Proteinases endoproteinase

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. 

P2.m. The cysteine proteinases are one of four major classes of endoproteinases that possess the ability to degrade intact glomerular basement membranes [144]. All nucleated cells produce cystatin at a stable rate. More than 99% is freely filtered by the glomerulus with little secretion or reabsorption. As a result, it has many of the ideal features for use as a marker of kidney function and eshmate of GFR. Serum cystatin C concentrations demonstrate a good inverse correlation with radionuclide derived measurements of GFR and has been shown in several studies to be superior to creatinine and comparable to iohexol clearances in estimating eGFR [145,146]. 

Both endoproteinases and chemicals can be used to cleave the protein, provided they are active at conditions optimal for complex formation. Table 4.5 provides a list of 13 commercially available proteinases which are useful for protein footprinting. The majority of chemicals reactive towards proteins suffers from the drawback of being reactive towards nucleic acids as well. Hydroxyl radicals produced from H202 in the vicinity of Fe2+ ions are, however, useful for protein surface predictions and for mapping DNA-13 and RNA binding sites (Fig. 4.1423) on proteins. 

A large collection of endoproteinases is available and they are crudely divided into two main groups, the specific and unspecific proteinases, depending on their level of specificity (summarised in Table 4.5). The specific proteinases, in general, yield few cleavages... 

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

On the other hand, this method has some deficiencies. First, it can not identify a single ammo acid as the labeled site in principle. Second, the mapping procedure becomes complicated if more than one labeled site exists between adjacent recognition sites of one proteinase. Third, if the labeled site overlaps with a recognition site of an endoproteinase, the mapping procedures again... 

If aggregation of proteins occurs, change the dialysis buffer to 2 taM Tris-HCl Chelator molecules such as EDTA should be completely removed, because endoproteinase Asp-N is a Zn-proteinase The dialysate can be stored at 4°C Add 2ME for long-term storage. 

Proteinase A Acid endoproteinase (carboxyl proteinase ) glycoprotein 30-45,000 I 2, 1 5 Vacuoles... 

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