Purification of multiple functional leaf-actin isoforms from Phaseolus vulgaris L.
Plant actins show diversity in their gene sequences, protein isovariants and tissue distribution in eukaryotes. Besides general difficulties with the isolation of proteins from plant material (i.e. the presence of a cell wall and high proteolytic activity), the actin concentration in any vegetative plant tissue is much lower than in cytoplasmic animal tissues. In this study, we adapted a deoxyribonuclease I-Sepharose affinity purification scheme and we were able to enrich and isolate multiple functional plant actin isovariants from common bean leaves (Phaseolus vulgaris). Urea (4 M) elution proved that the DNase I column was able to bind at least eight actin isoforms with pI values ranging from 5.5 to 5.9, as observed by two-dimensional Western blots. Three of the most acidic actin isoforms, with pI values of approximately 5.6-5.7, were eluted partially with 0.75 M urea. The purified actin was also able to bind leaf and rabbit muscle profilin, phalloidin and DNase I. Moreover, the protein could polymerize into filaments that contained the main isoforms eluted from the column. The average actin recovery using this procedure was approximately 4-8 microg from 20 g of fresh tissue, of which at least 80% was able to form filaments. This is the first report of the purification of multiple plant-actin isoforms that are functional by the criteria of both binding to other ligands and polymerization.
Díaz-Camino
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Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, U.N.A.M., Apartado Postal 510-3, Cuernavaca, Morelos 62250, México.
Villanueva
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MA
D000199:Actins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D002846:Chromatography, Affinity; D003285:Contractile Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007887:Fabaceae / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism; D010590:Phalloidine / Q000378:metabolism; D018515:Plant Leaves / Q000378:metabolism; D010946:Plants, Medicinal; D051304:Profilins; D020033:Protein Isoforms / Q000302:isolation & purification* / Q000378:metabolism* / Q000648:ultrastructure; D011817:Rabbits
D000199:Actins; D003285:Contractile Proteins; D008840:Microfilament Proteins; D051304:Profilins; D020033:Protein Isoforms; D010590:Phalloidine; D003850:Deoxyribonuclease I