title: Kappa-binding and degradation of [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9) in suspensions of guinea pig brain membranes.
abstract: Following incubation of [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9) with suspensions of guinea pig brain membranes, analysis of the supernatants by HPLC has shown that both peptides are degraded at 25 degrees C and at 0 degrees C. Bestatin and captopril reduce degradation at 0 degrees C but for a similar degree of protection at 25 degrees C arginine-containing dipeptides are also required. The effects of these peptidase inhibitors on the degradation profiles indicate that [3H]dynorphin A (1-8) has three main sites of cleavage: the Tyr1-Gly2, Arg6-Arg7, and Leu5-Arg6 bonds. With [3H]dynorphin A (1-9) as substrate the Arg7-Ile8 and Ile8-Arg9 bonds are also liable to cleavage. In binding assays, in contrast to the effects of peptidase inhibitors on the degradation of unbound [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9), bestatin and captopril have little effect on the binding characteristics of the tritiated dynorphin A fragments at the kappa-site at 0 degrees C. However, at 25 degrees C binding is low in the absence of peptidase inhibitors. When binding at mu- and delta-sites is prevented, the maximal binding capacities of [3H]dynorphin A (1-8), [3H]dynorphin A (1-9), and [3H](-)-bremazocine at the kappa-site are similar; [3H]dynorphin A (1-9) has 5-10 times higher affinity for the kappa-site than [3H]dynorphin A (1-8). Comparison of the effects of peptidase inhibitors on unbound dynorphin A fragments with their effects in binding assays suggests that the bound peptides are protected from the action of peptidases.
authors.forename: M G
authors.initials: MG
authors.lastname: Gillan
authors.forename: L E
authors.initials: LE
authors.lastname: Robson
authors.forename: A T
authors.initials: AT
authors.lastname: McKnight
authors.forename: H W
authors.initials: HW
authors.lastname: Kosterlitz
mesh_terms: D000818:Animals; D001575:Benzomorphans / Q000378:metabolism; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D004399:Dynorphins / Q000378:metabolism*; D006168:Guinea Pigs; D007700:Kinetics; D008566:Membranes / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D011957:Receptors, Opioid / Q000378:metabolism*; D017473:Receptors, Opioid, kappa; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001575:Benzomorphans; D010446:Peptide Fragments; D011480:Protease Inhibitors; D011957:Receptors, Opioid; D017473:Receptors, Opioid, kappa; D004399:Dynorphins; C026770:dynorphin (1-8); C040238:dynorphin A (1-9); C027248:bremazocine