Activation Of Other Proteases Research Articles

Domain structure of calpain: mapping the binding site for calpastatin.

The peptide EKLGERDDTIPPEYRELLEKKTGV was synthesized to mimic the central consensus sequence of calpastatin, the specific, endogenous inhibitor of the calpains (EC 3.4.22.17). The peptide competitively inhibits hydrolysis of casein by either micro- or milli-calpain but does not affect the activity of other proteases. This inhibitory peptide was preferentially cross-linked to milli-calpain in the presence of calcium using the heterobifunctional cross-linking reagent m-maleimidobenzoyl-N-hydroxysuccinimide ester. Cross-linking of the peptide was blocked by calpastatin. The site of cross-linking for the peptide within milli-calpain was localized using random chemical cleavage of the enzyme-peptide complex at cysteine residues. Calpain fragments were identified as amino-terminal fragments through reactivity with a peptide-specific antiserum or as non-amino-terminal fragments through incorporation of 14C from 14CN. Analysis of the control and cross-linked fragments, from experiments using both milli-calpain and micro-calpain, maps the chemical cross-linking site to cysteine-497 and localizes the binding site for the calpastatin-like peptide to this highly conserved region of domain III of calpains catalytic subunit.

A Coomassie Brilliant Blue G-250-Based Colorimetric Assay for Measuring Activity of Calpain and Other Proteases

The activity of cytosolic calcium-dependent neutral protease (calpain) is commonly measured using casein as a substrate. A novel, modified caseinolysis assay is now developed. It involves incubation of calpain with substrate casein, followed by removal of an aliquot to which Coomassie brilliant blue G-250 dye reagent is added. The assay is based on the observation that the dye interacts only with protein but not the proteolytic products (small peptides and amino acids). Unlike the existing caseinolysis assay, this novel assay does not require separation of substrate from products and measurement is done in the visible range (595 nm). It Is also more sensitive than existing colorimetric assays which use dye-conjugated protein substrates. The assay can also be used to measure the activity of other proteases such as trypsin and papain.

Salivary histatin as an inhibitor of a protease produced by the oral bacterium Bacteroides gingivalis

We examined the effect of histatin 5 from human parotid saliva on various proteases. Histatin 5 strongly inhibited a trypsin-like protease produced by Bacteroides gingivalis with an IC50 value of 55 nM. Clostripain was also inhibited (IC50=800 nM). Activities of other proteases were not affected significantly. Because B. gingivalis is a suspected periodontal pathogen and its proteolytic enzymes have been considered to be associated with periodontal tissue destruction, it is suggested that salivary histatins play a role as a preventive against periodontal disease.