Assays, with synthetic peptide substrates

Practical Aspects of Assays with Synthetic Peptide Substrates. 132... 

To investigate whether bevirimat functions as a PR inhibitor, several bioassays were conducted, including a cell-free fluorometric assay using a synthetic peptide substrate of PR and experiments using a recombinant form of the Gag precursor protein Pr55Gag. Both assays showed no effect of bevirimat on PR function compared with the PR inhibitor indinavir. Indeed, bevirimat at nanomolar concentrations could inhibit the replication of HIV-1 isolates resistant to PR inhibitors. 

In theory, at least, all of the above serine proteases can be assayed by selective specific tests employing synthetic peptide substrates. While these new tests are becoming routine research procedures (S16), the key question for the clinician and laboratory head is how to interpret these results in light of the extensive knowledge of patient testing with the above nonspecific clot-endpoint tests like the PT and APTT. This point will be examined more fully later. 

Some properties of phosphopeptides make them preferable to the native phosphoprotein substrates for use with phosphate detection systems. The values of for peptide substrates are two to three orders of magnitude larger than for protein substrates and allow setting assays with an appropriate substrate concentration using standard phosphate detection approaches. In addition, short synthetic peptides are inexpensive and easy to obtain. Nonetheless, although phosphopeptide substrates are clearly useful in exploring interactions in the immediate vicinity of the phosphatase active site, they are unable to probe distant (allosteric) sites. 

Primary screenings of candidate HDACIs are generally performed using in vitro assays consisting of sources of HDACs (i.e. cell extracts or rat liver) and substrates (i.e. radiolabeled acetylated histones or synthetic peptides with a fluorophor). Isoform selective studies are performed using purified recombinant, immunoprecipitated proteins using antibodies against selected HDACs and computer-based virtual prediction assays. 

To obtain the maximum rate of renin activity, saturating amounts of the renin substrate, angiotensinogen, should be present in the reaction system. In most procedures, however, the only substrate provided is that present in the test plasma, and its concentration can be quite variable. According to some investigators, PRA is best estimated when the plasma specimen is incubated with an excess of exogenous renin substrate prepared from nephrectomized human subjects, oxen, or sheep. This type of assay is usually known as a plasma renin concentration assay rather than PRA assay. Unfortunately the measured renin depends on the source and concentration of the renin substrate. Synthetic peptides that resemble the M-terminal portion of angiotensinogen have also been used as renin substrates, but these substances can be hydrolyzed by nonspecific plasma proteases. 

Goto et al. 1998). None of them probably reflects properly the enzyme activity over the real substrate, so it will be a matter of judgment and experience to select the most pertinent assay with respect to the actual use of the enzyme. Hydrolases are currently assayed with respect to their hydrolytic activities however, the increasing use of hydrolases to perform reactions of synthesis in non-aqueous media make this type of assay not quite adequate to evaluate the synthetic potential of such enzymes. For instance, the protease subtilisin has been used as a catalyst for a trans-esterification reaction that produces thiophenol as one of the products (Han et al. 2004) in this case, a method based on a reaction leading to a fluorescent adduct of thiophenol is a good system to assess the transesterification potential of such proteases and is to be preferred to a conventional protease assay based on the hydrolysis of a protein (Gupta et al. 1999 Priolo et al. 2000) or a model peptide (Klein et al. 1989). 

Bhatia and coworkers established synthetic urinary biomarkers for detection of disease-specific protease activity with independence from the need for expensive machinery and an ease of interpretation [173,174]. They used activatable nanoprobes, producing lu-inary biomarkers readily detectable using a paper strip assay similar to a pregnancy test. Ihe luinary biomarkers are PEG-coated iron oxide nanoworms densely conjugated with fluorescein-labeled thrombin and MMP substrates, with high peptide density both for sensitivity and photoinduced fluorescence quenching. 

The serine protease from Thermus caldophilus strain GK-24 [292] gave maximum activity at 90 °C in 20 min assays and had a broad pH optimum with casein as substrate. The enzyme showed hydrolytic activity on some small peptide substrates (e.g. CBZ-L-leu-L-tyr-NHj) and also possessed esterase activity. Hydrolysis of synthetic chromogenic peptides and esters is also a property of the recently described serine protease, caldolase, from Thermus strain ToK3 [293]. This enzyme contained 10% carbohydrate and four disulphide bonds, but neither calcium nor zinc were detected in the purified enzyme. Thermostability of the enzyme was high in 0.4 M NaCl, but in low ionic strength buffer rapid thermal denaturation occurred at 75 °C. Work has also been undertaken in this laboratory... 

The International Union of Biochemistry and Molecular Biology recommends that the term peptidase be used synonymously with the term peptide hydrolase (IUBMB, 1992). Thus, in this unit the term peptidase is used in reference to any enzyme that catalyzes the hydrolysis of peptide bonds, without distinguishing between exo- and endopeptidase activities. Peptidases may be assayed using native or modified proteins, peptides, or synthetic substrates. In this unit, the focus is on assays based on the hydrolysis of common, commercially available, protein substrates. Thus, the assays are not intended to be selective for a given peptidase they are designed to provide estimates of overall peptidase activity. Other units in this publication focus on synthetic or model substrates, which can be designed for the measurement of specific endo- and/or exopeptidase activities. 

Thioesterases preferentially cleave the thioester bond of acyl-CoA molecules to produce CoA and free fatty acid. E. coli contains two well-characterized thioesterases. Thioesterase I (encoded by tesA) is a periplasmic enzyme of 20.5 kDa, with a substrate specificity for acyl chains >12 carbon atoms. Thioesterase 1 hydrolyzes synthetic substrates used in the assay of chymotrypsin, which led to the initial conclusion that TesA was a protease ( protease F). However, the purified protein does not cleave peptide bonds. Thioesterase... 

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