Isolation of brush border vesicles from small intestine of Columba livia.

BERDASCO, CLARA; CAVIEDES-VIDAL E; CHEDIACK, JG

San Luis

Congreso; Reunión Anual de la Sociedad de Biología de Cuyo; 2014

UNSL

A variety of techniques has been employed for the isolation of small intestinal brush border membranes (BBMV), e.g. divalent cations as Ca++ or Mg++, differential centrifugation using sucrose density gradient and recently using polyethylene glycol (PEG) as precipitant. This study is aimed to improve a method to obtain brush border membrane vesicles (BBMV) from pigeon small intestines using polyethylene glycol (PEG) as precipitant. We tested different PEG concentrations and compared purity and enrichment of the obtained BBMV. The samples were obtained from adult birds. The intestine was cut and divided into two portions (proximal and distal) and the mucosa was scraped off with a glass slide. PEG 3350 solution was added (to give final concentrations of 5, 10, 15, 20 and 25%) and stirred for 15 min at 4°C. After several centrifugation bouts we obtain pellet fractions remained after PEG treatment and BBVM in the supernatant. The supernatant was centrifuged at 40,000g for 45 min and obtained BBMVs were resuspended in buffer HEPES-mannitol. We measure total protein content, activities of sucrase, maltase and aminopeptidase-N as marker enzymes of the apical cell membrane, Na+/K+ pump as basolateral cell membrane marker, and protein profile by SDS-PAGE. The procedures yielded a 20 fold enrichment of apical enzyme activities and remain activity of Na+/K+ pump in the BBMV in all PEG concentrations tested. Enrichment of some characteristics bands was found in BBMV profiles of SDS-PAGE. In conclusion PEG method allows obtaining BBMV with adequate purity. Supported by UNSL CyT 0814 to ECV and PIP N°0998 CONICET to JGC. Biología General, Celular y Molecular (BM) ? San Luis