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k ‐Binding and Degradation of [ 3 H]Dynorphin A (1–8) and [ 3 H]Dynorphin A (1–9) in Suspensions of Guinea Pig Brain Membranes

1985; Wiley; Volume: 45; Issue: 4 Linguagem: Inglês

10.1111/j.1471-4159.1985.tb05520.x

1471-4159

Maureen Gc Gillan, Linda E. Robson, Alexander T. McKnight, H. W. Kosterlitz,

Pain Mechanisms and Treatments

Abstract: Following incubation of [ 3 H]dynorphin A (1–8) and [ 3 H]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°C and at 0°C. Bestatin and captopril reduce degradation at 0°C but for a similar degree of protection at 25°C argininecontaining dipeptides are also required. The effects of these peptidase inhibitors on the degradation profiles indicate that [ 3 H]dynorphin A (1–8) has three main sites of cleavage: the Tyr 1 ‐Gly 2 , Arg 6 ‐Arg 7 , and Leu 5 ‐Arg 6 bonds. With [ 3 H]dynorphin A (1–9) as substrate the Arg 7 ‐Ile 8 and Ile 8 ‐Arg 9 bonds are also liable to cleavage. In binding assays, in contrast to the effects of peptidase inhibitors on the degradation of unbound [ 3 H]dynorphin A (1–8) and [ 3 H]dynorphin A (1–9), bestatin and captopril have little effect on the binding characteristics of the tritiated dynorphin A fragments at the k‐site at 0°C. However, at 25°C binding is low in the absence of peptidase inhibitors. When binding at μ‐ and δ‐sites is prevented, the maximal binding capacities of [ 3 H]dynorphin A (1–8), [ 3 H]dynorphin A (1–9), and [ 3 H](–)‐bremazocine at the k‐site are similar; [ 3 H]dynorphin A (1–9) has 5–10 times higher affinity for the k‐site than [ 3 H]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.