SUBCELLULAR COMPONENTS FROM HUMAN EPIDERMIS 101 Figure 3. Microphotograph of nuclei isolated frmn human leg epidermis after incubation with keratinase for 20 min and after homogeniza, tion in sucrose-citric acid solution and sucrose gradient centrifugation (X 600) The data in Table II indicate that keratinase had no apparent dele- terious effect on the activities of the marker enzymes of each particulate fraction after incubation for 20 and 30 min. The enzyme recovery was somewhat less than expected. This may be due to the low activity of each enzyme in epidermis, making contmnination of one fraction by another fraction difficult to measure accurately. Although NADPH-cytochrome c reductase activity was associated with the microsomal membranes, glucose-6-phosphate dehydrogenase, a mi- crosrenal enzyme, was found in the supernatant fraction. Electron mi- crographs of the microsomal pellets from an untreated sample of epi- dermis showed that the membranes were sufficiently fragmented to ac- count for the solubilization of glucose-6-phosphate dehydrogenase (Fig. 4). The NADPH-cytochrmne c reductase, however, was not solubilized. Another section through the microsomal pellet from an untreated sample of epidermis showed ribosomes only (Fig. 5). Keratinase-treated epi- dermis gave microsomal preparations similar to those of the untreated epidermis. Fragmentation of the microsomes may be due to autolysis (14), since one rarely obtains sufficient human skin for fractionation within a short time after surgery or autopsy. Even though keratinase loosens the epi-

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)