Photoaffinity labeling the substance P receptor using a derivative of substance P containing p-benzoylphenylalanine
| Autor: | White Cf, Cerpa R, Susan E. Leeman, Norman D. Boyd, Kaiser Et |
|---|---|
| Rok vydání: | 1991 |
| Předmět: |
GTP'
Photochemistry Phenylalanine Tachykinin peptides Submandibular Gland In Vitro Techniques Substance P Binding Competitive Biochemistry chemistry.chemical_compound Peptide synthesis Animals Binding site Gel electrophoresis chemistry.chemical_classification Guanylyl Imidodiphosphate Photoaffinity labeling Cell Membrane Affinity Labels Rats Inbred Strains Receptors Neurokinin-1 Rats Receptors Neurotransmitter Amino acid Dissociation constant chemistry |
| Zdroj: | Biochemistry. 30:336-342 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi00216a005 |
| Popis: | A novel photoreactive substance P (SP) analogue has been synthesized by solid-phase peptide synthesis methodology to incorporate the amino acid p-benzoyl-L-phenylalanine [L-Phe(pBz)] in place of the Phe8 residue of SP. [Phe8(pBz)]SP was equipotent with SP in competing for SP binding sites on rat submaxillary gland membranes and had potent sialagogic activity in vivo. In the absence of light, the 125I-labeled Bolton-Hunter conjugate of [Phe8(pBz)]SP bound in a saturable and reversible manner to an apparently homogeneous class of binding sites (Bmax = 0.2 pmol/mg of membrane protein) with an affinity KD = 0.4 nM. The binding of 125I-[Phe8(pBz)]SP was inhibited competitively by various tachykinin peptides and analogues with the appropriate specificity for SP/NK-1 receptors. Upon photolysis, up to 70% of the specifically bound 125I-[Phe8(pBz)]SP underwent covalent linkage to two polypeptides of Mr = 53,000 and 46,000, identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Quantitative analysis of the inhibitory effects of SP and related peptides on 125I-[Phe8(pBz)]SP photoincorporation indicated that the binding sites of the two photolabeled polypeptides have the same peptide specificity, namely, that typical of NK-1-type SP receptors. In addition, the labeling of the two polypeptides was equally sensitive to inhibition by guanyl-5'-yl imidodiphosphate, a nonhydrolyzable analogue of GTP. Further information on the relationship between the two labeled SP binding sites was provided by enzymatic digestion studies: the Mr = 46,000 polypeptide contains N-linked carbohydrates and is derived most likely from the higher molecular weight species by proteolytic nicking.(ABSTRACT TRUNCATED AT 250 WORDS) |
| Databáze: | OpenAIRE |
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