PURIFICATION AND CHARACTERIZATION OF THE CROWN GALL SPECIFIC ENZYME NOPALINE SYNTHASE

Nopaline synthase of sunflower (Helianthus annuus L.) crown gall tissue induced by Agrobacterium tu-mefaciens strain C58 or T37 (nopaline utilizers) was purified to homogeneity as judged by analytical disc gel electrophoresis. The native enzyme elutes from a column of Ultrogel AcA 34 as a single peak with an estimated molecular weight of 158000. The dissociated enzyme migrates on NaDodSO4-polyacryl-amide gels as a single band with a molecular weight of 40000. Thus, the native enzyme appears to be composed of four equal-weight subunits. Nopaline synthesizing activity is found exclusively in crown gall tissues induced by strains of A. tumefaciens that utilize nopaline (e.g., C58 and T37). We found the same tissue specificity for the purified protein that we believe represents nopaline synthase. The results of kinetic studies of the purified enzyme are consistent with a ter-bi rapid-equilibrium random-order mechanism. Nopaline synthase is probably responsible for the in vivo synthesis of both N2-(1, 3)-dicarboxypropyOarginine (nopaline) and N1-(l, 3-dicarboxypropyl)ornithine (ornaline) in crown gall tissues since substrate specificities and Km values do not change during purification. © 1979, American Chemical Society. All rights reserved.