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]. Soc. Cosmet. Chem., 22, 95-107 (Feb. 4, 1971) Problems Involved in the Isolation of Subcellular Components from Human Epidermis* MALAYA BHATTACHARAYA, Ph.D., and CHRISTOPHER CARRUTHERS, Ph.D.* Synopsis--The enzyme KERATINASE was shown to be capable of separating human epi- dermal cells, apparently by dissolving the intercellular keratin-like substances. Attempts were made to fractionate these keratinase-treated, as well as untreated, epidermal cells into their SUBCELLULAR COMPONENTS. The maximum yield of cell particulates was ob- tained after digestion of the intact EPIDERMIS with keratinase at 25øC for 20 or 25 minutes. Assay of the marker enzymes of the subcellular components showed that kerati- nase had no apparent deleterious effect on these enzyme activities. However, the micro- somal glucose-6-phoslahate dehydrogenase activity in both untrcated and keratinase-treated epidermal samples was localized into the supernatant fraction. This displacement of en- zyme activity •nay have been due to the fragmentation of the microsomal membranes re- sulting from either the delay in securing fresh skin or the force needed to disrupt epidermal cells. These two conditions also may have led to contamination of the mitochondria with such epidermal structures as desmosomes, fragqnented keratohyalin granules, and perhaps ]ysosomes. INTRODUCTION Several methods are available for the preparation of mammalian cell particulates. For soft, nonfibrous tissues, homogenization is best performed with a manual or a motorized Potter-Elvehjem, Dounce,* or other type of homogenizer. Difficulty arises, however, in the disruption * Supported in part by Grant No. CA07236 from the National Cancer Institute. * Department of Biochemistry Research, Roswell Park Memorial Institute, and the New York State Department of Health, Buffalo, N.Y. 14203. Reprint requests should be sent to Dr. Carruthers. $ Lab Glass, Inc., 1172 Northwest Blvd., Vineland, N. I. 08360. 95