57] Measurement of metabolite movement across the envelope and of the pH in the stroma and the thylakoid space in intact chloroplasts

Abstract

This chapter discusses the techniques for the measurement of metabolite movement across the envelope and of the pH in the stroma and the thylakoid space in intact chloroplasts. The silicone layer filtering centrifugation method enables a rapid separation of mitochondria and chloroplasts from the suspension medium by centrifugation through a layer of silicone oil into a denaturing agent, for example, HClO4 or NaOH. It is advantageous to carry out the centrifugation in capillary tubes from polypropylene of 400 μl content. The centrifugation tube is filled at the bottom with 20 μl of 1 M HClO4, followed by a layer of 70 μl of silicone oil and 200–300 μl of chloroplast suspension on top of this. If the uptake of bicarbonate is to be measured, the bottom layer consists of 20 μl of 2.5 M NaOH. There has to be a density gradient among the three layers. The determination of solute space is based on the measurement of that portion of the volume of the radioactively labeled supernatant which is found, after centrifugation, in the pellet fraction. Measurement of the uptake into the sorbitol-impermeable 3H2O space is also discussed in the chapter.