• Journal of Microbiology and Biotechnology
• /
• 제15권4호
• /
• pp.899-902
• /
• 2005

Peptide assimilation by Helicobacter pylori was investigated using L-phenylalanyl-3-thia-phenylalanine (PSP) as a detector peptide; the release of thiophenol upon enzymatic hydrolysis of PSP was spectrophotometrically detected with the aid of 5,5'-dithiobis[2-nitrobenzoic acid] (DTNB). By adding PSP to whole-cell suspension, thiophenol was produced progressively, resembling that found in Esherichia coli or Staphylococcus aureus. Interestingly, the rate of thiophenol production by H pylori in particular was markedly reduced when cells were pretreated with trypsin, indicating surface exhibition of peptidase. According to the competitive spectrophotometry using alanyl-peptides, H pylori did not appear to assimilate PSP through the peptide transport system. No discernible PSP assimilation could be ascertained in H pylori cells, unless provided with some additives necessary for peptidase activity, such as $Ni^{2+}\;or\;Mg^{2+}$ and an appropriate concentration of potassium or ammonium salts. These observations strongly suggest that, regardless of a presumptive peptide transport system, peptide assimilation of H. plori appears to be highly dependent upon milieu conditions, due to unique peptidase exhibition on the cell surface.

• Journal of Microbiology and Biotechnology
• /
• 제25권10호
• /
• pp.1629-1633
• /
• 2015

Peptide assimilation in Helicobacter pylori necessitates a coordinated working of the peptide transport systems (PepTs) and aminopeptidase (PepA). We found that H. pylori hydrolyzes two detector peptides, _L-phenylalanyl- _L-3-thiaphenylalanine (PSP) and _L-phenylalanyl- _L-2-sulfanilylglycine (PSG), primarily before intake and excludes their antibacterial effects, whereas Escherichia coli readily transports them with resultant growth inhibition. PSP assimilation by H. pylori was inhibited by aminopeptidase inhibitor bestatin, but not by dialanine or cyanide-m-chlorophenylhydrazone, contrary to that of E. coli. RT- and qRT-PCR analyses showed that H. pylori may express first the PepTs (e.g., DppA and DppB) and then PepA. In addition, western blot analysis of PepA suggested that the bacterium secretes PepA in response to specific inducers.

• Journal of Microbiology and Biotechnology
• /
• 제6권2호
• /
• pp.92-97
• /
• 1996

Portage kinetic constants of peptide transport can be measured by competitive spectrophotometry. The kinetic constants of L-Glu-L-Glu transport in Escherichia coli were ascertained using L-Phe-L-3-thia-Phe (PSP) as a detector. Since the production of thiophenol upon intracellular hydrolysis of PSP was competitively inhibited by L-Glu-L-Glu, it was able to compute the kinetic constants of L-Glu-L-Glu using this method. The resulted data were in agreement with the values obtained by the method of Michaelis-Menten kinetics. The potential of this method was examined against dipeptide transport systems in various microorganisms. These results strongly suggest that the overall properties of individual systems for dipeptide transports can be easily characterized by competitive spectrophotometry.

• 한국식품과학회지
• /
• 제29권4호
• /
• pp.630-634
• /
• 1997

N-glycanase처리에 의해 얻어진 1-amino-oligosaccharide를 FMOC-Cl로 표식 하는 방법을 확립하였다. 이 방법에 의해 얻어진 FMOC-표식 oligosaccharides는 기존의 방법보다 약 4배의 감도를 나타내었다. Amido 80 column을 사용한 분석에 의하여, Man 5-9 GlcNAc 2 amines의 5개의 성분은 각각 분리되어 용출 되었으며, 회수율은 기존의 방법과 거의 같았다. 1-amino-oligosaccharides는 0.05 pmol에서 1.5 pmol 사이에서 직선적인 관계를 나타내었으며, 2-aminopyridine에 의한 표식과 비교하였을 때, 안정된 화합물을 형성하고 있으며, 감도가 높아 미량의 oligosaccharides의 분석에 매우 적합한 것이 확인되었다.

• Bulletin of the Korean Chemical Society
• /
• 제35권5호
• /
• pp.1365-1374
• /
• 2014

Thirteen analogs of tridecapeptide ${\alpha}$-factor (WHWLQLKPGQPMY) of Saccharomyces cerevisiae with C- or N-terminal Trp extension and isosteric replacement by Aib at position 8 and 11, Trp at position 13, D-Ala at position 9, and Orn and Glu at position 6 were synthesized and assayed for their biological activity. Receptor binding assay was carried out using our newly developed spectrophotometric method with detector peptide 14. C- or N-terminal extended analogs, ${\alpha}$-factor-$[Trp]_n$ (n =1-5) 1-5 and $[N-Trp]_1$-${\alpha}$-factor 6, were all less active than native ${\alpha}$-factor and gradual decreases in both activity and receptor affinity were observed with greater Trp extension. Trp-substituted analog at position 13, $[Trp^{13}]{\alpha}$-factor 7, exhibited about 2-fold reductions in both activity and receptor affinity. Aib-substituted analogs, $[Aib^8]{\alpha}$-factor 8 and $[Aib^{11}]{\alpha}$-factor 9, showed 5- to 10-fold reduction in activity as well as 3-fold reduction in receptor affinity compared to native ${\alpha}$-factor. $[Orn^6]{\alpha}$-factor 10 demonstrated strong potency with a 7.0-fold increase in halo activity as well as 1.8-fold increase in receptor affinity compared to native ${\alpha}$-factor. For two double substituted analogs, [$Glu^6,{\small{D}}-Ala^9$]${\alpha}$-factor 12 showed the slightly decreased potency in halo activity compared to analog 10, whereas [$Orn^6,{\small{D}}-Ala^9$]${\alpha}$-factor 11 exhibited 15-fold higher halo activity as well as nearly 3-fold higher receptor affinity compared to native ${\alpha}$-factor.