Staphylococcus aureus V8 protease

Proteolysis of peptides and proteins by enzymes occurs in a selective or nonselective manner. Chymotrypsin, trypsin, lysyl endopeptidase, Staphylococcus aureus V8 protease, and en-dopeptidase Asp-N are frequently listed as selective enzymes, whereas thermolysin, pepsin, subtilisin, and elastase belong to the nonselective enzymes, although thermolysin preferentially cleaves peptide bonds before hydrophobic residues. 

Proteolytic modification has special importance for the improvement of solubility of proteins. This effect becomes significant even after very limited proteolysis. Hydrolysis of casein to DH of 2 and 6.7% with Staphylococcus aureus V8 protease increased the isoelectric solubility to 25 and 50%, respectively (Chobert et al., 1988a). However, it should be noted that the solubility profiles were not identical, due to a shift of the isoelectric point of the modified proteins. Solubility of a protein hydrolysate depends on the enzyme used (Adler-Nissen, 1986a). Protamex (a Bacillus proteinase complex) hydrolysates of sodium caseinate (DH 9 and 15%) displayed 85-90% solubility between pH 4 and 5 (Slattery and FitzGerald, 1998). 

Chobert, J.-M., Sitohy, M.Z., and Whitaker, J.R. 1988a. Solubility and emulsifying properties of caseins modified enzymatically by Staphylococcus aureus V8 protease. J. Agric. Food Chem. 36, 220-224. 

Staphylococcus aureus V8 protease deaves proteins specifically after glutamate residues, provided that the following residue is not proline, or a further glutamate or aspartate. In ammonium bicarbonate buffer at pH 7.8, the enzyme is very specific for glutamate in Na-phosphate buffer at the same pH it also cleaves adjacent to aspartate. V8 protease is added at a ratio of 1-2% (w/w) to the protein substrate. Reaction can be stopped by freezing, or by the addition of PMSF or a2-rtiacroglobu-lin. 

Achromobacter protease a-Chymotrypsin, subtilisins Carboxypeptidase Y Elastase Trypsin, Streptomyces griseus protease Staphylococcus aureus V8 protease Serine protease Serine protease Serine protease Serine protease Serine protease Serine protease -Lys-X -Trp(Tyr,Phe,Leu,Met)-X nonspecific -Ala(Ser,Met,Phe)-X -Arg(Lys)-X -Glu(Asp)-X... 

Streptavidin [16] proteases (pepsin, papain, staphylococcus aureus V8 protease) [17] antibodies and antibody-binding proteins such as Protein A, Protein G or Protein M [18] lectins such as concanavalin and jacaUn [19]. 

The band pattern after trypsin. Staphylococcus aureus V8 protease or N-chloro-succinimide digestion suggested extensive homology but also showed some differences in primar structure. 

Peptide mapping with Staphylococcus aureus V8 protease [which cleaves at the carbonyl-side of aspartic-and glutamic acid) of the isolated 27 kD and 25 kD polypeptides showed significant differences in the degradation products. 

The enzyme solution (in 62.5 mM Tris-HCl buffer, pH 6.8, containing 10% glycerol, 0.1 % SDS and 0.001 % bromophenol blue) is layered over the gel. Optimum enzyme concentrations are 2 ng papain or 8 ng Staphylococcus aureus protease V8 (Miles) per mm- of slot surface. It is better to dilute the enzyme solution so that the overlay produces a layer of about 3 mm high, in order to avoid possible artefacts if the gel is not perfectly horizontal. 

V8 protease, EC 3.4.21.19, Endoproteinase Glu-C, protease I, protease type XVII-B, an extracellular endopeptidase from the V8 strain of Staphylococcus aureus. It cleaves peptide bonds on the C-terminal side of glutamic acid and, to a lesser extent, of aspartic acid. The X-ray crystallographic structure of V8 protease was described in 2004 [G. R. Drapeau et al., J. Biol. Chem. 1972, 247, 6720 L. Prasad et al, Acfa Crystallogr. 2004, D60, 256]. 

The V8 protease of Staphylococcus aureus (MW 12 kd) is active at a pH between 3.5 and 9.5 and develops maximal activity at pH 4.0 and 7.8. At pH 4.0, the protease partially precipitates. In phosphate buffer, V8 protease cleaves peptide bindings on the carboxyl terminal side of aspartate or glutamate residues. In 50 mM ammonium bicarbonate buffer pH 7.8 or ammonium acetate buffer pH 4.0, on the other hand, the enzyme cleaves only behind glutamate residues. Below 40° C, the protease does not exhibit any self-digestion. Its watery solution can be frozen and thawed without activity loss. Divalent cations or EDTA have no effect on the enzyme activity. The enzyme also still works in 0.5% SDS. Diisopropyl fluorophosphate inhibits V8 protease. 

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