We have confirmed previous demonstrations of sodium gradient-stimulated transport of L-alanine, phenylalanine, proline, and beta-alanine, and in addition demonstrated transport of N-methylamino-isobutyric acid (MeAIB) and lysine in isolated rabbit kidney brush border vesicles. In order to probe the multiplicity of transport pathways available to each of these 14C-amino acids, we measured the ability of test amino acids to inhibit tracer uptake. To obtain a rough estimate of nonspecific effects, e.g., dissipation of the transmembrane sodium electrochemical potential gradient, we measured the ability of D-glucose to inhibit tracer uptake. L-alanine and phenylalanine were completely mutually inhibitory. Roughly 75% of the 14C-L-alanine uptake could be inhibited by proline and beta-alanine, while lysine and MeAIB were no more effective than D-glucose. Roughly 50% of the 14C-phenylalanine uptake could be inhibited by proline and beta-alanine; lysine was as effective as proline and beta-alanine, and the effects of pairs of these amino acids at 50 mM each were not cumulative. MeAIB was no more effective than D-glucose. We conclude that three pathways mediate the uptake of neutral L, alpha-amino acids. One system is inaccessible to lysine, proline, and beta-alanine. The second system carries a major fraction of the L-alanine flux; it is sensitive to proline and beta-alanine, but not to lysine. The third system carries half the 14C-phenylalanine flux, and it is sensitive to proline, lysine, and beta-alanine. Since the neutral, L, alpha-amino acid fluxes are insensitive to MeAIB, we conclude that they are not mediated by the classical A system, and since all of the L-alanine flux is inhibited by phenylalanine, we conclude that it is not mediated by the classical ASC system. L-alanine and phenylalanine completely inhibit uptake of lysine. MeAIB is no more effective than D-glucose in inhibiting lysine uptake, while proline and beta-alanine appear to inhibit a component of the lysine flux. We conclude that the 14C-lysine fluxes are mediated by two systems, one, shared with phenylalanine, which is inhibited by proline, beta-alanine, and L-alanine, and one which is inhibited by L-alanine and phenylalanine but inaccessible to proline, beta-alanine, and MeAIB. Fluxes of 14C-proline and 14C-MeAIB are completely inhibited by L-alanine, phenylalanine, proline, and MeAIB, but they are insensitive to lysine. Proline and MeAIB, as well as alanine and phenylalanine, but not lysine, inhibit 14C-beta-alanine uptake. However, beta-alanine inhibits only 38% of the 14C-proline uptake and 57% of the MeAIB uptake. We conclude that two systems mediate uptake of proline and MeAIB, and that one of these systems also transports beta-alanine.

The functional unit molecular size of the intestinal brush border membrane-bound Na+/glucose cotransporter was determined by radiation inactivation. Purified brush border membrane vesicles preserved in cryoprotectant buffer were irradiated (-1350C) with high-energy electrons from a 13-MeV (1 eV = 1.602 x 10-'9 J) linear accelerator at doses from 0 to 70 Mrad (1 rad = 0.01 Gy). After each dose, the cotransporter was investigated with respect to (i) Na'-dependent transport activity and (ii) immunologic blot analysis with antibodies against the cloned rabbit intestinal cotransporter. Increasing radiation decreased the maximal Na'-dependent cotransporter activity JfaX without affecting apparent Km. The size of the transporting functional unit was 290 ± 5 kDa. Immunologic blot analysis of brush border membranes gave a single band of Mr 70,000, which decreased in intensity with increased radiation dose and gave a target size of 66 ± 11 kDa. We conclude that activity of the intestinal Na+/glucose cotransporter in situ in the brush border membrane requires the simultaneous presence of four intact, independent, identical subunits arranged as a homotetramer.The intestinal brush border membrane Na+/glucose cotransporter is among the most thoroughly studied eukaryotic systems demonstrating secondary active (ion-coupled) transport. Biochemical and immunological experiments have identified the cotransporter as a polypeptide of Mr 70,000-75,000 on SDS/PAGE reducing gels (1-3), and the cloned cotransporter has a predicted molecular mass of 73 kDa (5, 6). However, information regarding structure-function relationships of the cotransporter in situ in the native membrane is lacking. In this study we used high-energy electron radiation inactivation to investigate the membrane-bound in situ size of the cotransporter. Our results indicate that the cotransporter functions in the membrane as a 290-kDa homotetramer comprised of four independent 73-kDa subunits. METHODS AND MATERIALSBrush border membrane vesicles were prepared from male New Zealand White rabbit small intestines by a 10 mM MgCl2 precipitation procedure (7,8). The membrane vesicles were enriched =15-fold in the brush border membrane marker enzymes alkaline phosphatase, leucine aminopeptidase, sucrase, and 'y-glutamyltranspeptidase. Vesicles (20 ,ug of protein per ,l) were equilibrated in a cryoprotectant buffer (9, 10) that contained 14% (vol/vol) glycerol, 1.4% (wt/vol) D-sorbitol, 150 mM KCI, and 5 mM Hepes-Tris (pH 7.5). Beliveau et al. (9, 10) have established that this buffer preserves glucose and phosphate transport, and Na+/H+ antiport in kidney brush border membrane vesicles. Aliquots of the vesicle suspension were frozen in 2-ml glass ampules (type 12012; Kimble, Toledo, OH). The sealed ampules were then kept at -80'C and transported on dry ice. The membranes were irradiated at -1350C with a beam of 13 MeV (1 eV = 1.602 x 10-1' J) electrons produced by a linear accelerator, as described elsewhere (11). After irradiation the ampules were held at -800C until...

To evaluate the effect of age on nutrient transport, the absorption rates of D-glucose, D-fructose, L-alanine, L-aspartate, L-leucine, L-lysine, L-proline, folic acid, and nicotinamide were determined in isolated jejunal tissues of young (6.7 mo old) and aged (23.7 and 27.0 mo old) mice (COBS:SFW). D-Glucose and D-fructose uptakes per milligram tissue were approximately 20-120% higher in the proximal jejunum and 15-50% higher in the distal jejunum of young mice. Amino acid and vitamin uptakes per milligram were also higher in young mice, but differences were not statistically significant. The number of Na(+)-D-glucose transporters per milligram tissue as estimated by specific phlorizin binding decreased with age. There was no age-related change in passive L-glucose permeability, in Kd of specific phlorizin binding, in transporter turnover rate, and in the molecular weight of the Na(+)-D-glucose transporter. Thus a reduction in D-glucose transporter site density fully accounts for the age-related decline in D-glucose transport rate per milligram small intestine.