Ctc

where C is the concentration of Sout (initially, the concentration of Sin = 0). Vmax, the rate of transport when all molecules of GLUT1 contain a bound S, occurs at an infinitely high Sout concentration. The lower the value of Km, the more tightly the substrate binds to the transporter, and the greater the transport rate at a fixed concentration of substrate. Equation 7-1 describes the curve for glucose uptake by erythrocytes shown in Figure 7-3 as well as similar curves for other uniporters.

For GLUT1 in the erythrocyte membrane, the Km for glucose transport is 1.5 millimolar (mM); at this concentration roughly half the transporters with outward-facing binding sites would have a bound glucose and transport would occur at 50 percent of the maximal rate. Since blood glucose is normally 5 mM, the erythrocyte glucose transporter usually is functioning at 77 percent of the maximal rate, as can be seen from Figure 7-3. GLUT1 and the very similar GLUT3 are expressed by erythrocytes and other cells that need to take up glucose from the blood continuously at high rates; the rate of glucose uptake by such cells will remain high regardless of small changes in the concentration of blood glucose.

In addition to glucose, the isomeric sugars d-mannose and d-galactose, which differ from d-glucose in the configuration at only one carbon atom, are transported by GLUT1 at measurable rates. However, the Km for glucose (1.5 mM) is much lower than the Km for d-mannose (20 mM) or d-galactose (30 mM). Thus GLUT1 is quite specific, having a much higher affinity (indicated by a lower Km) for the normal substrate d-glucose than for other substrates.

GLUT1 accounts for 2 percent of the protein in the plasma membrane of erythrocytes. After glucose is transported into the erythrocyte, it is rapidly phosphorylated, forming glucose 6-phosphate, which cannot leave the cell. Because this reaction, the first step in the metabolism of glucose (see Figure 8-4), is rapid, the intracellular concentration of glucose does not increase as glucose is taken up by the cell. Consequently, the glucose concentration gradient across the membrane is maintained, as is the rate of glucose entry into the cell.

The Human Genome Encodes a Family of Sugar-Transporting GLUT Proteins

The human genome encodes 12 proteins, GLUT1-GLUT12, that are highly homologous in sequence, and all are thought to contain 12 membrane-spanning a helices. Detailed studies on GLUT1 have shown that the amino acid residues in the transmembrane a helices are predominantly hydrophobic; several helices, however, bear amino acid residues (e.g., serine, threonine, asparagine, and glutamine) whose side chains can form hydrogen bonds with the hydroxyl groups on glucose. These residues are thought to form the inward-facing and outward-facing glucose-binding sites in the interior of the protein (see Figure 7-4).

The structures of all GLUT isoforms are quite similar, and all transport sugars. Nonetheless, their differential expression in various cell types and isoform-specific functional properties enable different body cells to regulate glucose metabolism independently and at the same time maintain a constant concentration of glucose in the blood. For instance, GLUT2, expressed in liver and the insulin-secreting p cells of the pancreas, has a Km of «20 mM, about 13 times higher than the Km of GLUT1. As a result, when blood glucose rises from its basal level of 5 mM to 10 mM or so after a meal, the rate of glucose influx will almost double in GLUT2-expressing cells, whereas it will increase only slightly in GLUT1-expressing cells (see Figure 7-3). In liver, the "excess" glucose brought into the cell is stored as the polymer glycogen. In islet p cells, the rise in glucose triggers secretion of the hormone insulin, which in turn lowers blood glucose by increasing glucose uptake and metabolism in muscle and by inhibiting glucose production in liver.

Another GLUT isoform, GLUT4, is expressed only in fat and muscle cells, the cells that respond to insulin by increasing their uptake of glucose, thereby removing glucose from the blood. In the absence of insulin, GLUT4 is found in in-tracellular membranes, not on the plasma membrane, and obviously is unable to facilitate glucose uptake. By a process detailed in Chapter 15, insulin causes these GLUT4-rich internal membranes to fuse with the plasma membrane, increasing the number of GLUT4 molecules on the cell surface and thus the rate of glucose uptake. Defects in this process, one principal mechanism by which insulin lowers blood glucose, lead to diabetes, a disease marked by continuously high blood glucose.

In contrast to GLUT1-GLUT4, which all transport glucose at physiological concentrations, GLUT5 transports fructose. The properties of other members of the GLUT family have not yet been studied in detail.

Transport Proteins Can Be Enriched Within Artificial Membranes and Cells

Although transport proteins can be isolated from membranes and purified, the functional properties of these proteins can be studied only when they are associated with a membrane. Most cellular membranes contain many different types of transport proteins but a relatively low concentration of any particular one, making functional studies of a single protein difficult. To facilitate such studies, researchers use two ap-

▲ EXPERIMENTAL FIGURE 7-5 Liposomes containing a single type of transport protein are very useful in studying functional properties of transport proteins.

Here, all the Integral proteins of the erythrocyte membrane are solubilized by a nonionic detergent, such as octylglucoside. The glucose uniporter GLUT1 can be purified by chromatography on a column containing a specific monoclonal antibody and then incorporated into liposomes made of pure phospholipids.

proaches for enriching a transport protein of interest so that it predominates in the membrane.

In one common approach, a specific transport protein is extracted and purified; the purified protein then is reincorporated into pure phospholipid bilayer membranes, such as liposomes (Figure 7-5). Alternatively, the gene encoding a specific transport protein can be expressed at high levels in a cell type that normally does not express it. The difference in transport of a substance by the transfected and by control nontransfected cells will be due to the expressed transport protein. In these systems, the functional properties of the various membrane proteins can be examined without ambiguity.

Other transport protein

Other transport protein

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