Purification and partial characterization of hepatocyte growth factor from plasma of a patient with fulminant hepatic failure.

Abstract

Human hepatocyte growth factor (hHGF) has been purified from plasma of a patient with fulminant hepatic failure. The purification procedure consists of five steps, heat treatment of plasma, ammonium sulfate precipitation, and chromatography on Affi-Gel Blue, heparin-Sepharose, and hydroxylapatite. Starting with 930 ml of plasma from a patient, 37 micrograms of hHGF has been obtained, representing a 209,000-fold purification and a 18% yield. Purified hHGF shows several bands with molecular weights between 76,000 and 92,000. The two major bands had molecular weights of 79,000 and 86,000. Each band shows growth-stimulating activity on cultured rat hepatocytes which is proportional to the intensity of the band. After reduction of the sample with 2-mercaptoethanol, SDS-PAGE yields two chains with molecular weights of 31,500-34,500 and 54,000-65,000. The effect of hHGF on DNA synthesis by hepatocyte is half-maximal at 3.5 ng/ml. Insulin-like growth factor -I and -II, fibroblast growth factor, platelet-derived growth factor and transforming growth factor-beta, which are known as growth factors, had no stimulatory effect on DNA synthesis in cultured rat hepatocytes. hHGF stimulates proliferation of cultured rat hepatocytes more effectively than human epidermal growth factor (hEGF) or insulin, and the effect of hHGF is additional or synergistic with the maximal effects of hEGF and insulin. Labeling index of cultures with these three growth factor (insulin, hHGF and hEGF) was 85%, showing that most hepatocytes were induced to proliferate. These results suggest that hHGF is a new growth factor which is different from hEGF.