RESUMEN
Cultured Thalictrum minus cells produce a benzylisoquinoline alkaloid, berberine, in the presence of benzyladenine, and excrete it into the culture medium. T. minus cells excluded berberine, even if berberine was exogenously added to the medium, without benzyladenine treatment. Similarly, T. minus cells excluded a heterocyclic dye (neutral red) and calcein AM, which is used as a fluorescent probe to detect the drug efflux pump activity by ABC transporters. The addition of several inhibitors of P-glycoprotein, a representative ABC transporter, induced the accumulation in of both berberine and calcein AM ATP-dependent manner. The expression of P-glycoprotein-like ABC transporter genes was also demonstrated. The involvement of ABC transporter in the secretion of berberine in T. minus cells is discussed.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Berberina/metabolismo , Thalictrum/citología , Thalictrum/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Secuencia de Aminoácidos , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Células Cultivadas , Electroforesis en Gel de Agar/métodos , Inhibidores Enzimáticos/farmacología , Fluoresceínas , Microscopía Fluorescente , Datos de Secuencia Molecular , Rojo Neutro , ARN de Planta/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Thalictrum/efectos de los fármacos , Factores de TiempoRESUMEN
The vanadate-induced nucleotide trapping technique, which has been conventionally used to characterize mammalian ATP-binding cassette (ABC) proteins, was applied to berberine-producing plant cell cultures, Thalictrum minus and Coptis japonica. One membrane protein at ca. 180 kDa was photoaffinity-labeled with 8-azido-[alpha-(32)P]ATP in the T. minus cells in the presence of vanadate, which was specifically induced by the addition of benzyladenine in a similar manner as the induction of berberine biosynthesis in these cell cultures, whereas three bands were observed in the C. japonica cells in the size region between 120 and 150 kDa corresponding to full-sized ABC protein. The benzyladenine-induced band in T. minus showed properties similar to those of human MDR1, including the recognition of berberine, which suggests that the ABC protein detected in T. minus takes this endogenous alkaloid as a putative substrate for transport. This is the first application of this technique to plant cells.