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PURIFICATION AND BIOLOGICAL CHARACTERIZATION OF HUMAN HEPATOPOETIN A: A POLYPEPTIDE GROWTH FACTOR FOR HEPATOCYTES
Citation:
Zarnegar, R. AND G. Michalopoulos. PURIFICATION AND BIOLOGICAL CHARACTERIZATION OF HUMAN HEPATOPOETIN A: A POLYPEPTIDE GROWTH FACTOR FOR HEPATOCYTES. U.S. Environmental Protection Agency, Washington, D.C., EPA/600/J-89/469.
Description:
We have previously reported that 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. e referred to this activity as Hepatopoietin A (HPTA) (6,7). t that time, however, complete purification of this growth factor had not been achieved. n the present report we describe the steps required for complete purification of HPTA from human plasma or rabbit serum. he purification involved sequential ammonium sulfate precipitation, heparin-affinity chromatography, anion exchange HPLC, and reverse-phase HPLC. he final purified product is a heterodimer consisting of a heavy and a light polypeptide chain with M.W. of 70,000 and 35,000 respectively as determined by SDS-PAGE under reducing conditions. nder non-reducing conditions, however, the purified HPTA migrated as single band on SDS-PAGE corresponding to a M.W. of 69,000. he mitogenic activity of HPTA was associated with this band when it was eluted from unstained SDS-polyacrylamide gels. el filtration HPLC under neutral isotonic conditions indicated that HPTA tends t form aggregates with M.W. of greater than 300,000. hromatofocusing indicated that HPTA is an acidic protein with a PI of about 5.5. he 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% SDS. he stimulation of DNA synthesis induced by HPTA was totally abrogated by TFGb and markedly reduced in the presence of heparin. e present biochemical as well as biological evidence that HPTA is a hepatocyte growth factor distinct from other known polypeptide mitogens such as EGF, TGFa, PDGF, FGF, and thrombin.