Purification of plant mitochondria by isopycnic centrifugation in density gradients of Percoll

Abstract

Mitochondria from potato tubers have been separated from contaminating organelles and membrane vesicles on self-generated Percoll gradients and in a relatively short time. The Percoll-purified mitochondria devoid of carotenoids and galactolipids showed no contamination with intact plastids, microbodies, or vacuolar enzymes. Percoll-purified mitochondria exhibited intact membranes and a dense matrix. The intactness of purified mitochondrial preparations was ascertained by the measurement of KCN-sensitive ascorbate cyt c-dependent O 2 uptake. When compared with washed mitochondria, Percoll-purified mitochondria showed improved rates of substrate oxidation, respiratory control, and ADP:O ratios. The recovery of the cyt oxidase was 70–90% and on a cyt oxidase basis the rate of succinate oxidation by unpurified mitochondria was equal to that recorded for Percoll-purified mitochondria. The great flexibility of purification procedure involving silica sols was extended from mitochondria to the isolation of intact peroxisomes.