INTRACELLULAR INSULIN BINDING IN TETRAHYMENA-PYRIFORMIS

Insulin, a classic vertebrate hormone, produces alterations in cellula r metabolism and growth in the ciliate Tetrahymena pyriformis, as well as an increase in insulin binding upon subsequent exposure, a phenome non known as hormonal imprinting, An antibody to a peptide correspondi ng to the alpha-subunit of the human insulin receptor (amino acid resi dues 657-670) was used to investigate the location and to partially ch aracterize immunoreactive proteins in insulin-exposed and non-insulin- exposed cells (control), Confocal microscopy revealed immunofluorescen t labeling of cilia, nuclei, vesicles and an oblong structure of unkno wn nature. Labeling of nuclei, mitochondria and ciliary microtubules w as seen with immunoelectron microscopy, Labeling was absent on the cel l and ciliary membranes by immunoelectron microscopy. Polyacrylamide g el electrophoresis revealed several differences in protein composition between control and insulin-exposed ciliary membrane extracts, especi ally in the 30-50 kDa range. Immunoblotting revealed 2 reactive protei ns in whole cell lysates but none were detected in ciliary membrane ex tracts or wheat germ agglutinin affinity column eluates of T. pyriform is whole cell preparations. Based on these findings it is unlikely tha t a cell surface structure similar to a mammalian insulin receptor exi sts in T. pyriformis.