Expression of size-selected mRNA encoding the intestinal Na/glucose cotransporter in Xenopus laevis oocytes.

Abstract

The expression of the rabbit intestinal brushborder Na/glucose cotransporter has been studied in Xenopus oocytes. Poly(A)I RNA isolated from the intestinal mucosa was injected into oocytes, and the expression of the transporter in the oocyte plasma membrane was assayed by measuring the Na-dependent phlorizin-sensitive uptake of methyl a-D-[1"Clglucopyranoside (MeGlc). Expression of the glucose carrier was detected 3-7 days after mRNA injection, and the rate of glucose transport was proportional to the amount of mRNA injected. mRNA (50 ng) increased the maximum velocity (V.)of MeGlc uptake by as much as 10-fold over background. The total mRNA was fractionated by preparative agarose gel electrophoresis and each fraction was assayed for its ability to induce transport activity. The mRNA encoding the Na/ glucose cotransporter was found in a single fraction of "z2.3 kilobases (kb), which contained 3% of the total mRNA. A similar mRNA fraction (2.0-2.6 kb) isolated from colon did not induce expression of this transporter. In vitro translation of the fractionated intestinal mRNA showed enhanced synthesis of two protein bands at 57 and 63 kDa. The mRNA encoding the cotransporter is smaller (2.3 kb) than that (2.6-2.9 kb) encoding the 55-kDa facilitated glucose carrier in human hepatoma cells and rat brain.Active glucose absorption across the small intestine is performed by enterocytes at the tip of the villus. Transport occurs in two stages: the first is intracellular accumulation of the sugar across the brush-border membrane by a sodium/ glucose cotransporter, and the second is facilitated diffusion of sugar out of the cell across the basolateral membrane.Recently, the sodium/glucose cotransporter has been identified as a 75-kDa polypeptide (1, 2), and some progress has been made in the characterization of this transport protein (3). The facilitated glucose carrier in the basolateral membrane is probably similar, if not identical, to the 55-kDa glucose carrier in human erythrocytes (see refs. 4-6).Intestinal glucose absorption exhibits adaptive regulation with diet, starvation, diabetes, gestation, lactation, and aging (7). For example, in diabetes mellitus, the maximal velocity (Vmax) of sodium-dependent glucose transport increases, and this is probably due to an increase in the number of sodium/glucose cotransporters. There is also a genetic component, and this is best exemplified by the rare hereditary disorder called primary glucose/galactose malabsorption (see ref. 8), which again is probably due to a defect in the cotransporter.As a first step in cloning the gene for the intestinal sodium/glucose cotransporter, we have attempted to express the carrier in Xenopus laevis oocytes and to identify the size of the mRNA encoding the 75-kDa protein. Amphibian oocytes translate foreign mRNAs very efficiently (9) and have been successfully exploited to express functional membrane transport proteins ranging from ion channels to ion exchangers (10-12). Here we demonstrate that Xenopus oocytes successfully translate i...

Glycyl-l-proline transport in rabbit enterocyte basolateral-membrane vesicles

“…It is absent from basolateral membranes [19,25,26]. The 75 kDa Na+/glucose symporter is also specific to the brush-border membrane [28,29]. The major histocompatibility antigen (anti-RLA Class 1) is uniquely located on the basolateral membrane [19,25].…”

“…After electrotransfer on to cellulose nitrate sheets, the potential of the proteins to interact with the monoclonal antibody 23B921 raised against the 140 kDa antigen [19], the polyclonal antibodies raised against the major Vol. 269 histocompatibility antigen (anti-RLA class I) [30,31] and polyclonal antibodies raised against the Na+/glucose symporter [28,29] was assessed. As shown in Fig.…”

Arabidopsis POLYOL TRANSPORTER5, a New Member of the Monosaccharide Transporter-Like Superfamily, Mediates H+-Symport of Numerous Substrates, Including myo-Inositol, Glycerol, and Ribose

“…The encoded SMIT1 protein shows significant homology to the animal Na þ /glucose symporter SGLT1 (Hediger et al, 1987), but both Na þ symporters do not share significant homology with the plant MST-like genes. Energy-dependent inositol transporter genes (ITR1 and ITR2) that are likely to be H þ -symporters have also been cloned from yeast (Nikawa et al, 1991).…”

The human kidney low affinity Na+/glucose cotransporter SGLT2. Delineation of the major renal reabsorptive mechanism for D-glucose.

“…cRNA was in vitro transcribed from clone Hu 14 (SGLT2) using T3 RNA polymerase ( 18). Xenopus laevis oocyte expression studies ( 19) were performed as described previously (20) with minor modifications. Briefly, collagenase-treated and manually defolliculated oocytes were injected with 50 nl of water or cRNA (50 ng/oocyte).…”