biostudies-literatureSchepetkin IANational Institute of Food and Agriculture (NIFA)3132https://www.ebi.ac.uk/biostudies/studies/S-EPMC12567500biostudies-literatureUnknown14(20)Leaf senescence is a developmental process that allows nutrients to be remobilized and transported to sink organs. Previously, papain-like cysteine proteases (PLCPs) have been found to be highly expressed during leaf senescence in different plant species. In this study, we analyzed active PLCPs in barley (<i>Hordeum vulgare</i> L.) leaves during the terminal stage of natural senescence. Anion exchange chromatography of protein extracts from barley leaves, harvested six weeks after anthesis, followed by activity assays using the substrates Z-FR-AMC and Z-RR-AMC, revealed a single prominent peak corresponding to active PLCPs. This hydrolytic activity was completely inhibited by E-64, a potent and irreversible inhibitor of cysteine proteases. Fractions enriched for PLCP activity were affinityPlants (Basel, Switzerland)Activity-Based Profiling of Papain-like Cysteine Proteases During Late-Stage Leaf Senescence in Barley.PMC125675002023-67014-39568Schepetkin IAFischer AMfalseActivity-Based Profiling of Papain-like Cysteine Proteases During Late-Stage Leaf Senescence in Barley.Leaf senescence is a developmental process that allows nutrients to be remobilized and transported to sink organs. Previously, papain-like cysteine proteases (PLCPs) have been found to be highly expressed during leaf senescence in different plant species. In this study, we analyzed active PLCPs in barley (<i>Hordeum vulgare</i> L.) leaves during the terminal stage of natural senescence. Anion exchange chromatography of protein extracts from barley leaves, harvested six weeks after anthesis, followed by activity assays using the substrates Z-FR-AMC and Z-RR-AMC, revealed a single prominent peak corresponding to active PLCPs. This hydrolytic activity was completely inhibited by E-64, a potent and irreversible inhibitor of cysteine proteases. Fractions enriched for PLCP activity were affinity2025-01-01T00:00:00Z2025 Oct2026-05-15T03:22:11.306Z2026-05-15T03:13:05.653ZS-EPMC125675004115769110.3390/plants14203132