Characterization of a rapid cellular-fractionation technique for hepatocytes. Application in the measurement of mitochondrial membrane potential in situ.

摘要

A rapid cellular-fractionation technique [ Hoek , Nicholls & Williamson (1980) J. Biol. Chem. 255, 1458-1464] was further characterized by using hepatocytes. Of the mitochondrial marker-enzyme activity, 80% was routinely separated from 71-98% of the total cell activities of marker enzymes for plasma membranes, Golgi-membranes, endoplasmic reticulum, lysosomes and cytosol. The mitochondria were contaminated with 53% of cell nuclei. [3H]Triphenylmethylphosphonium ion (TPMP+) was added to hepatocytes in an attempt to measure cellular transmembrane electrical potentials. After rapid cell fractionation the electrical potential between mitochondria in situ and the incubation medium was found to be 202 mV. This value was slightly increased when hepatocytes were treated with oligomycin, but substantially decreased by oligomycin plus an uncoupler of oxidative phosphorylation. Although estimates of TPMP+ binding were obtained, substantial difficulties prevented the accurate measurement of the electrical potential across the plasma membrane. It is concluded that TPMP+ may be employed to demonstrate the integrity of mitochondria during the fractionation procedures. However, the cation is inadequate for the determination of the separate components of the electrical potential between the mitochondrial matrix and the incubation medium.