102 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table II Enzyme Activities in Subcellular Fractions of Human Epidermis a Condition Enzymes Glucose-6- NADPH- phosphate Cytochromc Dehydro- c Reductase genase in Cytochrome in Micro- Supernatant Oxidase in Re- somes Re- Fraction Re- Mito- covery (units/ covery (units/ covery chondria v (•)c mg N) • (•)c mg N) a (%)6 Epidermis, without 0.141 q- 65 0.106 q- 77 0.051 q- 68 keratinase 0.029 0. 007 0. 009 Epidermis, treated 20 0.172 q- 75 0.112 q- 80 0.059 q- 72 rain with keratinase 0. 043 0. 013 0. 004 Epidermis, treated 30 0.167 q- 81 0.111 q- 82 0.056 q- 70 min with keratinase 0. 027 0. 014 0. 008 "Average mean enzyme activity q- S.D. of 5 experiments. b Activity is expressed as Alog [ferrocytochrome c] per min per mg nitrogen. • Recovery of the enzyme in particular fraction is expressed as the percentage of total enzyme activity found in homogenate. a One unit of enzyme is defined as that amount of enzyme which causes an absorbancy change of 1.0 per rain with a 1-cm light path under the standard assay conditions. Figure 4. Electron micrograph of a microsomal pellet prepared from human epidermis, showing mostly fragqnented and some intact membranes (X 53,000)

SUBCELLULAR COMPONENTS FROM HUMAN EPIDERMIS 103 Figure 5. Electron micrograph of a microsomal pellet prepared from human epidermis, showing ribosomes (X 80,000) derreal cells, the force necessary to disrupt these cells may well facilitate fragmentation, particularly in epidermis which is not fresh. The opti- mum pH of keratinase (8.0) used for the determination of the enzyme activity is rather close to that of tris buffer (7.6), used for the isolation of microsomes from rat liver (15) in which case there was no fragmentation. The cytochrome oxidase activity was low and relatively the same in mitochondrial fractions obtained from untreated and keratinase-treated epidermis. Electron micrographs of the mitochondrial pellets showed the presence of partially intact and fragmented mitochondria along with other particulate material (Fig. 6). This contamination, as well as frag- mentation of the mitochondria, would account for the low activity of cytochrome oxidase. Bagatell et al. (16) and Rosett et al. (17) have iso- lated mitochondria from fresh rat epidermis. These preparations were also heavily contaminated with other tissue components. Due to the presence of desmosomes, keratohyalin granules, and perhaps lysosomes and other particulate matter such as keratin, the isolation of pure mito- chondria from epidermis poses a considerable challenge. The very nature of epidermis, containing basal cells, differentiating cells in the process of keratinization, cells in the process of dying, as well as the granular and keratin layers, complicates the isolation of mitochondria in pure form.