By Liza A. Pon and Eric A. Schon (Eds.)
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CoQ-Depleted and CoQ-Reconstituted Mitochondria Coenzyme Q can be extracted from BHM preparations using organic solvents. CoQ-depleted mitochondria are usually reconstituted with CoQ homologues and analogues. Polar solvents, such as acetone, were first used for CoQ extraction, but they irreversibly damage Complex I; thus nonpolar solvents are preferred, but they can extract neutral lipids, such as CoQ, only from dry material. The extraction is performed on lyophilized BHM preparations using a modification of the method of Szarkowska (1966).
The mitochondrial pellet is washed once with Solution A and finally resuspended in the same buVer. , 2000). E. Isolation of Mitochondria from Frozen Tissues Generally, mitochondrial isolations are performed on fresh tissues. Under these conditions, isolated mitochondria are pure and suitable for most of the biochemical assays and for further modifications of mitochondria to be used in particular 1. Isolation and Subfractionation of Mitochondria 21 assays. However, in cases when fresh tissues are not available, it is often necessary to prepare mitochondria from frozen tissues.
IM, inner membrane; IMS, intermembrane space; and OM, outer membrane. collapse the membrane potential), can also be used to detect ÁpH in ETPH preparations (Fig. 3). C. Broken Submitochondrial Particles SMP are broken membrane fragments that can react with exogenously added cytochrome c. They are not coupled, but they have good rates of individual enzymatic activities, such as NADH-coenzyme Q (CoQ) oxidoreductase and ubiquinolcytochrome c oxidoreductase. , 1993). A, in a Braun (Labsonic U) sonicator equipped with a 9-mm tip at 150 W for a total of 5 min, in 30-sec bursts followed by 30-sec oV, thus allowing the preparation to cool down.