Synthesis and biological properties of p-azidophenylalanine13-β-melanotropin,a potent photoaffinity label for MSH receptors

p-Azidophenylalanine¹³-α-melantropin (Pap¹³]-α-MSH) was synthesized in homogeneous solution by the fragment condensation method, and its biological activity was determined in three different assay systems. The pigment-dispersing activity relative to α-MSH was 65%, measured with melanophores of Rana pipiens or of Xenopus laevis tadpoles. The tyrosinase-stimulating activity was 50%, determined with cultured mouse melanoma cells. UV. irradiation of solutions containing ≤10^?4_MPap¹³]-α-MSH at 338 nm (intensity: 10^?3 W · cm^?2) led to complete photolysis of the photolabel within <20 min. Under these conditions Pap¹³]-α-MSH was covalently inserted into MSH-receptors which produced a longlasting pigment dispersion in Xenopus melanphores (see 3]). The extent of this prolonged stimulation depended on the hormone concentration used during photolysis. 1.8·10^?9_M Pap¹³]-α-MSH which produced a full initial response failed to prolong the effect, whereas 1.2·10^?8_M hormone caused irreversible stimulation. It appears that only about 10% of the initially occupied receptors were covalently labelled because the log dose response curve was shifted to ～ 10x higher concentration after a 200 min wash period: EC₅₀ immediately after photolysis was 6 · 10^?10_M; after 200 min EC₅₀ increased to ～8·10^?9_M.