PURIFICATION OF A HEAD-ACTIVATOR RECEPTOR FROM HYDRA

Head activator (HA) is a neuropeptide conserved from hydra to humans. It acts in the development of neuronal cells and is, in hydra, an impo rtant factor in head regeneration. Here we report the solubilization a nd purification of one head activator receptor (K-d approximate to 1 n M) from a multiheaded mutant of Chlorohydra viridissima using HA affin ity chromatography. Functional solubilization of the HA receptor from hydra membranes was best performed with Triton X-100 or Chaps. The add ition of salt or urea and the protein concentration were important par ameters in determining the yield of solubilized receptor. For affinity chromatography HA was coupled to Sepharose. The length of the spacer was optimized with respect to binding of the solubilized HA receptor. After rigorous washing a 200-kDa protein was eluted from HA Sepharose but not from control Sepharoses coupled to bradykinin or without pepti de. Ligand binding was preserved in the eluate from the HA Sepharose, and a 200-kDa protein could be photoaffinity labeled. The 200-kDa prot ein was shown to be glycosylated mainly of the N-linked type. By Edman degradation of the purified protein sequence information was obtained for the N-terminus and after protease digestion for several internal peptides.