- Differences in the hydrolysis of enkephalin congeners by the two domains of angiotensin converting enzyme
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The hydrolysis of enkephalin (Enk) congeners by the isolated N- (N-ACE) and C-domain of angiotensin I converting enzyme (ACE) and by the two-domain somatic ACE was investigated. Both Leu5- and Met5-Enk were cleaved faster by the C-domain than by N-ACE; rates with somatic ACE were 1600 and 2500 nmol/min/nmol enzyme with both active sites being involved. Substitution of Gly2 by D-Ala2 reduced the rate to 1/3rd to 1/7th of that of the Enks. N- ACE cleaved Met5-Enk-Arg6-Phe7 faster than the C-domain, probably with the highest turnover number of any naturally occurring ACE substrate (7600 min- 1). This heptapeptide is also hydrolyzed in the absence of Cl-, but the activation by Cl- is unique; Cl- enhances the hydrolysis of the heptapeptide by N-ACE but inhibits it by the C-domain, yielding about a 5- fold difference in the turnover number at physiological pH. This difference may result in the predominant role of the N-domain in converting Met5-Enk- Arg6-Phe7 to Enk in vivo.
- Deddish, Peter A.,Jackman, Herbert L.,Skidgel, Randal A.,Erdoes, Ervin G.
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- Investigations of the in-vitro metabolism of three opioid tetrapeptides by pancreatic and intestinal enzymes
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The metabolism of three opioid tetrapeptides, Tyr-D-Arg-Phe-Nva-NH2, Tyr-D-Arg-Phe-Phe-NH2 and Tyr-D-Ala-Phe-Phe-NH2, was investigated in the presence of pure pancreatic enzymes (trypsin, chymotrypsin, elastase, carboxypeptidase A and carboxypeptidase B), as well as in the presence of pure carboxylesterase and aminopeptidase N. The cleavage patterns of the pure pancreatic enzymes were then compared with those found in rat and human jejunal fluid. Metabolism was also studied in homogenates from different intestinal regions (duodenum, jejunum, ileum and colon) and in enterocyte cytosol from rats. The effect of various protease inhibitors was investigated in the jejunal homogenate. The parent peptides were assayed by high-performance liquid chromatography and metabolites were identified by means of liquid chromatography-mass spectrometry. Of the pure enzymes, the quickest hydrolysis of the peptides was observed for the pancreatic enzymes chymotrypsin, trypsin and carboxypeptidase A. In most cases they formed the corresponding deamidated tetrapeptides (chymotrypsin and trypsin) or tripeptides with a missing C-terminal amino acid (carboxypeptidase A). Regional differences in intestinal metabolism rates were found for all three peptides (P A showed that lumenal pancreatic proteases might be a clear metabolic obstacle in oral delivery even for small peptides such as these tetrapeptides.
- Krondahl, Eva,Von Euler-Chelpin, Hans,Orzechowski, Achim,Ekstroem, Gunilla,Lennernaes, Hans
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- Use of an internal reference for the quantitative HPLC-UV analysis of solid-phase reactions: A case study of 2-chlorotrityl chloride resin
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Here we evaluated the use of internal reference compounds for the rapid assessment of reactions performed in solid-phase. An internal reference compound (commercially available) was bound to the resin, together with the substrate, and cleaved with the products after completion of the reaction. The peak area of the reference compound in the HPLC-UV chromatograms can be correlated directly with those of other compounds present in the reaction mixture, thereby allowing a quantitative interpretation of the chromatograms with respect to conversion and yield. The usefulness of this method was demonstrated by optimization of a protocol for the synthesis of proline-based tripeptides.
- Spengler, Jan,Fernandez-Llamazares, Anna-Iris,Albericio, Fernando
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supporting information
p. 229 - 234
(2013/07/11)
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- A novel L-amino acid ligase from Bacillus subtilis NBRC3134, a microorganism producing peptide-antibiotic rhizocticin
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L-Amino acid ligase catalyzes the formation of an α-peptide bond from unprotected L-amino acids in an ATP-dependent manner, and this enzyme is very useful in efficient peptide production. We performed enzyme purification to obtain a novel L-amino acid lig
- Kino, Kuniki,Kotanaka, Yoichi,Arai, Toshinobu,Yagasaki, Makoto
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body text
p. 901 - 907
(2009/11/30)
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