The problem of a growing resistance of bacteria and other microorganisms to conventional antibiotics gave rise to a search for new potent antimicrobial agents. Insect antimicrobial peptides (AMPs) seem to be promising novel potential anti-infective therapeutics. The dipeptide β-alanyl-tyrosine (β-Ala-Tyr) is one of the endogenous insect toxins exhibiting antibacterial activity against both Gram-negative and Gram-positive bacteria. Prior to testing its other antimicrobial activities, it has to be prepared in a pure form. In this study, we have developed a capillary zone electrophoresis (CZE) method for analysis of β-Ala-Tyr isolated from the extract of the hemolymph of larvae of the fleshfly Neobellieria bullata by reversed-phase high-performance liquid chromatography (RP-HPLC). Based on our previously described correlation between CZE and free-flow zone electrophoresis (FFZE), analytical CZE separation of β-Ala-Tyr and its admixtures have been converted into preparative purification of β-Ala-Tyr by FFZE with preparative capacity of 45.5 mg per hour. The high purity degree of the β-Ala-Tyr obtained by FFZE fractionation was confirmed by its subsequent CZE analysis.

• Keywords
• antimicrobial peptides, beta-alanyl-tyrosine, capillary zone electrophoresis, free-flow zone electrophoresis, peptide analysis, peptide purification,
• MeSH
• Anti-Infective Agents chemistry   isolation & purification   MeSH
• Dipeptides chemistry   isolation & purification   MeSH
• Electrophoresis methods   MeSH
• Hemolymph chemistry   MeSH
• Larva chemistry   MeSH
• Sarcophagidae chemistry   MeSH
• Chromatography, High Pressure Liquid MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• alanyltyrosine MeSH Browser
• Anti-Infective Agents MeSH
• Dipeptides MeSH

Rhomboid proteases are increasingly being explored as potential drug targets, but their potent and specific inhibitors are not available, and strategies for inhibitor development are hampered by the lack of widely usable and easily modifiable in vitro activity assays. Here we address this bottleneck and report on the development of new fluorogenic transmembrane peptide substrates, which are cleaved by several unrelated rhomboid proteases, can be used both in detergent micelles and in liposomes, and contain red-shifted fluorophores that are suitable for high-throughput screening of compound libraries. We show that nearly the entire transmembrane domain of the substrate is important for efficient cleavage, implying that it extensively interacts with the enzyme. Importantly, we demonstrate that in the detergent micelle system, commonly used for the enzymatic analyses of intramembrane proteolysis, the cleavage rate strongly depends on detergent concentration, because the reaction proceeds only in the micelles. Furthermore, we show that the catalytic efficiency and selectivity toward a rhomboid substrate can be dramatically improved by targeted modification of the sequence of its P5 to P1 region. The fluorogenic substrates that we describe and their sequence variants should find wide use in the detection of activity and development of inhibitors of rhomboid proteases.

• Keywords
• enzyme kinetics, enzyme mechanism, fluorescence resonance energy transfer (FRET), intramembrane proteolysis, membrane reconstitution, rhomboid protease, substrate specificity, transmembrane domain,
• MeSH
• Fluorescent Dyes chemistry   MeSH
• Kinetics MeSH
• Liposomes MeSH
• Peptides metabolism   MeSH
• Peptide Hydrolases metabolism   MeSH
• Fluorescence Resonance Energy Transfer MeSH
• Substrate Specificity MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fluorescent Dyes MeSH
• Liposomes MeSH
• Peptides MeSH
• Peptide Hydrolases MeSH