The aim of this project is to isolate and study the endogenous polypeptide from rat liver that arrests growth of normal hepatocytes in a reversible (cytostatic), rather than irreversible (cytotoxic), fashion. The first objective of this work is to arrive at a purification scheme for isolation of this hepatic proliferation inhibitor (HPI), so that sufficient material is then available for structural and biological studies. Structural studies will allow comparison with known polypeptide factors, antibody production and gene cloning. Biological studies will focus on determination of such things as mechanism of action, detection of receptor, and interaction with growth factor pathways. A preparative scheme now appears to be in hand to allow acquisition of sufficient amounts of HPI for the structural and biological studies mentioned above to be carried out. The purification scheme for this molecule, initially a modification of previous methodology, has evolved into an essentially entirely new procedure. The steps now used in the isolation of HPI from rat liver involve a two step homogenization of tissue, acidification of the homogenate, centrifugation, neutralization of the supernatant, centrifugation, ammonium sulfate precipitation and ethanol precipitation. This is followed by a series of column chromatographic steps involving phenyl sepharose, gel filtration, cation and anion exchange FPLC, and weak anion exchange HPLC. Approximately a 200,000-fold purification of HPI was achieved from this series of steps. The bioassay for the elution position of HPI during these chromatographies was the one previously developed in this laboratory and involves a simple and rapid method, with the use of 96-well microtiter plates for cell culture, a tritiated thymidine uptake assay using a PhD cell harvester, and an assay for total cell DNA content using a viable fluorescent dye and an automatic microtiter plate fluorescence reader. HPI has an ED 50 in approximately nanomolar concentrations, is stable to acid, DTT reduction, heat (50 degrees C), and is a single polypeptide chain of 25K daltons.
