Fate of corticotrophins in an isolated adrenal-cell bioassay and decrease of peptide breakdown by cell purification.
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.
D000311:Adrenal Glands / Q000166:cytology / Q000378:metabolism*; D000324:Adrenocorticotropic Hormone / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001681:Biological Assay; D002417:Cattle; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D002852:Chromatography, Ion Exchange; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D011956:Receptors, Cell Surface; D012710:Serum Albumin, Bovine; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013997:Time Factors; D014316:Tritium; D014357:Trypsin; D014443:Tyrosine
D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D014316:Tritium; D012710:Serum Albumin, Bovine; D014443:Tyrosine; D000324:Adrenocorticotropic Hormone; D014357:Trypsin