Abstract
Degradation of CCK-8, CCK-4, and related peptides by a crude synaptosomal fraction of rat brain was investigated by monitoring the tryptophan fluorescence of reaction products after HPLC fractionation. At 20°C, the half disappearance time was 52 min for CCK-8, 35 min for unsulphated CCK-8, 20 min for unsulphated CCK-7, 6 min for Tyr(SO 3H)-Trp-Met-Asp-Phe-NH 2, and 3 min only for CCK-4. Caerulein was much more resistant than CCK-8,and Boc-CCK-4 and Aoc-CCK-4 remained stable for at least 3 h. The apparent K m for CCK-8 and CCK-4 was 40 μM and maximal activity on CCK-8 was observed at pH 7.0. Zn 2+ was strongly inhibitory. The protease inhibitors puromycin and bacitracin, the metal chelator 1,10-phenanthroline, and the sulphydryl blocking agents N-ethylmaleimide and p-chloromercuribenzoate greatly reduced the release of tryptophan from CCK-8. Puromycin inhibition of CCK-8 degradation provoked the accumulation of a CCK-7-like peptide, and that of CCK-4 degradation was of a competitive type ( K i = 2 μM). The CCK-8 degrading activity of brain synaptosomes was present in the cytosol as well as in synaptic membranes.
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