RESUMO
A protein that inhibits the replication of a malignant human mammary cell line, MCF-7, was purified to apparent homogeneity from human plasma-derived serum (PDS). Serum fractionation was accomplished by molecular sieve chromatography on Sephadex G-100 and Sephadex G-100 superfine columns. Further purification was accomplished by ion-exchange chromatography, with the use of DEAE-Sephacel followed by preparative polyacrylamide gel electrophoresis (PAGE) under nondenaturing conditions. Active fractions analyzed by sodium dodecyl sulfate (SDS)-PAGE showed one band with an apparent molecular weight of 53,000. Assays for the stability of those PDS fractions with the greatest growth-inhibitory activity and specificity for MCF-7 cells revealed that activity was retained during incubation at pH 2-10. Exposure to 6 M urea also had no effect on inhibitory activity. The activity was significantly reduced by incubation at 70 degrees C for 1 hour, exposure to 0.1% SDS, and treatment with chymotrypsin. Isoelectrofocusing showed two isoelectric points, 5.1 and 6.8.
