14 research outputs found
Mass spectra of analogue III (M.W = 1590.9 Da; Cm = 103.0 µm) and IX (M.W = 1463.7 Da; Cm = 134.0 µm) recorded before incubation and after 4 hours, 24 hours and 48 hours incubation with SDS-activated yeast 20S proteasome ([E] = 0.18 µm).
<p>Mass spectra of analogue III (M.W = 1590.9 Da; Cm = 103.0 µm) and IX (M.W = 1463.7 Da; Cm = 134.0 µm) recorded before incubation and after 4 hours, 24 hours and 48 hours incubation with SDS-activated yeast 20S proteasome ([E] = 0.18 µm).</p
Inhibitory activities of monocyclic SFTI-1(<b>I</b>) and its analogues at pH 8.1 and 37°C.
<p>*- the K<sub>i</sub> values were calculated using the online IC<sub>50</sub>-to-<i>K<sub>i</sub></i> converter tool assuming competitive inhibition (BotDB Database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089465#pone.0089465-Shibatani1" target="_blank">[22]</a>); N/D – not determined (very low activity). The highest activities (the lowest IC<sub>50</sub> values) are in bold. Z-LLL-CHO was used as the positive control (mean±S.E.M., n = 3).</p
Like other canonical inhibitors, SFTI-1 interacts with a cognate enzyme (i.e. with trypsin) <i>via</i> its solvent-exposed binding loop between Cys<sup>3</sup> and Cys<sup>11</sup>.
<p>The central peptide bond between Lys<sup>5</sup> (P<sub>1</sub>) and Ser<sup>6</sup> (P<sub>1</sub>′) is known as a reactive site <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089465#pone.0089465-Luckett1" target="_blank">[17]</a>. The adjacent residues placed on the left side of this bond are marked with non-prime P (P<sub>2</sub>, P<sub>3</sub>, … P<sub>n</sub> etc), whereas, on the right side, with prime P (P<sub>2</sub>′, P<sub>3</sub>′, … P<sub>n</sub> etc) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089465#pone.0089465-Schechter1" target="_blank">[18]</a>. Corresponding enzyme substrate binding pockets are called non-prime (S<sub>1</sub>, S<sub>2</sub>, S<sub>3</sub>, …, S<sub>n</sub>) and prime (S<sub>1</sub>′, S<sub>2</sub>′, S<sub>3</sub>′, …,S<sub>n</sub>′) sites, respectively.</p
Inhibition of T-L activity of latent human 20S proteasome by Z-LLL-CHO, monocyclic SFTI-1 and its analogues ([E] = 2.7 nm; [S] = 52 µm).
<p>Inhibition of T-L activity of latent human 20S proteasome by Z-LLL-CHO, monocyclic SFTI-1 and its analogues ([E] = 2.7 nm; [S] = 52 µm).</p
Assays in 50m Tris-HCl buffer without SDS (A) analogue V; (B) analogue IX.
<p>Both peptides stimulate ChT-L activity of latent yeast 20S proteasome; [E] = 0.9 nm; [Suc-LLVY-AMC] = 5.2 µm; incubation time 30 min. at 37°C. Data for analogue III are not presented.</p
The rate of substrate Suc-LLVY-AMC hydrolysis (a release of AMC molecule) with control and inhibitor V- treated human 20S versus the substrate concentration (A), the enzyme concentration was kept constant at 2.7 nm; the double reciprocal Lineweaver-Burk plot (B).
<p>The rate of substrate Suc-LLVY-AMC hydrolysis (a release of AMC molecule) with control and inhibitor V- treated human 20S versus the substrate concentration (A), the enzyme concentration was kept constant at 2.7 nm; the double reciprocal Lineweaver-Burk plot (B).</p
Inhibition of human 20S C-L activity by Z-LLL-CHO, BPTI monocyclic SFTI-1 and its analogues ([E] = 2.7 nm; [S] = 80 µm).
<p>Inhibition of human 20S C-L activity by Z-LLL-CHO, BPTI monocyclic SFTI-1 and its analogues ([E] = 2.7 nm; [S] = 80 µm).</p
Inhibition of chymotrypsin-like (ChT-L) and caspase-like (C–L) activities of SDS-activated human 20S ([E] = 2.7 nm) by BPTI and Z-LLL-CHO (A).
<p>Inhibition of trypsin-like (T-L) activity of latent human 20S ([E] = 2.7 nm) (<b>B</b>). Incubation time was about 30 min. at 37°C.</p
Three Wavelength Substrate System of Neutrophil Serine Proteinases
Neutrophil serine proteases, including elastase, proteinase
3,
and cathepsin G, are closely related enzymes stored in similar amounts
in azurophil granules and released at the same time from triggered
neutrophils at inflammatory sites. We have synthesized new fluorescence
resonance energy transfer (FRET) substrates with different fluorescence
donor–acceptor pairs that allow all three proteases to be quantified
at the same time and in the same reaction mixture. This was made possible
because the fluorescence emission spectra of the fluorescence donors
do not overlap and because the values of the specificity constants
were in the same range. Thus, similar activities of proteases can
be measured with the same sensitivity. In addition, these substrates
contain an N-terminal 2-(2-(2-aminoethoxy)ethoxy)acetic acid (PEG)
moiety that makes them cell permeable. Using the mixture of these
selected substrates, we were able to detect the neutrophil serine
protease (NSP) activity on the activated neutrophil membrane and in
the neutrophil lysate in a single measurement. Also, using the substrate
mixture, we were in a position to efficiently determine NSP activity
in human serum of healthy individuals and patients with diagnosed
Wegener disease or <i>microscopic polyangiitis</i>
Inhibition of human 20S ChT-L activity by the selected peptides, [E] = 4.4 nm (0.5 µg/200 µL); [S] = 52.4 µm.
<p>Inhibition of human 20S ChT-L activity by the selected peptides, [E] = 4.4 nm (0.5 µg/200 µL); [S] = 52.4 µm.</p