed a smaller s20,w. Analytic sedimentation equilibrium experiments revealed a tetramerdimer dissociation with a dimer molecular weight of 69,000 ± 3000. The molecular weight on calibrated Sephadex G-200 and Bio-Gel P-200 was 97,000 and 93,000, respectively, which may indicate reversible dissociation. The hydrolysis of 5-diazo-4-oxonorvaline was used to determine the sedimentation coefficient of the active species. Sedimentation of the enzyme in 5-diazo-4-oxonorvaline showed complex patterns with ultraviolet optics due to protein absorbance. A new double sector cell was devised which allows layering of enzyme into both sectors simultaneously, cancelling the absorbance of the enzyme. The s20,w value for the species which degraded 5-diazo-4-oxonorvaline was 7.6 ± 0.2 S. By matching zone sedimentation and active enzyme experiments, enzyme species smaller than tetramer were shown to have 4% or less of the activity of the tetramer.