Purification and subunit structure of hepatocyte growth factor from rat platelets

Abstract

A hepatocyte growth factor (HGF) that stimulates DNA synthesis of adult rat hepatocytes in primary culture was purified as a homogeneous material from platelets of 1000 rats by a four‐step procedure: stimulation of its release from platelets by thrombin, cation‐exchanger fast protein liquid chromatography (FPLC) on a Mono S column, heparin‐Sepharose CL‐6B chromatography, and reverse‐phase HPLC on a C₄ column. The purified HGF stimulated DNA synthesis of adult rat hepatocytes in primary culture at 1 ng/ml and was maximally effective at 5 ng/ml, being about twice as potent as EGF at this concentration. HGF did not stimulate DNA synthesis of Swiss 3T3 cells. It was found to be a beat‐ and acid‐labile protein that was inactivated by reduction with dithiothreitol. The purified HGF had a molecular mass of 82 kDa, as estimated by SDS‐PAGE, and was found to be a heterodimer which dissociated into a large subunit of 69 kDa and a small one of 34 kDa by SDS‐PAGE under reducing conditions. These biological and chemical properties showed that HGF was not identical with any known growth factors, including platelet‐derived growth factor (PDGF).