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Synthesis of prolactin was measured in a clonal strain of functional pituitary cells in culture by incubating the cells with [3H]leucine; labeled prolactin was separated from the other labeled proteins by specific immunoprecipitation followed by dissociation with sodium dodecyl sulfate and disc gel electrophoresis. Accumulation of labeled prolactin in the cells was linear for at least 1 hour; labeled prolactin was released into the culture medium after 1 hour. Treatment with 28 nm thyrotropin-releasing hormone for 3 days increased prolactin accumulation in the medium 2.5-fold, and the increased synthesis of prolactin completely accounted for the increased accumulation. Treatment with 5 x 10-6 m hydrocortisone for 7 days decreased prolactin accumulation in the medium 2- to 5-fold, and the decrease in synthesis completely accounted for the decreased accumulation. Therefore the long term (3 to 7 days) effects on these cells of thyrotropinreleasing hormone and hydrocortisone result only in a change in the synthesis of prolactin and not in its rate of degradation. Pulse-chase experiments, using 10 µg per ml of cycloheximide or 10 mm leucine to block further incorporation of [3H]leucine into prolactin, show that there is no detectable degradation of prolactin under the usual conditions of culture. Incubation with 0.1 mm leucine does not prevent incorporation of radioactive leucine into prolactin and results in a 1.7-fold increase in labeled prolactin within 4 hours after the start of the chase. This result suggests there are two intracellular pools of leucine in GH cells, one of which does not appear to equilibrate readily with the medium.

The serological activity of the S-100 protein from bovine brain is reduced if the protein is aggregated by disulfide bond formation, the sulfhydryl groups are blocked by p-mercuribenzoate, or the activity is destroyed by incubation at high temperatures. Chelating agents, which bind metals that can oxidize sulfhydryl groups, protect the serological activity from heat denaturation. Blocking the sulfhydryl groups with p-mercuribenzoate stabilizes the reduced serological activity to further inactivation by heat. The serological activity, whether reduced or destroyed, can be recovered in all cases by incubation with 2-mercaptoethanol at 60°. The serological activity of S-100 from extracts of different portions of the brain is not the same, but incubation with 2-mercaptoethanol abolishes the differences.