Fate of corticotrophins in an isolated adrenal-cell bioassay and decrease of peptide breakdown by cell purification

Abstract

1. The fate of corticotrophins in a trypsin-dispersed rat adrenal-cell assay system was investigated with a view to establishing whether differences in the rate of inactivation might contribute to potency differences observed between analogues. 2. Corticotrophin-(1-24)-tetracosapeptide and to a lesser extent synthetic 1-39 corticotrophins were found to be inactivated during incubation with cell suspension. 3. Peptide fragments were isolated by using [[(3)H(2)]Tyr(23)]corticotrophin-(1-24)- tetracosapeptide as a marker. The fragments indicate a peptidase with a predominantly tryptic specificity. 4. The peptidase is present in the extracellular fluid and is released from cells when they are damaged. 5. Cells were fractionated on an albumin gradient. Cells from the zona fasciculata and the zona intermedia or reticularis were present in fractions which produced fluorogenic steroids in response to corticotrophin. 6. Purification of the cells by centrifugation through albumin decreased degradation by peptidases, so that if the assay is carried out with a dilute suspension of purified cells peptide breakdown should not affect the observed potencies of adrenocorticotrophin analogues. 7. No binding of [[(3)H(2)]Tyr(23)]corticotrophin-(1-24)- tetracosapeptide to cells could be detected at low concentrations of the peptide. This indicated that less than 120 receptors/cell are occupied during stimulation by a dose that would elicit approx. 80% of the maximal response.

Levels of Corticotrophin Analogues in the Blood After Infusion Into Rats

“…However, in an isolated adrenal cell system (Lowry, McMartin & Peters, 1973) the 1-24 peptide has been found to be several times more potent than the full sequence and it has been shown that the potency difference is not a result of different rates of inactiva¬ tion of the peptides in the assay system (Bennett, Bullock, Lowry, McMartin & Peters, 1974).…”

Adenylate cyclase activity in Y1 mouse adrenocortical tumor cells: some properties of the enzyme associated with purified plasma membrane fractions

“…These acidic groups may interact with the basic core at residues 15-18 and impede the binding of ACTH to its receptor, thus providing a possible explanation for the observed differences in affinity between ACTH1-24 and ACTH1-39. Additional evidence for the interaction of these two domains is provided by the observation that ACTH1-39 is more resistant to proteolysis than is ACTH1-24; possibly, amino acids 25-39 protect the natural peptide against proteolytic degradation (30). These observations suggest that further investigations into the relative effects of ACTH 1-24 and ACTH are warranted.…”

Isolated guinea pig adrenocortical cells in vitro: Morphology and steroidogenesis in control and ACTH‐treated cultures

“…ACTH has been shown to be rapidly degraded in vitro by both plasma (Purvis and Sirett, 1968) and adrenocortical cells (Voigt et al, 1974). Its degradation is enhanced by the presence of both (Voigt et al, 1974), is more rapid with denser cell populations, and may be due to proteases released into the medium from damaged (dying) cells (Bennett et al, 1974). Thus it is possible that even though we used maximal dosages of ACTH, significant degradation occurred in our cultures over 2 days, particularly at the high cell densities.…”