Purification and biological characterization of human hepatopoietin A, a polypeptide growth factor for hepatocytes

We have previously reported the presence of a high molecular weight polypeptide growth factor in the plasma of normal human or rat serum which stimulates DNA synthesis in primary cultures of normal rat hepatocytes. We referred to this activity as hepatopoietin A (HPTA) (Michalopoulos, G., Houck, K. A., Dolan, M. L., and Luetteke, N. C. Control of hepatocytes replication by two serum factors. Cancer Res., 44: 4414-4419, 1984; Thaler, J., and Michalopoulos, G. Hepatopoietin A. Partial characterization and trypsin activation of a hepatocyte growth factor. Cancer Res., 45: 2545-2549, 1985). At that time, however, complete purification of this growth factor had not been achieved. In the present report we describe the steps required for complete purification of HPTA from human plasma or rabbit serum. The purification involved sequential ammonium sulfate precipitation, heparin-affinity chromatography, anion-exchange high-performance liquid chromatography (HPLC), and reversed phase HPLC. The final purified product is a heterodimer consisting of a heavy and a light polypeptide chain with molecular weights of 70,000 and 35,000, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Under nonreducing conditions, however, the purified HPTA migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular weight of 69,000. The mitogenic activity of HPTA was associated with this band when it was eluted from unstained sodium dodecyl sulfate-polyacrylamide gels. Gel filtration HPLC under neutral isotonic conditions indicated that HPTA tends to form aggregates with molecular weights of greater than 300,000. Chromatofocusing indicated that HPTA is an acidic protein with an isoelectric point value of about 5.5. The mitogenic activity of HPTA was sensitive to heat, trypsin, and 2-mercaptoethanol, but relatively resistant to exposure to 1 N acetic acid, 2 M guanidine-HCl, and 0.1% sodium dodecyl sulfate. The stimulation of DNA synthesis induced by HPTA was totally abrogated by transforming growth factor-beta and markedly reduced in the presence of heparin. We present biochemical as well as biological evidence that HPTA is a hepatocyte growth factor distinct from other known polypeptide mitogens such as epidermal growth factor, transforming growth factor-alpha, platelet-derived growth factor, fibroblast growth factor, and thrombin.