Protease sensitivity assays

Kinase substrates can become resistant to the actions of proteases due to their phosphorylations. Thus, the fluorescence quench assays (described in Chapter 2 covering protease assays) can be used to measure kinase activity. The assays can be viewed as coupled because they require a second enzyme to convert a product or substrate into a detectable signal. With kinase assays, the formation of phosphopeptide inhibits the protease action on the peptide and the signal remains quenched and therefore decreased (Rodems et al., 2002). Inhibiting the kinase results in increases in protease sensitivity and in signal. 

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Plumper E, Bradley PJ, Johnson PJ (2000) Competition and protease sensitivity assays provide evidence for the existence of a hydrogenosomal protein import machinery in Trichomonas vaginalis. Mol Biochem Parasitol 106 11-20 Pool MR (2005) Signal recognition particles in chloroplasts, bacteria, yeast and mammals (review). Mol Membr Biol 22 3-15... 

Due to their response mechanism the polyion-selective electrodes are not sensitive to the small fragments of polyionic macromolecules. Thus, if an enzyme cleaves the polyionic molecule these sensors can be used for detection of enzyme activity. Polycation protamine is rich in arginine residues that make it a suitable substrate for protease-sensitive electrochemical assays. Real-time detection of trypsine activity was demonstrated with the protamine-selective electrode as a detector [38],... 

Proteins with N-terminal signal peptides or C-terminal membrane anchor regions and proteins where the N terminus contributes to the biological properties of the protein require special attention to achieve a satisfactory fusion A number of variants may need to be made and assayed. A protease-sensitive site may be designed into the amino acid sequence at the fusion point for eventual separation of the desired gene product from the fusion. 

A succinylated casein derivative that has nearly all its amines blocked can be used as a substrate in protease assays (Hatakeyama etal., 1992). As the casein is degraded by a protease, free amines are created from a-chain cleavage and release of a-amino groups. The creation of amines can be monitored by an amine detection reagent such as trinitrobenzene sulfonic acid (TNBS Section 4.3). The procedure forms the basis for a highly sensitive assay for protease activity. 

A protease-free assay using Suc-Ala-Ala-Pro-Phe-DFA (DFA is 2,4-difluoroani-line) was also developed, but its sensitivity at 246 nm is low (<0.006 absorbance unity) for a typical peptide concentration of 21 pmol L-1 [56], The same problem limits the applicability of the assay reported by Fischer and coworkers based on the pH sensitivity of the secondary amide bonds. A pH jump (i.e. from 2.0 to 7.3) led to a slight variation of absorbance at 220 nm which reflects the pH-dependent CTI (Fig. 8.8). However, this assay cannot be used routinely with oligopeptides, although it gives kinetic data that cannot be easily obtained by other methods [21]. 

Many studies show that the use of PCR-based molecular methods to amplify PSA-mRNA as molecular marker offer a sensitive assay for the detection of extraprostatic PSA synthesizing cells suitable for monitoring and detection of micrometastases and circulating tumor cells originated from prostatic carcinoma. The use of PSA as a molecular target proved to be more sensitive than the amplification of other prostate-specific tissue markers like prostate-specific membrane antigen (PSMA) or human glandular kallikrien (a member of the kallikrien family of serine proteases with trypsin like activity). 

To determine if higher plants produce nonpeptide inhibitors of Candida-secreted aspartic proteases (SAPS), it was necessary to develop a sensitive and specific primary bioassay suitable for screening large numbers of plant extracts, to have access to a unique and diverse plant collection to evaluate for the presence of inhibitors, and to have available purified recombinant Saps for use in a secondary assay to corroborate any observed activity. 

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