Steady-state analysis of alpha-melanotropin in the pars intermedia of Anolis carolinensis: effect of background adaptation.

The steady-state levels of alpha-melanotropin-stimulating hormone (alpha-MSH)-related peptides were examined in the pars intermedia of the reptile Anolis carolinensis as a function of background adaptation. After a 7-day period, the content of immunoreactive alpha-MSH-related material in the pars intermedia of light-adapted animals was approximately fourfold higher than that of animals maintained on a dark background for the same period. The immunoreactive alpha-MSH-related material present in the pars intermedia of light-adapted and dark-adapted animals was separately analyzed by gel filtration chromatography, reverse-phase HPLC, and cation-exchange chromatography. For light-adapted animals the major form of alpha-MSH had an apparent molecular weight of 1.5 kDa and a net charge of +4 at pH 3.5. Following reverse-phase HPLC this material eluted as a single peak of immunoreactivity with a retention time distinct from that of both mammalian ACTH(1-13)amide and N-acetyl-ACTH(1-13)amide. For dark-adapted animals a peak of alpha-MSH-sized material with an apparent molecular weight of 1.5 kDa was also detected. Following reverse-phase HPLC analysis this material eluted as an apparent single peak of immunoreactivity with a retention time distinct from that of the mammalian standards. Subsequent analysis of this major HPLC peak by cation-exchange chromatography revealed the presence of at least two forms of immunoreactive alpha-MSH. These forms differed in relative proportions. The major peak of immunoreactivity had a net charge of +4, whereas the minor peak had a net charge of +3. The +3 immunoreactive form was not detected to any appreciable degree in light-adapted animals.