Recent work from this laboratory has demonstrated the presence of a structurally and functionally different
ornithine decarboxylase (ODC) in mouse epidermal
tumors induced by a two-stage protocol involving initiation with
7,12-dimethylbenzanthracene (DMBA) and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). In this report, the enzymatic properties of ODC present in DMBA-initiated
chrysarobin-promoted
papillomas are compared to the
enzyme induced by
chrysarobin in normal epidermis. Analyses of 13 individual
tumor extracts indicated each had an elevated level of ODC activity compared to uninduced normal epidermis. Addition of
GTP to the
enzyme assay caused a marked stimulation of ODC activity in nine of 13
tumor extracts but had no effect on
chrysarobin-induced epidermal ODC.
Enzyme kinetic analyses indicated that
GTP lowered the atypically high apparent Km values for L-
ornithine of the
papilloma enzyme to values typical of epidermal ODC. The K 1/2 for
GTP activation of
papilloma ODC was approximately 7 x 10(-9) M. When a series of
nucleotides was tested, only
GTP, the non-hydrolysable analog
GTP gamma S,
dGTP and
GDP were capable of significant activation at 1 microM, while other derivatives including GMP,
ATP and
CTP were less effective. The ability of the
tumor enzyme to bind
GTP was confirmed by the results of
GTP-agarose chromatography, in which the
papilloma enzyme (but not
chrysarobin-induced epidermal ODC) bound to this affinity column and could be eluted by
GTP. While some differences were observed in the properties of ODC from
chrysarobin-promoted versus TPA-promoted
papillomas, the major conclusion of this study is that both agents cause the appearance of a functionally altered ODC in the majority of
papillomas produced by a two-step protocol.