General methods were developed and applied to the biosynthesis and purification of products of activated lymphocytes available in minute quantities. The activity studied here was the migration inhibitory factor (MIF) produced by purified protein derivative (PPD)- or concanavalin A (Con A)-stimulated lymphocytes obtained from one guinea pig or less. The methods selected yielded results in terms of two chemical parameters characteristic of the molecules involved, namely Kd on Sephadex G-75 and isoionic point, pI, on isoelectric focusing.

When supernatants were fractionated on G-75 columns, there were several areas even in control supernatants which produced migration inhibition relative to medium controls. However, in PPD- and Con A-stimulated supernatants, at least one peak of MIF activity was found solely in the stimulated cultures, with a Kd of 0.15. A double-labeling technique was used to characterize the proteins of this peak. Control, unstimulated cultures were labeled with [14C]leucine and stimulated cultures were labeled with [3H]leucine. After mixing the supernatants and G-75 filtration, a major "ratiolabeled" broad peak. i.e. one with increased 3H/14C ratio, was found. When a narrow portion of this peak about Kd 0.15, containing most of the MIF activity, was subjected to analytical isoelectric focusing, all of the label was associated with proteins of lower net charge than albumin. A unique ratiolabeled peak was found in PPD- and Con A-stimulated fractions with a pI of approx. 5.3. A micropreparative isoelectric focusing technique was developed and yielded MIF activity in the same region as the major ratiolabeled peak. Further study will be required to ascertain whether the ratiolabeled protein is MIF. By following the Kd, pI, and 3H/14C labeling ratio, at least 14 products of activated lymphocytes, synthesized either de novo or in increased amounts, could be distinguished.

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