Proteases/proteinases cleavage

Proteases (proteinases, peptidases, or proteolytic enzymes) are enzymes that break peptide bonds between amino acids of proteins. The process is called peptide cleavage, a common mechanism of activation or inactivation of enzymes. They use a molecule of water for this, and are thus classified as hydrolases. 

Proteases (endopeptidases or proteinases) commonly used for specific cleavage of proteins are summarised in Table 6.2. Trypsin is almost always used as an enzyme of first choice it is highly specific and stable, has an appropriate pH-optimum and is commercially available in high purity and quality. When the results obtained are ambiguous, or the trypsin cannot be used for any other reason, a different protease can be easily chosen. In all experiments, described here, the trypsin cleavage was applied. 

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. 

Another possible mechanism for kallikrein action in physiology and pathobiology is the activation of proteinase-activated receptors (PARs). PAR is a recently described family of G-protein-coupled receptors with seven transmembrane domains that are stimulated by cleavage of their N-termini by a serine protease rather than by ligand-receptor interaction [184-186]. Four PARs have been identified so far, of which PARI, PAR3,... 

During last decades the domains C-2 symmetry (the dyad rotation symmetry) of low-B palindrome was established in many enzymes (chymotrypsin, trypsin, aspartyl proteinases, HIV-1 protease, carboxypeptidase A, phospholipase A-2 ribonuclease, etc.) (Lumry, 2002 and references therein). It is proposed that the pair domain closure causes constrain of pretransition state complex that activates cleavage or formation of chemical bonds. Thus control of strong bonds by the cooperation of many matrix or knots bonds takes place. As an example, in the active site of carboxypeptidase A the zinc ion is attached to one of the catalytic domains by histidine 69 and glutamine 72 and connected by hystidine 196 to the second domain. Similar structures were found in the chymotrypsin and pepsin active sites where protons are driven under compression of the domains closure. 

Since aiAT represents the archetype for the serpin (serine-proteinase inhibitor) superfamily of proteins, it is possible that similar oxidative or proteolytic mechanisms may function in the inactivation of other serpins that are important in controlling the inflammatory cascade. Some serpins contain a readily oxidised reactive-centre methionine residue (e.g. plasminogen activator-inhibitor [108] and a2-antiplasmin [109]), whilst all serpins (including antithrombin III [110] and protease nexin I [111]) contain an exposed loop which is susceptible to cleavage by proteinases. 

A number of proteinase assays are based on a fluorescein-labeled peptide. Hopkins et al. (1991) used a 14-mer synthesized with and without fluorescein as a label to assay rhinovirus 3c protease. The label did not affect the rate of hydrolysis. The sequence of the peptide containing fluorescein was M-E-A-L-F-Q-G-P-L-Q-Y-K-E(fluorescein)-L, and cleavage occurred after the first six residues. 

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

Among the replicative differences between eukaryotic cells (of the viral host) and the pathogenic virions is that the latter need virus-dependent proteinases to produce the mature infectious virus. That is, HIV protease is an essential enzyme utilized by the virus to process proteins in order to reproduce itself. Retroviruses (and RNA viruses) produce the structural and enzymic proteins as large multidomain polyproteins. The specific cleavage of these various domains into the final products is a posttranslational event. These end-products enable the virion to develop into the actual infectious agent (Kransslich and Wimmer, 1988). 

It is well understood that caspases are critical for the process of apoptosis. Caspases are cysteinyl-containing active center proteases with specificity for protein cleavage after aspartyl residues. Thus the term caspase is derived from (ysteinyl-containing aij artate-specific proteinase. The caspases are responsible for many of the hallmarks of apoptosis defined in Table 18.1, mediated through their cleavage of specific polypeptide substrates. The caspases (e.g., caspase-2, -3, -6, -7, -8, -9, -10, and -12 in the mouse) are not the only proteases involved in PCD, as calpains have also been shown to play... 

The cleavage of the molecular cross-links between cellular proteins by the proteolytic enzyme allows the antigen to return to its normal conformation. For the enzymatic unmasking different proteolytic enzymes can be used such as pronase, protease type XXIV, pepsin, proteinase K, ficin or trypsin. 

---