Epidermal aminopeptidase activity and metabolism as observed in an organized HaCaT cell sheet model

Abstract

Metabolism studies on organized HaCaT keratinocyte cell sheets are reported. Cells were grown on porous membranes to form organized cell sheets of several cell layers, which were considered as a model of viable epidermis. Metabolism was studied by reflection kinetics, with the top side of the cell sheets in contact with a donor solution and the basal side closed by an impermeable backing layer. Metabolite formation was followed by HPLC of substrate and metabolite in the donor. For comparison, studies with homogenized HaCaT cells were also performed. Model substrates were amino acid amides of 4-methoxy-2-naphthylamine (MNA) (i.e., Ala-MNA, Arg-MNA, Glu-MNA, and Leu-MNA). Also Leu-enkephalin was studied as a model peptide. Except for Glu-MNA, all substrates were metabolized in both the organized cell sheet and in the homogenates. In homogenate studies, saturation of the metabolic reaction was reached at <100 nmol mL(-1) substrate, whereas metabolism in organized cell sheets was below saturation (up to 500 nmol mL(-1)) except for Leu-enkephalin that showed saturation at >100 nmol mL(-1). In homogenates, substrate inhibition was found with Leu-MNA (> approximately 20 nmol mL(-1)) but not with Ala-MNA and Arg-MNA, both of which showed saturation. Differences of homogenates versus organized cell sheets are due to the intact organization and enzyme compartmentation of the cell sheets as opposed to the loss of organization and compartmentation in homogenates. Also, diffusion of substrate into cell sheets may be rate limiting.