Photoaffinity labeling of the hormone binding site of neurophysin.
The chemical structure of the hormone binding region of the neurophysins has been investigated by photoaffinity labeling with the photolabile tripeptide, L-[methyl-3H]Met-L-Tyr-p-azido-L-Phe amide. Photolysis of the photoaffinity tripeptide in the presence of bovine neurophysin I and II and a human neurophysin II led to approximately equal extents of covalent incorporation of radioactivity into protein. Photolabeled bovine neurophysin II was fractionated into binding site derivatized protein and nonbinding site derivatized protein by affinity chromatography, with results of amino acid and radiolabel analysis of the hormone binding site blocked protein indicating that 1 mol of tripeptide was covalently incorporated/mol of protein. Tyrosine 49 was the only protein amino acid modified in the binding site photolabeling reaction as assessed by peptide mapping of the performic acid oxidized and trypsin-digested photolabeled protein using reverse phase high performance liquid chromatography. Modification of the single neurophysin tyrosine also was found by amino acid analysis of performic acid oxidized photolabeled bovine neurophysin II. The covalent bond formed in neurophysin upon photolysis was cleaved by either exhaustive acid hydrolysis or reduction-carboxymethylation without loss of the protein amino acid residues and by performic acid oxidation with loss of both protein and tripeptide tyrosine residues. These overall data indicate that tyrosine 49 is the probable site for specific covalent attachment of the photoaffinity tripeptide. Assuming that the tripeptide binding site is the high affinity hormone binding site reported for the neurophysins, this conclusion argues that tyrosine 49 is close to or within this site.
D000345:Affinity Labels / Q000494:pharmacology*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001386:Azides; D001665:Binding Sites; D002417:Cattle; D009481:Neurophysins / Q000302:isolation & purification / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology*; D010446:Peptide Fragments / Q000032:analysis; D010902:Pituitary Gland / Q000378:metabolism*; D010903:Pituitary Gland, Anterior / Q000378:metabolism*; D014357:Trypsin
D000345:Affinity Labels; D000596:Amino Acids; D001386:Azides; D009481:Neurophysins; D009842:Oligopeptides; D010446:Peptide Fragments; C033690:methylmethionyl-tyrosyl-4-azido-phenylalanine amide; D014357:Trypsin