A COMPARISON OF PEPTIDASE ACTIVITIES AND PEPTIDE METABOLISM IN CULTURED MOUSE KERATINOCYTES AND NEONATAL MOUSE EPIDERMIS

One of the barriers to transdermal delivery of peptides is the metabolic activity of the epidermis. To define this metabolic activity, aminopeptidase activity and Leu-enkephalin metabolism were measured in the epidermis obtained from neonatal mouse skin and in cultured mouse keratinocytes. Aminopeptidase activity was measured fluorometrically using leucine, tyrosine, lysine, and aspartic acid derivatives of beta-naphthylamine as substrates. Similarities in substrate kinetic values (K(m) and V(max)) and substrate specificity of the enzyme(s) in homogenates prepared from neonatal mouse skin epidermis and cultured mouse keratinocytes strongly suggest that the keratinocytes in culture express the same aminopeptidase(s) with the same relative activity as in neonatal skin. The K(m) and V(max) values for aminopeptidase(s) with different substrates in epidermis homogenates are as follows: leucine beta-naphthylamide (11-mu-M and 38 nmol . min-1 . mg-1), tyrosine beta-naphthylamide (21-mu-M and 18 nmol . min-1. mg-1), and lysine beta-naphthylamide (11-mu-M and 35 nmol . min-1 . mg-1). Aspartic acid beta-naphthylamide and glutamic acid beta-naphthylamide were not hydrolyzed by these homogenates at pH 7.4 (37-degrees-C). Leu-enkephalin hydrolysis by the homogenates from cultured mouse keratinocytes and neonatal mouse epidermic gave similar K(m) (32 and 24-mu-M), V(max) (9.77 and 7.55 nmol . min-1 . mg-1) and K(i) (223 and 194-mu-M) values. In addition, the cellular homogenates gave similar metabolite profiles for Leu-enkephalin.