Tissue and intracellular localization of indican and the purification and characterization of indican synthase from indigo plants

Indican (indoxyl beta-D-glucoside) was found to accumulate only in green leaves of the indigo plant, and not in any other tissues. Comparisons of the indican content of protoplasts and vacuoles showed that indican was stored only in the vacuole of the cell. Indican content appeared and increased with the appearance and growth of leaves. In mature plants, the younger leaves contained larger amounts of indican than the older ones. Cell extracts of young leaves of indigo plant catalyzed the synthesis of indican from UDP-glucose and indoxyl. Indican synthase was extracted and purified from young leaves. The enzyme was separated into two fractions by anion-exchange chromatography. The enzyme in the fraction which was eluted by 0.1M NaCl had a molecular weight of 53,000 by SDS-PAGE. Optimum pH of the enzyme was at about 10.0, indicating that the enzyme is likely localized in a different intracellular compartment from that of indican storage. The enzyme showed normal Michaelis-Menten kinetics and a K-m value of 0.13 mM for UDP-glucose.