[Isolation and properties of mitochondria from leaves of Spinacia oleracea and Beta vulgaris].

From mature green leaves of Spinacia oleracea and Beta vulgaris a fraction has been obtained which is enriched in mitochondria. Washed leaves were crushed in a meat-mill in an isotonic or slightly hypertonic medium containing sucrose, EDTA, MgSO4, bovine serum albumin, mercaptoethanol, KH2PO4, and HEPES as a buffer substance. After squeezing through nylon the suspension was centrifuged first at 1500xg, and following removal of the sediment a second time at 25000xg. After resuspension in a washing medium, the pellet was centrifuged in a swing-out rotor for 30 min at 35000xg on a density gradient generated from a silica sol ("Ludox HS 40"). A temperature of 2°C was maintained during the whole procedure.Whereas the usual isolation techniques are unsatisfactory for the preparation of intact leaf cell mitochondria, the above procedure enabled us to obtain a fraction from the gradient which had the following properties. The respiration rate was 29.5 μatoms O/mg protein-N/h with α-ketoglutarate as a substrate. Phosphorylation rates as calculated from the phosphate measurements and P/O ratios were low. This has been interpreted as a consequence of ATPase activity in the fraction. In polarographic studies ADP/O ratios of 1.32-2.15 with succinate as a substrate were measured. Respiratory control was also observed. This indicates the presence of tightly coupled mitochondria in the fraction.The fine structure of the mitochondria was shown to be intact as far as electron microscopical evidence can be used as a criterion. The procedure appears to be suitable for isolating active mitochondria and intact chloroplasts from green leaves. It may permit the study of interrelationships between these organelles.