Mitochondria and nuclei of pig and human epidermis: isolation and lipid composition

Pig and human epidermis were mechanically disrupted with a Polytron homogenizer and mitochondria and nuclei were isolated from homogenates in buffered sucrose and 2.5% citric acid solution, respectively, by differential and density-gradient centrifugation. Examination of the mitochondrial preparations by electron microscopy and the assay of several marker enzymes indicated that they consisted mostly of mitochondria with some microsomal contamination. The nuclear preparations were substantially free from contaminants as judged by particle counts with the aid of phase and electron microscopy. The lipid compositions of the mitochondria from both species were characterized by a high concentration of cardiolipin and low concentration of cholesterol and sphingomyelin and an absence of glycosphingolipids as compared with the lipid composition of the whole epidermal cell. The lipid compositions of the nuclei were characterized by a high proportion of phosphatidyl choline and an absence of glycosphingolipids. The neutral lipids accounted for an unusually high proportion of the total lipids especially in human epidermal nuclei. Although the technical difficulties in isolating subcellular organelles from mammalian epidermis limit the yield and quality of the mitochondria, these preparations as well as those of nuclei are superior to preparations reported previously and are pure enough for a valid comparison with other membrane systems of the epidermal cell.