Microplate Assay to Study Carboxypeptidase A Inhibition in Andean Potatoes

TELLECHEA MARIANA; JAVIER GARCIA PARDO; COTABARREN, JULIANA; LUFRANO, DANIELA; LAURA BAKAS; FRANCESC X. AVILÉS; OBREGÓN, WALTER D.; LORENZO JULIA; SEBASTIÁN TANCO

BIOPROTOCOL

BIOPROTOCOL

Lugar: Sunnyvale; Año: 2016 vol. 6

2331-8325

Metallocarboxypeptidases (MCP) are zinc-dependent exopeptidases that catalyze thehydrolysis of C-terminal amide bonds in proteins and peptides. They are involved in a wide range ofphysiological processes and have recently emerged as relevant drug targets in biomedicine (Arolas etal., 2007). In this context, the study and discovery of new MCP inhibitors from plants constitute avaluable approach for the development of new therapeutic strategies. Herein we describe a simple andaccessible microplate method for the study of the specific and dose-response carboxypeptidase Ainhibitory activities present in Andean potato tubers. Our protocol combines an extraction methodoptimized for small protein inhibitors in plant tissues, with the measurement of enzyme kinetics using amicroplate reader. These instruments are capable of reading small sample volumes, for many samplesin a very short time-frame, therefore reducing the time and costs of high-throughput screeningexperiments. Although this protocol describes the study of Andean potatoes, our approach is alsoapplicable to the analysis other plant samples.Keywords: Metallocarboxypeptidase, Carboxypeptidase A, Inhibitor, Inhibitory activity, Microplateassay, Potatoes[Background] In higher plants, small proteinaceous protease inhibitors are wound-induced moleculesproduced as a part of its defense system against insect attack (Graham et al., 1981; Villanueva et al.,1998). Among the studied inhibitors, only two are specific for MCP, i.e., the potato carboxypeptidaseinhibitor (PCI) and its close homolog found in tomato plants (TCI). Over the last few decades, thepresence of MCP inhibitors in Solanaceae has been extensively reported, revealing potato (Solanumtuberosum) as one of the most important sources of MCP inhibitors (Hass et al., 1979; Obregón et al.,2012; Lufrano et al., 2015). In humans, MCP action is exquisitely regulated and dysregulation of itsfunction might lead to disease or even to cell death (Arolas et al., 2007). In fact, MCP have beenassociated with human pathologies such as acute pancreatitis (Appelros et al., 1998), diabetes (Cool etal., 1997), several types of cancer (Ross et al., 2009; Sun et al., 2016; Abdelmagid et al., 2008; Tsakiris