Fragmentation of proteins by S. aureus strain V8 protease

Abstract

Staphylococcus aureus strain V8 protease is a serine endopeptidase which cleaves peptide bonds at the carboxyl side of Glu and Asp. Specific cleavage at Glu has previously been achieved in ammonium bicarbonate whereas in sodium phosphate cleavage at both Glu and Asp was observed. However, it is shown here that bicarbonate does not restrict the specificity to Glu‐X bonds, it simply inhibits the enzyme. The degradation of a mixture of oxidized insulin and glucagon proceeds similarly in the two buffers, although faster in phosphate.

Diffuse Pharmacophoric Domains of Vasoactive Intestinal Peptide (VIP) and Further Insights into the Interaction of VIP with the N-terminal Ectodomain of Human VPAC1 Receptor by Photoaffinity Labeling with [Bpa6]-VIP

“…6). This model is also consistent with the idea that crucial basic residues present in the central part of VIP including Arg 14 , Lys 15 , and Lys 21 (9) may interact with the important acidic residues in the electronegative groove in the N-terminal ectodomain of the receptor (7). Development of new photoaffinity probes with Bpa substitutions at other positions between Phe 6 and Tyr 22 of VIP should be done to further strengthen this model.…”

“…3) obtained after photoaffinity labeling of the wild-type receptor was digested with endopeptidase Glu-C (Fig. 3), which under our experimental conditions cleaves proteins at the C-terminal side of Glu residues with the notable exception of the Glu-Pro sequence (15). Endopeptidase Glu-C treatment of the 30-kDa band generated a 25-kDa band representing the receptor segment 67-108 that contains two N-glycosylation sites on Asn 69 and Asn 100 (Table I).…”

“…Endopeptidase Glu-C digestion of proteins in this material was obtained by incubation for 6 h at 25°C in 25 mM ammonium carbonate, 0.01% SDS, 5% acetonitrile, pH 7.8, as described (15). Trypsin cleavage of proteins was performed for 2 h at 37°C in 0.1 M MOPS buffer, pH 7.0.…”

Photoaffinity Labeling Demonstrates Physical Contact between Vasoactive Intestinal Peptide and the N-terminal Ectodomain of the Human VPAC1 Receptor

“…2) were digested with endoproteinase V8, which under our experimental conditions cleaves proteins at the C-terminal side of Glu residues with the notable exception of Glu-Pro sequence (21). As expected, considering the presence of Glu residues in the Trp 67 -Met 137 receptor fragment, this cleavage further decreased the apparent molecular mass of the labeled band (Fig.…”

“…V8 endoproteinase digestion of proteins in this material was obtained by incubation for 6 h at 25°C in 25 mM ammonium bicarbonate, 0.01% SDS, 5% acetonitrile, pH 7.8, as described previously (21).…”