Physical Properties and Subunit Structure of Xanthosine 5′-Phosphate Aminase

Abstract

Abstract Xanthosine 5'-phosphate aminase (xanthosine 5'-phosphate:ammonia ligase (AMP), EC 6.3.4.1) was purified from Escherichia coli B-96. The purity of the enzyme preparation was established by its behavior in disc gel electrophoresis, ultracentrifugation, and gel filtration. The extinction coefficient (E1%280) was determined to be 11.2 cm-1 with the use of the analytical ultracentrifuge as a differential refractometer. The diffusion coefficient (D020,w) of 5.09 x 10-7 cm2 s-1 was obtained by the measurement of free diffusion in the analytical ultracentrifuge. Differential sedimentation equilibrium provided a value of 0.739 ml per g for the partial specific volume (v). The sedimentation coefficient (s020,w) was estimated to be 5.91 x 10-13 s. The molecular weight of the enzyme was determined by sedimentation equilibrium measurements to be 126,000 ± 4,000. Disc gel electrophoresis in sodium dodecyl sulfate, sedimentation equilibrium in guanidine hydrochloride, and NH2-terminal analysis indicated that xanthosine 5'-phosphate aminase is a dimer composed of identical subunits. The molecular weight of the subunit was determined to be 63,000 ± 3,000. Possible molecular dimensions for the enzyme consistent with its hydrodynamic properties are discussed.