An improved technique for isolation of brush border vesicles (BBMV) from avian small intestine.

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

San Luis

Congreso; Reunión Científica Anual de la Sociedad de Biología de Cuyo; 2012

Sociedad de Biología de Cuyo

This study presents an improved method of brush border membrane (BBM) preparation from the avian small intestine using polyethylene glycol (PEG) precipitation. This technique is useful in physiology because small intestinal brush border membranes contain hydrolytic enzymes and nutrient transporters that are important for the digestion and absorption. Samples were obtained from adult house sparrows (Passer domesticus) with a body mass around 27 g. The small intestine was excised and divided in three portions (proximal, medial and distal to the pyloric valve), and the mucosa was scraped with a glass slide. A 2% homogenate of the mucosa was prepared in 2 mM Tris–HCl buffer (pH 7.1). A solution of PEG 3350 was added to obtain a final concentration of 7% and stirred for 15 min at 4°C. This was centrifuged at 7,500g for 15 min, the supernatant was respun at 12,000g for 15 min and finally supernatant was respun at 40,000g for 45 min. The pellet was washed twice with buffer and finally suspended in the same buffer. In the BBMVs were measured total protein content and tested the activity of marker enzymes, sucrase, maltase (for proximal-medial portion) and N-aminopeptidase (for distal portion). Isolated BBMVs were relatively pure as judged by an 8 to 20 folds enrichment of marker enzyme activities. Supported by NSF 1025886 to ECV and WHK, by UNSL CyT 9502 and PIP N°0998 CONICET and CyT-UNSL 0110 to JGC.