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This paper reports a study to find small peptide substrates for the important virulence factor of Yersinia pestis, plasminogen activator, Pla. The method used to find small substrates for this protease is reported along with studies examining the ability of these peptides to inhibit activity of the enzyme. Through the use of parallel synthesis and positional scanning, small tripeptides were identified that are viable substrates for the protease.
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Y. pestis contains a unique, 9.5-kb plasmid pPCP that expresses plague plasminogen activator (Pla) which is responsible for fibrinolytic and coagulase activities. Pla expression is associated with the marked ability of Y. pestis to colonize the vicera and thus cause lethal infection upon administration by peripheral (i.e. intradermal (i.d.), subcutaneous (s.c.) or intraperitoneal) routes of infection. The importance of Pla for plague pathogenesis was verified with isogenic Pla mutants of epidemic Y. pestis strains KIM and CO92, which showed an up to 10 fold reduced virulence by the s.c. routes., Recently, it was confirmed that bubonic plague, but not septicemic, depends on Pla to facilitate the rapid dissemination of bacteria from the site of i.d. infection. Moreover, it was shown that Pla allows Y. pestis to replicate rapidly in the airways, thus playing the essential role in causing pneumonic plague. The invasive properties of Pla which is a cell surface-located protease are likely due to the ability of this enzyme to induce fibrinolysis and degrade extracellular matrix and basement membranes., The activation of plasminogen to plasmin while degrading the plasmin inhibitor α2-antiplasmin by Pla is the mechanism used by Y. pestis to progress from the peripheral sites into the circulation and systemic infection., This paper reports work to identify inhibitors of Pla through parallel synthesis of small peptidic substrates for the enzyme.
A thermally stable enzyme used in PCR to amplify a nucleic aci ..
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Truncated versions of the initial six-residue substrate were examined to probe the length of peptide necessary, as well as to examine the role of the fluorogenic and quencher molecules. Versions of the hexapeptide substrate with and without the EDANS group were evaluated as substrates and as inhibitors in the fibrinolytic assay. A derivative of the peptide without the fluorophore EDANS but with the quencher DABCYL did not inhibit the activation of plasminogen at the concentration in the range of 100-350 µM but was still a substrate for the enzyme. Every truncated version except the dipeptide DABCYL-Arg-Arg-OH, were substrates for Pla (). Apparently EDANS plays a role in increasing inhibitory activity. We are in the process of examining the origin of this effect through the incorporation of molecules with structures similar to EDANS. This effect was observed when EDANS was attached by either the side chain or main chain carboxyl of the peptide (1 and 6, ). Synthesis of the hexapeptide omitting DABCYL also resulted in the loss of the inhibitory activity, indicating that both fluorophore and quencher play a role in the inhibition of Pla. As a control, both free EDANS and DABCYL where examined and found not to inhibit the enzyme. As shown in the length of the peptide can be reduced to a trimer DABCYL-Arg-Arg-Ile-EDANS and maintain activity.
Having established a “minimal” length of the substrate a positional scan approach, similar to the work of Houghten, was used to identify individual peptides. In this approach one amino acid position is held constant while the others are varied. The Fmoc-Glu(EDANS)-Gly-Wang or Fmoc-Glu(EDANS)-Ala-Wang resin was divided into 20 equal portions, and each portion was coupled to an individual amino acid followed by coupling with an isokinetic mixture of eighteen amino acids. This provides each vessel with a mixture of dimers with the known amino acid at position 1 and the other eighteen amino acids represented at position 2. This process was then repeated for positions 3, 4 and 5 and then the final introduction of DABCYL. Each peptide mixture was cleaved from the support and then tested in a reaction with Pla to determine which amino acid in the first position resulted in the hydrolysis. Fluorescence was detected using a plate reader at excitation and emission wavelengths of 360 and 460 nm, respectively. The amino acid in position 1 that resulted in fluorescence was then held constant and the second position is scanned in the same manner as the first. This process is then repeated for the third, fourth and fifth positions. (It should be noted that while the original paper reports isokinetic ratios for Boc-protected amino acids and usage of 10 eq, we used Fmoc-based strategy and 5 eq during coupling.) Despite the limitation of a positional scan approach, the method provided substrates for Pla enzyme, with a sequence Arg-Arg-Ile-Asn-Arg being selected as the “best”. The substrate based on this sequence was resynthesized on Sieber amide resin to eliminate influence of Gly/Ala, and the resulting DABCYL-Arg-Arg-Ile-Asn-Arg-Glu(EDANS)-NH2 was confirmed to be a fluorogenic substrate for Pla.
4A can go into the cytoplasm to be used in protein synthesise
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The original approach undertaken was to synthesize, on solid support, ensembles of fluorogenic peptides based on loop 5 of Pla, a region of the enzyme that is known to undergo self cleavage, and then screen those libraries for fluorescence (). Six-residue peptides, overlapping by one amino acid were synthesized with the DABCYL/EDANS quencher/fluorophor system chosen for this work. In theory, peptides in the mixture that are substrates for Pla would be hydrolyzed resulting in the separation of the quencher (DABCYL) from the fluorophor (EDANS) and fluorescence of the bead. The targeted libraries synthesized on the PEGA1900 support resulted in no fluorescence when submitted to the purified enzyme. Additionally, targeted libraries based on the region of plasminogen that is known to be cleaved by Pla were also screened, resulting in no fluorescence. Some potential reasons for the lack of a signal are: the enzyme and the solid support the peptides were attached to may not be compatible in terms of porosity or hydrophobicity; the enzyme may not accept small substrates or substrates that do not have a particular secondary structure.
In addition to providing information about the substrate specificity of the enzyme this work has provided a series of small fluorogenic peptides that are currently being used as leads for the development of inhibitors of the protease Pla. Additionally such fluorogenic substrates are being used to develop a high-throughput screen for small molecule inhibitors of Pla.
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review biology 2 photosynthesis Study Sets and …
The smooth endoplasmic reticulum plays a major role in synthesizing by means of enzymes embedded in these smooth membranes. It produces the and used in membrane formation, and along with the membrane proteins produced by the rough ER it can synthesize more membrane for itself, for the Golgi complex, the , , and others.
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