Subcellular Localization of Alkaloids and Dopamine in Different Vacuolar Compartments of Papaver bracteatum

Abstract

Fractionation of Papaver bracteatum Arya II Lindl. latex on Renografin step gradients revealed that 43% of the dopamine was compartmentalized along with .alpha.-mannosidase (40%) in vacuoles sedimenting in the 2% fraction. Twenty-two percent of the dopamine was in the supernatant, but a corresponding amount (18%) of .alpha.-mannosidase was also present suggesting vacuole breakage during isolation. By subcellular fractionation of protoplasts from cultured P. bracteatum cells, the 1,000g sedimenting organelles have been identified as the major site of accumulation of the morphinan alkaloid, thebaine (99 .+-. 0.8%), and the benzopheanthridine alkaloid, sanguinarine (96 .+-. 3%). Although the 1,000g pellet also contained 33 .+-. 4% of the total alkaloid precursor, dopamine, and half of the total vacuolar marker enzyme, .alpha.-mannosidase, 62 .+-. 10% of the amine was localized in the 100,000g supernatant. A differential distribution of the alkaloids was discovered upon resolution of the lysed protoplasts on Renografin step gradients. Over 40% of the dopamine was in the supernatant with 15% in a 2% Renografin band. The remainder was evenly distrbuted in denser fractions of the gradient. The 4 to 8% interface, previously found to contain the largest amount of thebaine and small amounts of sanguinarine and dopamine, has been shown to be enriched in vacuoles by electron microscopy. Using a histofluorescence method, dopamine compartmentation in vacuoles of intact cultured cells was corroborated. In summary, dopamine, sanguinarine, and thebaine occur in vacuoles of different densities. A large fraction of the total dopamine in cultured cells was found in the 100,000g supernatant along with 37% of the .alpha.-mannosidase suggesting that the amine may be sequestered in more fragile vacuoles than the alkaloids. The possibility that some dopamine may be cytosolic cannot be ruled out.