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 important factor in head regeneration. Here we report the solubilization and purification of one head activator receptor (Kd approximately 1 nM) from a multiheaded mutant of Chlorohydra viridissima using HA affinity chromatography. Functional solubilization of the HA receptor from hydra membranes was best performed with Triton X-100 or Chaps. The addition of salt or urea and the protein concentration were important parameters 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 peptide. Ligand binding was preserved in the eluate from the HA Sepharose, and a 200-kDa protein could be photoaffinity labeled. The 200-kDa protein 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.
Franke
I
I
Zentrum für Molekulare Neurobiologie, Universität Hamburg, Germany.
Buck
F
F
Hampe
W
W
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D006031:Glycosylation; D006801:Humans; D006829:Hydra / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D009479:Neuropeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives; D018000:Receptors, Peptide / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility
D009479:Neuropeptides; D010446:Peptide Fragments; D018000:Receptors, Peptide; C069362:head activator receptor, Hydra; C032522:head activator peptide; D011761:Pyrrolidonecarboxylic Acid