Endoproteinase Glu

Prabakaran, S., Tepp, W. and DasGupta, B.R., Botulinum neurotoxin types B and E purification, limited proteolysis by endoproteinase Glu-C and pepsin, and comparison of their identified cleaved sites relative to the three-dimensional structure of t q)e A neurotoxin, Toxicon, 39, 1515-1531, 2001. 

Glutamyl endopeptidase [EC 3.4.21.19] (also known as staphylococcal serine proteinase, V8 proteinase, protease V8, and endoproteinase Glu-C), a member of the peptidase family S2B, catalyzes the hydrolysis of Asp-Xaa and Glu-Xaa peptide bonds. In appropriate buffers, the specificity of the bond cleavage is restricted to Glu-Xaa. Peptide bonds involving bulky side chains of hydrophobic aminoacyl residues are hydrolyzed at a lower rate. 

TPCK-treated trypsin, TLCK-treated chymotrypsin, and endoproteinase-Glu-C (Staphlococcal V8 protease) are all obtainable from Sigma (Poole, UK)... 

The introduction of a glutamic acid (E bold), after each RADI 6 repeat was made to allow cleavage by endoproteinase Glu-C, which cleaves C-terminal to glutamate. A cellulose binding domain (CBD) (Table 1) was selected as the affinity tag, as CBDs bind strongly and specifically to cellulose which is a relatively cheap and abundant purification matrix. The main problem encountered was the low level of peptide recovered. Theoretically, 1 g of fusion protein should give 267mg of peptide, but only 10.1 mg of peptide was recovered after RP-HPLC. 

Figure 1. Peptide maps of PVDF-bound soy bean trypsin inhibitor (190 pmol) digested with endoproteinase Glu-C in the presence of 50 pi of A) 1% RTX-IciO/lOO mM Tris pH, 8.0, B) 1% octylglucopyranoside/100 mM Tris pH, 8.0, and C) 1% heptylglucopyranoside/100 mM Tris, pH 8.0 as described in Materials and Methods. Digestion in the presence of 1% decylglucopyranoside was not analyzed by HPLC due to the presence of micelles.

In summary, octylglucopyranoside can be substituted for RTX-100 with only a slight decrease in peptide recoveries. Decylglucopyranoside may be used in place of RTX-100 when acetonitrile is added to the digestion buffer, but is not useful for endoproteinase Glu-C. Heptyl and nonyl glucopyranosides are not suitable alternatives as there was no peptides recover with the former and micelle formation occurred with the latter. 

GSE is potentially less costly and more stable in comparison to V8 protease. The commercial detergent Alcalase is a rich source of GSE, from which it can be purified. GSE has the same Pi specificity as the protease from the V8 strain of Staphylococcus aurens (V8 protease, endoproteinase Glu-C).t ... 

Fig. 2. Biochemical probes for NM conversion in vitro. The N and M regions of Sup35 have distinct sensitivities to the proteases chymotrypsin (CHY) and endoproteinase Glu-C (V8) CHY sites are exclusively found in N, while V8 sites are limited to M. V8 Anti-Sup35 Western blot of NM alone (—) or following digestion with V8 (arrow). NM was removed from an assembly reaction over a time course, and each sample was then treated with protease for the same amount of time. CHY Anti-Sup35 Western blot of NM alone (—) or following digestion with CHY (arrow) as for V8. SDS Anti-Sup35 Western blot of NM following incubation in 2% SDS at either 100°C or 25°C for 10 min. Samples were taken over the same time course as for V8 and CHY.

Glutamyl endopeptidase. Staphylococcal serine proteinase. V8 proteinase. Protease V8. Endoproteinase Glu-C. 3.4.21.19 Preferential cleavage Asp-I-Xaa, Glu-I-Xaa. 

Endoproteinase Glu-C Origin Staphylococcus aureus strain V8 Fluka Lab... 

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]. 

A protein of the sequence Phe-Tyr-Ser-Met-Asp-Ile-Leu-Lys-Trp-Val-Ala-Met-Glu-Val-Asp-Tyr-Gly-Pro-Arg-Thr-Asn-Pro-Glu-Ala-Leu-Phe-Ile-Tyr-Arg-Thr-Ser-Met-Tyr-Trp-Gln-Leu-Met is treated first with endoproteinase Glu-C and then with trypsin. How many peptide fragments will be generated by this treatment, and what will be their molecular mass ... 

Reference method A reference method has been established that involves cleavage of hemoglobin into peptides by the endoproteinase Glu-C, followed by separation and quantitation of the hexapeptides by LC-electrospray ionization mass spectrometry or LC-capillary electrophoresis. 

Enzyme stock preparations To make 1 ml trypsin, chymotrypsin, or endoproteinase Glu C stock solution, dissolve 1 mg enzyme in 1 ml UHQ water. Distribute the solution in 50-//1 portions in 0.5-ml Eppendorf tubes, lyophilize in a vacuum centrifuge, and store at —20°C. To make 0.2 ml endoproteinase Asp-N stock solution, dissolve 10 //g enzyme in 200 //I UHQ water. Distribute in 10-/xl portions, lyophilize, and store as above. To make 100 //I carboxypeptidase Y, A, or M stock solution, dissolve 100 //g enzyme in 100 //I UHQ water. Distribute in 10-(A portions, lyophilize, and store as above. 

Also suited for the specific enzymatic hydrolysis of peptide chains is the endoproteinase Glu-C from Staphylococcus aureus V8. It cleaves Glu-X bonds (ammonium carbonate buffer pH 7.8 or ammonium acetate buffer pH 4.0) as well as Glu-X plus Asp-X bonds (phosphate buffer pH 7.8). The most important chemical method for selective cleavage uses cyanogen bromide (BrCN) to attack Met-X-hnkages (Reaction 1.86). Hydrolysis of proteins with strong acids reveals a difference in the rates of hydrolysis of peptide bonds depending on the adjacent amino acid side chain. Bonds involving amino groups of serine and threonine are particularly susceptible to hydrolysis. This effect is due to... 

With plasmin, only 18 h digest samples were analyzed. With Endoproteinase Glu-C, only 30 minute digest samples were analyzed. 

In a global analysis of histone H2A and H2B variants derived from Jurkat cells by MS, nine histone H2A and 11 histone H2B subtypes were identified, some of which had only been postulated before at the DNA level [391]. This was achieved by combining MS with HPLC separations and enzymatic proteolysis using endoproteinase Glu-C, endoproteinase Arg-C, and trypsin. With regard to modification status, e.g., the two main H2A variants, H2A.o and H2A.C, as well as H2A.1 were foimd either acetylated at Lys-5 or phos-phorylated at Ser-1. For the replacement histone H2A.z, acetylation at Lys-4 and Lys-7 was observed. The main histone H2B variant, H2B.a, was found acetylated at Lys-12, -15, and -20. In an other study, a direct, top-down MS approach was applied to identify H2B isoforms isolated from asynchronous HeLa cells using ESI-FT-ICR MS and BCD for MS/MS analysis of intact protein molecular ions (Fig. 22) [59]. These cells were foimd to express H2B.A, H2B.B, H2B.E, H2B.F, H2B.J, H2B.K, H2B.Q, and H2B.T. 

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