Filtering centrifugation through two layers of silicone oil: a method for the kinetic analysis of rapid metabolite transport in organelles.

Kinetic studies of ATP uptake in amyloplasts from sycamore (Acer pseudoplatanus L.) have been performed with a newly developed method of centrifugation through a double layer of silicone oil; the results are compared with the frequently used method of centrifugation through a single layer. The present technique employs two separate silicone layers: the upper one prevents mixing of the organelle preparation with the incubation layer, which contains the metabolites, and the lower one prevents mixing of the incubation and pelleting layers. Incubation of the organelles takes place in the incubation layer after the upper layer of silicone inverts during centrifugation. Depending on the speed of centrifugation, incubation periods as short as 1 sec and as long as 2 min can be achieved. High reproducibility and accuracy, acquisition of multiple data in a single centrifugation, and maintenance of structural integrity and metabolic activity of the organelles make the technique advantageous for the analysis of metabolite transport in amyloplasts and other kinds of organelles.