Isolation of chestnut chloroplasts: membrane potentials of chestnut and spinach thylakoids

Typical chestnut thylakoid extracts isolated by mechanical disruption of leaf tissues had an equivalent of 0.28 kg m⁻³ chlorophyll (Chl) which is six times less than in thylakoids obtained from spinach, although Chl content in leaves was only half as small. According to optical microscopy, the vesicles showed a good integrity, exhibiting at 21 °C a high capacity of photon-induced potential membrane generation, which was demonstrated by the almost full 9-amino-6-chloro-2-methoxyacridine fluorescence quenching in a hyper-saline medium containing 150 mM KCl and having osmotic potential of −1.5 MPa. The half-time of the thylakoid potential generation was 11.7 s with the time of dissipation around 8.9 s. In such conditions, spinach thylakoids showed an increased swelling and also differences in the half-time generation which was almost four times faster than was observed in chestnut. However, when spinach thylakoids were incubated in a typical hypo-saline medium without KCl with osmotic potential −0.8 MPa, no additional swelling was observed. Consequently the half-time of potential dissipation was 35 s. Studies with nigericin suggested a chestnut thylakoid ΔpH significantly smaller than that observed in spinach, which was confirmed by the measurements of the ATP driven pumping activity.