Intact epidermal cell assay for ornithine decarboxylase activity.
A procedure measuring the ornithine decarboxylase (ODC) activity and polyamine formation of intact neonatal mouse epidermal cells in culture has been developed and tested. Basal cells prepared from neonatal mouse epidermis were plated on round 15-mm Lux coverslips, placed in Costar 24 well culture clusters and grown at 32 degrees C in M-199 + 13% fetal bovine serum. Before assay the cells were rendered permeable to ornithine 14C and ODC inhibitors using the buffer described by Berger et al. [3]. The slides, covered with adhering cell layers, were then placed in vials, covered with assay buffer and assayed intact for ODC activity. The ODC reaction was terminated by addition of citric acid to the buffer and the amount of 14CO2 released was determined by scintillation counting of a center well filled with trapping agent. The baseline ODC activity of the intact cells was 500-1,000 pmol 14CO2/mg protein/45 min. The validity of this ODC assay procedure using intact neonatal mouse keratinocytes was tested by use of three specific ODC inhibitors and by measuring the formation of polyamines from uniform labeled ornithine. The results indicated that authentic ODC activity was measured and preserved in this intact neonatal mouse epidermal cell assay. This technique holds promise for future studies of epidermal cell regulation of ODC and polyamine synthesis and studies of the multiple ornithine metabolites and conjugates formed, using a highly manipulable in vitro system.
D000818:Animals; D002262:Carboxy-Lyases / Q000032:analysis*; D002478:Cells, Cultured; D000078404:Epidermal Cells; D004817:Epidermis / Q000201:enzymology; D051379:Mice; D008810:Mice, Inbred C57BL; D009955:Ornithine Decarboxylase / Q000032:analysis*; D012867:Skin / Q000166:cytology / Q000201:enzymology*
D002262:Carboxy-Lyases; D009955:Ornithine Decarboxylase