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We have demonstrated the presence of ACTH-potentiating factors in porcine thymus which contained neither biologically active nor radioimmunoassayable ACTH. The factors were acidic and heat stable in nature, and were present in heterogeneous forms having various molecular weight. Purification of the factors with small molecular weights was performed using reversed-phase high-performance liquid chromatography following gel filtration and cation-exchange chromatography. A purified factor exerted dose-dependent ACTH-potentiating activity. Loss of the ACTH-potentiating activity of purified factors by treatment with carboxypeptidase Y indicated their peptidic nature. ACTH-potentiating activity was readily observable after preincubating the adrenal cells with the thymic extract. The enhancement of ACTH-induced steroidogenesis by the factors was observed in the presence of bacitracin at a concentration to prevent ACTH degradation. Therefore, the existence of potentiating mechanisms other than inhibition of ACTH proteolysis was indicated.

High molecular weight ACTH fractions, obtained through gel filtration of boiled rat anterior pituitary extract, induced a marked increase in corticosterone production from isolated rat adrenal cells in the presence of low concentrations of ACTH-(1-24). This indicates the presence of heat-stable factors augmenting the steroidogenic action of ACTH in the rat anterior pituitary. We also noted that these factors potentiated the activity not only of ACTH-1(1-24) but also of ACTH-(1-8). The ACTH-potentiating factors in rat anterior pituitary extract are possibly present in heterogeneous forms according to their molecular weights (8,000, 10,000 and 15,000), their mobility in ion-exchange chromatography and their content in RIA-ACTH activity. Of these three forms, the former comigrated with biological ACTH activity. The remaining two forms were free of it. Since the effect of potentiating factors on modified ACTH-(1-9), shown to be less susceptible to proteolytic degradation from ACTH-(1-24), was similar to the effect on ACTH-(1-24), it is suggested that potentiation was not due to an inhibition of ACTH proteolysis.