Induction and Isolation of Oxalate Binding Protein in Rat Intestinal Brush-Border Membrane
The role of pyridoxine in oxalate metabolism is well documented. To understand the precise role of pyridoxine in regulation and induction of the oxalate transport carrier, the uptake of 14C-oxalate in the intestinal brush-border-membrane vesicles (BBMV) (1, 2) was undertaken. Male weanling rats were maintained on a pyridoxine-deficient diet for 42 days. At the end of the experimental period, the pyridoxine status was assessed by measuring erythrocyte alanine transaminase and stimulation by pyridoxal phosphate (PALP-stimulation index). Starting from the ligament of Treitz, 30 cm of intestine was excised, washed with cold saline, and everted on a steel rod. Mucosa was scraped and BBMV were prepared (1, 2). Oxalate uptake by BBMV prepared from normal and pyridoxine-deficient rats revealed an induction of a biphasic carrier-mediated transport, characteristic for oxalate uptake in this deficiency. The BBMV from pyridoxine-deficient intestines were solubilized by treatment with non-ionic detergent, sodium deoxycholate, which was then removed by dilution below the critical micellar concentration, followed by ultracentrifugation. The protein pellet was incubated with 14C-oxalate. The oxalate-bound protein (OBP) was separated on Sephadex G-75 and Sephadex G-150 columns, and eluted with a 0.2-M phosphate buffer, pH 7.0. The effluent was monitored for protein and β-counts and the oxalate-bound peak was identified. It is postulated that this OBP may be involved in the carrier-mediated phenomenon of oxalate uptake in pyridoxine deficiency.
KeywordsSodium Deoxycholate Critical Micellar Concentration Cold Saline Protein Pellet Pyridoxal Phosphate
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