The goal of this project is to isolate and study the endogenous protein from rat liver that causes reversible, cytostatic inhibition of liver cell proliferation. Research efforts have been focused on purifying this hepatic proliferation inhibitor (HPI) so that a number of structural and biological studies can be carried out. The strategy previously developed for isolating HPI, involving extraction from liver, ammonium sulfate and ethanol precipitation, and chromatography by phenyl sepharose, gel filtration, and high resolution cation- and anion-exchange FPLC, was initially applied. HPI from this procedure was highly active, but not homogeneous. Work was directed at obtaining completely homogeneous preparations of HPI. The first approach involved further purification steps on the HPI obtained as described above. Weak anion- and cation-exchange and reverse phase HPLC, chromatography and hydrophobic interaction FPLC, and denaturing and nondenaturing one-dimensional electrophoresis were explored in this effort. In the second approach, a new set of chromatographic steps was substituted for the four column steps previously used. The new purification procedure involves chromatography by DEAE-cellulose, gel filtration and high resolution chromatofocusing and hydrophobic interaction FPLC. The HPI obtained by this means has about 100-1000 times the specific activity of HPI obtained by the earlier methods, with an estimated ED 50 in the range of 50-500 pg/ml.
