Alkaloid Sequestration by Papaver somniferum Latex

Abstract

The 1000 x g organelles of Papaver somniferum latex have been shown to accumulate rapidly, large quantities of alkaloids (0.05- 1 mg/mg organelle protein), particularly morphine, thebaine, codeine and papaverine and have been found to exhibit a surprising degree of specificity for the accumulation of these alkaloids. The uptake of alkaloid is independent of temperature and does not show a requirement for ATP 10-3 ᴍ, Mg2+ 10-3 ᴍ, or KCl 10-3 ᴍ. It is unaffected by known ATPase inhibitors (chlormadinone acetate 2.5 × 10-5 ᴍ, N,N-dicyclohexylcarbo- diimide 5 × 10-4 ᴍ and sodium vanadate 10-4 ᴍ) but showed considerable increase in morphine accumulation in the presence of 4-chloromercumbenzoate 5 x 10-4 ᴍ, while N-methylmaleimide 5 × 10-4 ᴍ had no effect, and carbonylcyanide 4-(trifluoromethoxy)phenylhydrazone 5 × 10-4 ᴍ caused a reduction (ca. 30%) of morphine uptake. An acid medium, pH 4.5-5.5, also results in decreased morphine uptake.

Dopamine Accumulation in Papaver somniferum Latex

“…In this respect, dopam ine accum ulation by latex 1 0 0 0 x 0 organelles appeared to be sim ilar to that of the mammalian storage vesicles [3], although in marked contrast to the accum ulation of m orphine by the latex 1 0 0 0 x g organelles which is apparently temperature insensitive [2 ].…”

“…Discontinuous gradients were prepared by sequen tially layering 200 m M phosphate buffer pH 7.0 containing sucrose 60% (7 ml) 50% (12 ml) 40% (12 ml) 30% (3 ml) in a 37 ml centrifuge tube. The level of dopamine which accumulates in the latex 1000x g organelles which sediment at the 50% -60% interface (Band B, [2]) on the discontinuous sucrose gradient is less by a factor of ten than the ac cumulated alkaloids in the same band. Levels of dopamine which remain in the supernatent from the lOOOx# organelle preparation [1] are very much higher than the level of alkaloid; which may result either from the production of dopam ine from dopa by dopadecarboxylase [1 2 ] in excess of uptake by the lOOOx# organelles [2], or from the rupture of 1 0 0 0 x 0 organelles during handling.…”

“…The level of dopamine which accumulates in the latex 1000x g organelles which sediment at the 50% -60% interface (Band B, [2]) on the discontinuous sucrose gradient is less by a factor of ten than the ac cumulated alkaloids in the same band. Levels of dopamine which remain in the supernatent from the lOOOx# organelle preparation [1] are very much higher than the level of alkaloid; which may result either from the production of dopam ine from dopa by dopadecarboxylase [1 2 ] in excess of uptake by the lOOOx# organelles [2], or from the rupture of 1 0 0 0 x 0 organelles during handling. This last pos sibility, if correct, suggests that dopam ine is ac cumulated in separate organelles from the alkaloids, since the ratio of alkaloid to dopam ine in the supernatant is much lower than the same ratio in the 1 0 0 0 x g fraction.…”

“…1). The sharp fall off in activity at alkaline pH was in contrast to the situation with morphine accum ulation where the latex 1 0 0 0 x 0 organelles lost their ability to accumulate morphine, as well as to take up neutral red, below pH 4.5 but retained the ability to ac cumulate morphine to a pH of at least 8.5 [2]. Additions of 20 mM ascorbate and 50 mM DIECA to buffer C in a separate set o f experim ents at pH greater than 7.5 clearly demonstrates that the loss in activity is not attributable to oxidation of the dopamine at alkaline pH.…”

“…This last pos sibility, if correct, suggests that dopam ine is ac cumulated in separate organelles from the alkaloids, since the ratio of alkaloid to dopam ine in the supernatant is much lower than the same ratio in the 1 0 0 0 x g fraction. accumulation of dopam ine by the neurotransm itter storage vesicles in mammals, where accum ulation is linear with time for only the first four m inutes of exposure [3], as well as with the rapid accum ula tion of alkaloids by latex 1 0 0 0 x g organelles [2 ].…”