Year-end Sale % OFF 
Abstract
To investigate whether the C-terminal loop 13 of rabbit sodium/glucose cotransporter SGLT1 is involved in the recognition of the substrate d-glucose, isolated loop 13 (amino acids (aa) 541-638) was immobilized to a lipid bilayer. Interactions were investigated by surface plasmon resonance spectroscopy using an antibody directed against the late part of the loop (aa 606-631) or the glucoside transport inhibitor phlorizin. Specific binding of the antibody to the loop could be detected. The number of bound antibodies decreased upon the addition of d-glucose but not upon the addition of l-glucose. Phlorizin also significantly lowered the number of bound antibodies. Binding of phlorizin to the loop could also be demonstrated directly. Binding of phlorizin was, however, reduced to a similar extent upon the addition of either d-glucose or l-glucose, indicating their unspecific competition with the inhibitor's sugar moiety. Thus, the presence of a stereospecific glucose interaction site in the late part of the loop and a second, but non-stereospecific, sugar binding site on the same loop was assumed. To investigate whether the early part of loop 13 contains this non-stereospecific sugar binding site, peptides containing aa 541-598 were expressed in Escherichia coli and purified. Both d-glucose and l-glucose quenched the peptides tryptophan fluorescence and reduced the Trp accessibility to acrylamide to a similar degree. In view of the recently proposed transmembrane orientation of loop 13, the two binding sites may be part of the extracellular (stereospecific) and intracellular (non-stereospecific) sugar interaction sites of SGLT1.
Highlights
In the human kidney two transporters accomplish proximaltubular glucose resorption on the luminal side of tubule cells; whereas the low affinity sodium/glucose cotransporter SGLT23
Is located in the early proximal kidney tubule, the high affinity sodium/glucose cotransporter SGLT1 can be found in the late proximal tubule [1]
SGLT1 and SGLT2 are secondary active sodium/glucose symporters, i.e. they use the electrochemical sodium gradient across the membrane, which is maintained by the basolateral Naϩ/Kϩ-ATPase, to accumulate their substrates within the cell
Summary
Preparation of Lipid Vesicles and Solutions—For preparation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) vesicles, 2 mg of POPC was dissolved in 2 ml of CHCl3. Stock solutions of sugars and inhibitors were freshly prepared before experiments Dilution of these components with glutathione-containing HEPES buffer to the desired final concentrations (10 mM sugar, 0.5 mM phlorizin, 0.3 mM phloretin, 0.3 mM arbutin) was done immediately before injection into the BIACORE. 3.5 g/ml PAN3-2 or 0.5 mM phlorizin was injected into FC2 to get a reference value for unblocked interaction between immobilized loop 13 and the antibody or the inhibitor, respectively. Sugar or inhibitor (acting as blocking reagent of PAN3-2 or phlorizin binding, respectively, during experiments) was injected in FC1-2 to interact with unaffected loop 13 in FC1 and to show if it can remove already bound antibody/phlorizin from peptides in FC2.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
