Necessary conditions for isolation of tightly coupled higher plant mitochondria

Abstract

A number of parameters associated with the isolation of plant mitochondria were carefully examined in the hope of optimizing the isolation technique. Dark-grown mung bean (Phaseolus aureus var. Jumbo) hypocotyls were used as the source of mitochondria. Necessary conditions for the isolation of tightly coupled plant mitochondria include: (a) gentle tissue disruption; (b) rigorous exclusion of contaminating particles from the mitochondrial fraction; (c) the use of an optimal grinding medium containing a high concentration of mannitol, 10 mm phosphate or triethanolamine buffer to maintain the pH above 7.2, ethylenediaminetetraacetic acid at 1 mm level, cysteine at a low concentration (0-5 mm), and a high concentration of bovine serum albumin (0.1-1.0%); and (d) the use of the proper wash and suspending medium consisting of mannitol, buffer, and bovine serum albumin at the same concentration as in the grinding medium.