Shah P K, Borchardt R T
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence 66045.
Pharm Res. 1991 Jan;8(1):70-5. doi: 10.1023/a:1015882323677.
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 (Km and Vmax) 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 Km and Vmax values for aminopeptidase(s) with different substrates in epidermis homogenates are as follows: leucine beta-naphthylamide (11 microM and 38 nmol.min-1.mg-1), tyrosine beta-naphthylamide (21 microM and 18 nmol.min-1.mg-1), and lysine beta-naphthylamide (11 microM 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 epidermis gave similar Km (32 and 24 microM). Vmax (9.77 and 7.55 nmol.min-1.mg-1) and Ki (223 and 194 microM) values. In addition, the cellular homogenates gave similar metabolite profiles for Leu-enkephalin.
