Isolation of Active Mitochondria from Apples

Abstract

IN recent studies on the physiology of ripening in fruits, attention has been focused on the possible relationship between phosphorylative processes and the climacteric rise in respiration^1–3. Since many respiratory enzymes are located in the mitochondria, the isolation of these subcellular units in an active state is of great importance in such investigations. Although the apple is the classic object for studies in fruit physiology, attempts to prepare particulate fractions with mitochondrial activity from apples have not been successful. Lieberman⁴ isolated particles with cytochrome oxidase activity by grinding apples in a medium containing tris-hydroxy-methyl aminomethane at pH. 9.2. By using a homogenizing medium buffered at pH 7 with 0.5 M phosphate, Neal and Hulme⁵ obtained apple particles which oxidized succinate and malate to a slight extent, but only in the presence of the phenolic compound protocatechuic acid. Pearson and Robertson² isolated apple particles that had a high endogenous oxygen uptake and that showed some oxidative activity with succinate, citrate and malate. Pearson and Robertson's² particles were obtained after a single centrifugation of the homogenate and possibly contained contaminating phenolic compounds. These compounds may have contributed in part to the endogenous oxygen uptake and, in the light of Neal and Hulme's⁵ results, may also have been responsible for the oxidation of the added acids. In all these studies, stress has been laid on the necessity for counteracting the high acidity of the tissue when preparing mitochondria from apples.