Purification of alpha 1-microglobulin produced by human hepatoma cell lines. Biochemical characterization and comparison with alpha 1-microglobulin synthesized by human hepatocytes.
alpha 1-Microglobulin (alpha 1m) was determined by radio-immunoassay in the supernatants of five human hepatoma cell lines. High amounts of alpha 1m were produced by PLC/PRF/5, intermediate ones by Hep G2 and Hep 3B and very low ones by Malhavu and SK Hepl. alpha 1m isolated from hepatoma cell lines PLC/PRF/5 or Hep G2 supernatants displayed the same physicochemical properties as that purified from human urines: the apparent molecular mass was 26 kDa and the pI from 5.6 to 6.4 as measured after two-dimensional polyacrylamide gel electrophoresis in denaturating conditions; for the native molecule the pI was estimated to be 4.0-4.9. Both urinary and hepatoma alpha 1m migrate as a diffuse band in the alpha zone in agarose gel at pH 8.6 in non-denaturing conditions and present a brown chromophore covalently associated with the molecule. After biosynthetic labelling with [35S]methionine, proteins extracted from hepatoma cell line PLC/PRF/5 and from isolated hepatocytes of human liver were separated by two-dimensional PAGE and transferred to a nitrocellulose membrane. alpha 1m was identified and found to be identical in both cases. However, when compared with the alpha 1m isolated from cell supernatants, less charge heterogeneity but also minor additional spots of higher molecular mass were observed.
D000510:Alpha-Globulins / Q000096:biosynthesis / Q000302:isolation & purification*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D002460:Cell Line; D002845:Chromatography / Q000379:methods; D005455:Fluorescent Antibody Technique; D006801:Humans; D007158:Immunologic Techniques; D008099:Liver / Q000378:metabolism*; D008113:Liver Neoplasms / Q000378:metabolism*; D011506:Proteins / Q000032:analysis
D000510:Alpha-Globulins; D011506:Proteins; C016001:alpha-1-microglobulin