Purification and Biochemical Characterization of Human Lymphocyte Mitogenic Factor (LMF)

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Abstract
Supernatants of human T lymphocytes stimulated by TT antigen release two factors that induce mitogenesis in autologous and allogeneic B lymphocytes. These factors are precipitated by 60% ammonium sulfate and 50% ethanol, and are both destroyed by heating to 70°C for 5 min. By equilibrium ultracentrifugation there was a peak of mitogenic activity in the fraction with a specific gravity of 1.3147 corresponding to a partial specific volume of 0.761. After ultrafiltration through an Amicon XM50 membrane, the concentrate was chromatographed on a Sephadex G-200 column. Mitogenic activity was found only in the post-albumin fraction. When the postalbumin fraction was run on an isoelectrofocusing column, two distinct mitogenic factors were identified. The major peak of mitogenic activity (LMF) had a pI of 6.68 ± 0.05 and the minor peak (MMF) had a pI of 7.27 ± 0.05. Amino acid analysis of LMF identified it as a protein and PAGE showed that LMF probably was a tetramer with a m.w. of 80,000.