Reconstitution of a partially purified Na+-dependent D-glucose transport system from rat jejunal brush border membranes.

The Na+-dependent D-glucose transport system of rat jejunal brush border membranes was partially purified and reconstituted into functional proteoliposomes. Brush border membrane vesciles isolated from villous cells were first extracted with 0.3% cholate to remove extrinsic proteins and the insoluble residual pellet was reextracted with 1.2% cholate. The 1.2% cholate-extracted soluble fraction was then further purified by hydroxylapatite and Concanavalin A affinity chromatography in tandem. When the HLP-unadsorbed-ConA-unadsorbed fraction was reconstituted into proteoliposomes, it showed a characteristic Na+-coupled, phlorizin inhibitable, D-glucose transport activity that was 3 fold higher than that of the reconstituted proteoliposomes of the 1.2% cholate-extracted fraction. This partially purified fraction also displayed the simplest polypeptide composition pattern among all the membrane fractions analysed in SDS-polyacrylamide gels.

Coupled sodium-chloride transport by rabbit ileal brush-border membrane vesicles.

Uptake of Na and Cl into brush-border membrane vesicles isolated from rabbit ileal epithelial cells was investigated with a rapid filtration technique using 22Na and 36Cl as tracers. The rank order of anion dependence for Na uptake in the absence of anion gradients was SCN greater than NO3 greater than gluconate. The sequence of cation specificity for Cl uptake was Na congruent to Li greater than K greater than choline. The transport of Na and Cl were both inhibited by harmaline, furosemide, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, and 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid. Carrier mediation of Cl-stimulated Na transport was suggested by the competition for 22Na uptake with increasing concentrations of unlabeled Na in the presence of Cl but not when gluconate was the counterion. Chloride-dependent Na uptake had an apparent Km of 4.5 mM and a Vmax of 20 nmol . mg prot-1 . 15 s-1. Na-H exchange and Cl-OH (or HCO3) exchange were also demonstrated in these vesicles. These findings confirm the presence of an electrically neutral carrier-mediated, Na-Cl-coupled transport process in the apical cell membrane of rabbit ileal epithelial cells. The nature of the coupling of Na to Cl transport, i.e., NaCl symport or a process that combines Na-H antiport with Cl-OH (or HCO3) antiport, remains to be determined.

Reconstitution of a partially purified Na+-independent D-glucose transport system from rat jejunal basolateral membranes.

1. Basolateral membranes of rat small intestine were first solubilized in a 0.6% cholate buffer and then the insoluble fraction was reextracted with a 1.2 or 1.6% cholate buffer. 2. Proteoliposomes reconstituted from the 1.2 or 1.6% cholate-extracted membrane fraction demonstrated characteristic Na+-independent D-glucose transport of the native basolateral membrane vesicles: inhibitable by mercuric chloride and D-galactose. 3. To further purify this D-glucose transport system, the 1.6% cholate-extracted membrane fraction was chromatographed on either hydroxylapatite, concanavalin A, wheat-germ lectin or castor bean lectin-120 affinity gels. 4. Proteoliposomes reconstituted from the membrane proteins adsorbed on hydroxylapatite and subsequently passed through agarose-castor bean lectin-120 showed a 12-fold enrichment of Na+-independent D-glucose transport activity over that of the native membrane vesicles. 5. SDS-electrophoretic analysis showed that the protein composition of the hydroxylapatite-castor bean lectin-120 treated fraction was much simpler than that of both 1.6% cholate-extracted fraction and the native membrane vesicles.

Effect of caffeine, theophylline and nicotine on D-glucose and folate transport in rat jejunal brush border membrane vesicles.

1. Nicotine at 10 mM, but not caffeine or theophylline, reduced by 20% the overshoot of the Na+-dependent D-glucose transport in rat jejunal brush border membrane vesicles. 2. Since nicotine did not affect the transport of Na+, its inhibition on Na+-dependent D-glucose transport must be due to a direct effect upon the D-glucose transport system. 3. Folate transport in these membrane vesicles was found to a be a free diffusion process at pH 7.4. 4. Neither caffeine, theophylline nor nicotine has any effect on folate transport.

Partial purification of the Na+-dependent D-glucose transport system from renal brush border membranes.

A membrane extract enriched with the Na+ -dependent D-glucose transport system was obtained by differential cholate solubilization of rat renal brush border membranes in the presence of 120 mM Na+ ions. Sodium ions were essential in stabilizing the transport system during cholate treatment. This membrane extract was further purified with respect to its Na+-coupled D-glucose transport activity and protein content by the use of asolectin-equilibrated hydroxylapatite. The reconstituted proteoliposomes prepared from this purified fraction showed a transient accumulation of D-glucose in response to a Na+ gradient. The observed rate of Na+-coupled D-glucose uptake by the proteoliposomes represented about a sevenfold increase as compared to that of the reconstituted system derived from an initial 1.2% cholate extract of the membranes. Other Na+-coupled transport systems such as L-alanine, alpha -ketoglutarate and phosphate were not detected in these reconstituted proteoliposomes.

Bile-salt inhibition of sodium ion-coupled D-glucose and L-alanine accumulation by brush-border-membrane vesicles from hamster jejunum.

The effects of bile salts on Na+-coupled accumulation of D-glucose and L-alanine by brush-border-membrane vesicles isolated from hamster jejunum were investigated. The approximate percentage inhibition of Na+-coupled D-glucose accumulation produced by various bile salts at a concentration of 1 mM were: deoxycholate and chenodeoxycholate, 60%; glycine and taurine conjugates of deoxycholate and chenodeoxycholate, 40--50%; lithocholate, 45%; cholate and its glycine and taurine conjugates, less than 10%. Inhibition of Na+-coupled accumulation of D-glucose was rapid, reversible and not due to dissolution of the vesicles. Na+-coupled accumulation of L-alanine was also inhibited by deoxycholate. Deoxycholate but not cholate enhanced (1) the rate of Na+ influx, (2) the rate of influx of D-glucose and L-alanine in the absence of a Na+ gradient and (3) the rate of efflux of D-glucose and L-alanine from vesicles preloaded with this sugar or amino acid. Deoxycholate-stimulated efflux of D-glucose was not blocked by phlorizin, which completely prevented efflux in the absence of this bile salt. These results suggest that selected bile salts inhibit Na+-coupled accumulation of D-glucose and L-alanine by enhancing the rate of dissipation of the Na+ gradient required for substrate accumulation. In addition, bile salts may also decrease D-glucose and L-alanine accumulation by increasing the rate of efflux of these substrates across the brush-border plasma membrane.

Intestinal microvilli: responses to feeding and fasting.

Membrane-bound bodies are formed individually and in groups from the plasma membrane surrounding the microvilli in cells of hamster small intestine. These bodies are vesiculate structures, are slightly smaller in diameter than the microvilli from which they are released, and retain at least a portion of glycocalyx. The production of the membrane-bodies occurs regularly, perhaps continuously in vivo, and appears to be related to the feeding cycle. The brush border of jejunal cells from animals which had been feeding ad lib contained more membrane-bodies andhad taller microvilli than comparable cells from those which had been fasted for 48 hours. Since membrane-bodies are also found among the microvilli of cells which have no digestive function, we propose that their formation and release may be of general significance in the normal functioning of active epithelial cells.

Sodium ion-coupled uptake of taurocholate by intestinal brush-border membrane vesicles.

The uptake of taurocholate was studied in membrane vesicles isolated from brush borders of hamster jejunum and ileum. When an extra- to intra-vesicular gradient of Na+ ions was present ileal vesicles took up 10 times more taurocholate than did jejunal vesicles. Accumulation of taurocholate by ileal vesicles was transient and was due to transport of this bile salt into an osmotically active intravesicular space rather than simple binding. Uptake of taurocholate was specifically dependent on Na+ ions; NaCl and Na2SO4 were capable of supporting accumulation, whereas KCl, LiCl and mannitol were not. Na+-coupled uptake of taurocholate into ileal vesicles was inhibited by other trihydroxy bile salts, by preloading the vesicles with Na+ and by simultaneous flow of glucose into the vesicles. Similarly, vesicular uptake of glucose was inhibited by simultaneous uptake of taurocholate. These results demonstrated that brush-border membrane vesicles prepared from ileum possess an Na+-coupled co-transport system for taurocholate that is similar to the active bile-salt transport system present in the intact ileum.

Incorporation of D-glucose-, L-alanine- and phosphate-transport systems from rat renal brush-border membranes into liposomes.

An extract of soluble proteins was prepared from a rat kidney brush-border membranes by Triton X-100 solubilization followed by centrifugation for 1 h at 100000g. Its protein composition was markedly different from that of the brush-border membranes. Proteoliposomes were formed by co-sonication of the Triton X-100-free extract with a naturally occurring mixture of phospholipids extracted from rat kidney. These proteoliposomes were shown to contain Na+-stimulated D-glucose-, L-alanine- and phosphate-transport systems.

Uptake of 3-O-methyl-(14)C-D-glucose by a unicellular blue-green alga.

The uptake of 3-O-methyl-(14)C-D-glucose, a non-metabolizable sugar, by autotrophically grown Synechococcus cedrorum was studied at low sugar concentrations in the incubation medium (0.71-11.36 µM), in the light and in the dark. Optimum sugar accumulation against a concentration gradient occurred within dark-treated "starved" cells that were incubated in the light. This phenomenon was greatly inhibited by metabolic inhibitors; it was much less when sugar uptake was observed in the dark. Control cells incubated in the light accumulated 3-O-methyl-(14)C-D-glucose against a concentration gradient only at lower sugar concentrations (0.71-2.84 µM) and to a lesser extent than the dark-treated cells. Sugar uptake against a concentration gradient by the control cells was completely inhibited in the dark. The results indicate that the morphologically simple unicellular blue-green alga, S. cedrorum, is under certain conditions capable of obtaining a sugar from its medium by an active transport process.

Active sugar transport by the small intestine. The effects of sugars, amino acids, hexosamines, sulfhydryl-reacting compounds, and cations on the preferential binding of D-glucose to tris-disrupted brush borders.

Tris-disrupted and intact brush border membrane preparations from mucosa of hamster jejunum were capable of preferentially binding actively transported D-glucose in a similar manner. Density gradient centrifugation of the Tris-disrupted brush borders indicated that D-glucose was bound to a fraction containing the cores or inner material of the microvilli. The properties of this binding were examined with the Tris-disrupted brush border preparation. Actively transported sugars competitively inhibited preferential D-glucose binding, whereas no effect was observed with nonactively transported sugars. Neither actively nor nonactively transported amino acids affected D-glucose binding. D-Glucosamine, which is not actively transported, was inhibitory to preferential D-glucose binding as well as to the active transport of D-glucose by everted sacs of hamster jejunum. No inhibitory effect was observed with the same concentration of D-galactosamine. Preferential D-glucose binding was also inhibited by sulfhydryl-reacting compounds, Ca(2+), and Li(+) ions. On the other hand, Mg(2+) was shown to be stimulatory and Na(+), NH(4) (+), and K(+) had no effect on this phenomenon. The results of these experiments suggest that preferential D-glucose binding to brush borders is related to the initial step in active sugar transport by the small intestine.