| dc.description.abstract | We synthesized short chromogenic peptidyl-Arg-p-nitroanilides containing either (Gal beta)Ser or (Glc alpha,beta)Tyr at P-2 or P-3 sites as well as O-acetylated sugar moieties and studied their hydrolysis by bovine trypsin, papain, human tissue kallikrein and rat tonin. for comparison, the susceptibility to these enzymes of Acetyl-X-Arg-pNa and Acetyl-X-Phe-Arg-pNa series, in which X was Ala, Phe, Gin and Asn were examined. We also synthesized internally quenched fluorescent peptides with the amino acid sequence Phe(8)-His-Leu-Val-Ile-His-Asn(14) of hu man angiotensinogen, in which [GlcNAc beta]Asn was introduced before Phe(8) and/or after His(13) and ortho-aminobenzoic acid (Abz) and N-[2-, 4-dinitrophenyl]-ethylenediamine (EDDnp) were attached at N- and C-terminal ends as a donor/receptor fluorescent pair. These peptides were examined as substrates for human renin, human cathepsin D and porcine pepsin. the chromogenic substrates with hydrophilic sugar moiety increased their susceptibility to trypsin, tissue kallikrein and rat tonin. for papain, the effect of sugar depends on its position in the substrate, namely, at P-3 it is unfavorable, in contrast to the P-2 position that resulted in increasing affinity, as demonstrated by the higher inhibitory activity of Ac-(Gal beta)Ser-Arg-pNa in comparison to Ac-Ser-Arg-pNa, and by the hydrolysis of Ac-(Glc alpha,beta)Tyr-Arg-pNa. On the other hand, the acetylation of sugar hydroxyl groups improved hydrolysis of the susceptible peptides to all enzymes, except tonin. the P-4', glycosylated peptide [Abz-F-H-L-V-I-H-(GlcNAc beta)N-E-EDDnp], that corresponds to one of the natural glycosylation sites of angiotensinogen, was shown to be the only glycosylated substrate susceptible to human renin, and was hydrolysed with lower K-m and higher k(cat) values than the same peptide without the sugar moiety. Human cathepsin D and porcine pepsin are more tolerant to substrate glycosylation, hydrolysing both the P-4' and P-4 glycosylated substrates. | |
