Abstract
Abstract 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.
Highlights
Moving boundary velocity experiments showed that most of the native enzyme sediments as the tetramer
Moleczdar Weeight of Subunits-The electrophoretic migration of proteins denatured in sodium dodecyl sulfate has been shown to be a linear function of the logarithm of the subunit molecular weight [4]
In order to confirm that the sedimentation coefficients were not seriously in error due to theoretical approximations, a computer program was constructed which computed both the moments tamic dehydrogenase, trypsin, E. coli asparaginase, and lysozyme as standards, the glutaminase-asparaginase has a migration identical with E. coli asparaginase which corresponds to a molecular weight of 33,000 f 200 g per mole (N = 6)
Summary
Acinetobaclet 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 Analytic sedimentation equilibrium experiments revealed a tetramerdimer dissociation with a dimer molecular weight of 69,000 f. Sedimentation of the enzyme in 5-diazo-4-oxonorvaline showed complex patterns with ultraviolet optics due to protein absorbance. 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. Preliminary physical studies of this enzyme revealed a lower molecular weight by gel filtration than by sedimentation velocity. Dissociation of the enzyme into active smaller fragments mav increase its effectiveness by increasing the distribution of the enzyme in the animal host. During the course of these investigations, we have devised some new ultracentrifuge techniques which may be applicable to other enzymes
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