Ornithine Cyclase (Deaminating): II. PROPERTIES OF THE HOMOGENEOUS ENZYME

Abstract

Abstract Ornithine cyclase from Clostridium sporogenes was purified to electrophoretic and ultracentrifugal homogeneity. The pure cyclase had a sedimentation coefficient (s20,w) of 5.6 S measured by high speed sedimentation velocity centrifugation, and a molecular weight of 81,000 calculated from high speed equilibrium sedimentation. Amino acid analysis indicated a molecular weight of 80,000 and a partial specific volume of 0.733 cm3 per g for the protein. Gel electrophoresis on polyacrylamide in the presence of sodium dodecyl sulfate indicated the enzyme was made up of two subunits of equal size each with a molecular weight of 41,500. The light absorption spectrum had a major absorption peak at 277 nm, and calculations based on the absorbance values at 280 and 260 nm indicated the presence of approximately 1 mol of pyridine nucleotide per mol of cyclase. Assay of the pure enzyme for bound NAD+ demonstrated the presence of 1 mol of bound NAD+ per mol of enzyme. The activity of pure enzyme was stimulated by NAD+ and by either ADP or ATP. Chromatography of the pure enzyme on DEAE-cellulose columns dramatically increased the dependence of the cyclase activity on added NAD+. It was inhibited severely by oxygen and by several sulfhydryl group inhibitors.