ABSTRACT
The photolabile peptide, L-methionyl-L-tyrosyl-p-azido-L-phenylalaninamide, was synthesized by solution methods. This peptide, as well as the analogous species containing tritiated methionine, were found to bind reversibly and specifically, in the dark, to bovine neurophysin II. The dissociation constant, stoichiometry, and pH-dependence of this noncovalent interaction are typical of those properties for hormone (oxytocin) and hormone-like ligand binding to neurophysin II. Under photolytic conditions, methionyl-tyrosyl-p-azidophenylalaninamide causes irreversible inhibition of the noncovalent ligand binding activity of neurophysin II. This inactivation was achieved to the extent of about 90%. Both the dark and light (photolytic) interactions of the photolabile peptide with neurophysin II indicate its reaction at the hormone binding site of the protein and thus its potential use to identify amino acid residues at this site by covalent photoaffinity labelling.
Subject(s)
Neurophysins , Receptors, Cell Surface , Animals , Cattle , Peptides/chemical synthesis , Phenylalanine/analogs & derivatives , PhotolysisSubject(s)
Azides , Phenylalanine/analogs & derivatives , Endoplasmic Reticulum , Glutathione , Humans , Indicators and Reagents , PhotochemistryABSTRACT
The syntheses are described of p-guanidino-L-phenylalanine and some of its derivatives. alpha-N-(p-Toluenesulphonyl)-p-guanidino-L-phenylalanine methyl ester is an excellent substrate of bovine trypsin (EC 3.4.21.4) (Km 57 micron; kcat. 320s-1 at pH 7.4-8.0) and a very poor substrate of human thrombin (EC 3.4.21.5) (Km 190 micron, kcat. 0.2s-1) and bovine chymotrypsin (EC 3.4.21.1). The ester inhibits thrombin clotting activity. It also inhibits the amidase and esterase activities of human thrombin, this inhibition being of the mixed type. The inhibition constant, K1, of the order of 1 micron, increases with increasing inhibitor concentration. This suggests that the enzyme binds the inhibitor at multiple sites. The importance of the residue at the P1 position [notation of Berger & Schechter (1970) Philos. Trans. R. Soc. London Ser. B 257, 249-264] in determining the selectivity of a substrate or quasi-substrate among trypsin-like enzymes is borne out. p-Guanidino-L-phenylalanine may have a use in the synthesis of selective peptide inhibitors of thrombin.
Subject(s)
Arginine/analogs & derivatives , Thrombin/metabolism , Trypsin/metabolism , Animals , Cattle , Guanidines/chemical synthesis , Guanidines/metabolism , Humans , Kinetics , Phenylalanine/analogs & derivatives , Phenylalanine/chemical synthesis , Phenylalanine/metabolism , Thrombin/antagonists & inhibitors , Tosyl Compounds/metabolismSubject(s)
Histidine , Peptides/chemical synthesis , Acylation , Amino Acid Sequence , Chemical Phenomena , Chemistry , EstersSubject(s)
Ethers, Cyclic , Peptides/chemical synthesis , Tryptophan , Acylation , Catalysis , Chlorobenzenes , Fluorides , KetonesSubject(s)
Gastrointestinal Hormones/chemical synthesis , Peptides/chemical synthesis , Amino Acid Sequence , Animals , Biodegradation, Environmental , Chromatography, Paper , Chromatography, Thin Layer , Chymotrypsin , Countercurrent Distribution , Dogs , Electrophoresis, Paper , Gastrointestinal Hormones/isolation & purification , Peptides/isolation & purification , SwineABSTRACT
An octacosapeptide with the entire sequence proposed for the newly isolated vasoactive intestinal polypeptide was synthesized and had the expected biological properties. Synthetic peptides corresponding to the C-terminal hendeca-, tetradeca-, pentadeca-, and docosapeptide sequences of the vasoactive intestinal polypeptide showed activities that increased with increasing chain length.
